U.S. patent application number 14/213520 was filed with the patent office on 2014-09-18 for biocompatible hydrogel polymer matrix for delivery of cells.
This patent application is currently assigned to Medicus Biosciences LLC. The applicant listed for this patent is Medicus Biosciences LLC. Invention is credited to Syed H. ASKARI, George Horng.
Application Number | 20140271767 14/213520 |
Document ID | / |
Family ID | 51527906 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140271767 |
Kind Code |
A1 |
ASKARI; Syed H. ; et
al. |
September 18, 2014 |
BIOCOMPATIBLE HYDROGEL POLYMER MATRIX FOR DELIVERY OF CELLS
Abstract
Provided herein are biocompatible hydrogel polymer matrices,
which are prepared from biocompatible pre-formulations. The
biocompatible pre-formulations comprise at least one nucleophilic
compound, at least one electrophilic compound, and at least one
cell. The biocompatible hydrogel polymer matrix is bioabsorbable
and releases the cell at a target site, achieving a controlled
delivery. The biocompatible hydrogel polymer matrix provides a
solid support conducive for cell viability and functionality. The
cells may grow on the hydrogel polymer surface of inside the
hydrogel polymer matrix.
Inventors: |
ASKARI; Syed H.; (San Jose,
CA) ; Horng; George; (Millbrae, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medicus Biosciences LLC |
San Jose |
CA |
US |
|
|
Assignee: |
Medicus Biosciences LLC
San Jose
CA
|
Family ID: |
51527906 |
Appl. No.: |
14/213520 |
Filed: |
March 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61785477 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
424/422 ;
424/486; 424/93.7 |
Current CPC
Class: |
A61L 2430/34 20130101;
A61L 26/008 20130101; A61K 31/505 20130101; A61L 2300/404 20130101;
A61L 2430/24 20130101; A61K 31/635 20130101; A61K 31/573 20130101;
A61P 17/02 20180101; A61L 27/18 20130101; A61K 31/728 20130101;
A61L 27/3604 20130101; A61L 2400/06 20130101; A61P 19/02 20180101;
A61K 49/0409 20130101; A61K 35/28 20130101; A61L 2300/206 20130101;
A61K 31/00 20130101; A61L 27/3695 20130101; A61K 49/0457 20130101;
A61K 33/00 20130101; A61L 27/52 20130101; A61P 29/00 20180101; A61K
9/06 20130101; A61K 49/0404 20130101; A61K 31/785 20130101; A61K
31/795 20130101; A61K 9/0024 20130101; A61K 31/155 20130101; A61L
26/0019 20130101; A61L 27/54 20130101; A61K 45/06 20130101; A61K
47/10 20130101; A61L 2300/41 20130101; A61L 26/0066 20130101; A61K
47/34 20130101; A61K 47/00 20130101; A61K 31/155 20130101; A61K
2300/00 20130101; A61K 31/573 20130101; A61K 2300/00 20130101; A61K
31/635 20130101; A61K 2300/00 20130101; A61K 31/505 20130101; A61K
2300/00 20130101; A61K 31/728 20130101; A61K 2300/00 20130101; A61K
33/00 20130101; A61K 2300/00 20130101; A61L 27/18 20130101; C08L
71/02 20130101 |
Class at
Publication: |
424/422 ;
424/93.7; 424/486 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 35/28 20060101 A61K035/28 |
Claims
1. A fully synthetic, polyglycol-based biocompatible hydrogel
polymer matrix comprising a fully synthetic, polyglycol-based
biocompatible hydrogel polymer comprising at least one first
monomeric unit bound through at least one amide, thioester, or
thioether linkage to at least one second monomeric unit, wherein
the polymer forms the matrix that encapsulates: (a) at least one
cell; and (b) a culture medium which supports the growth of the at
least one cell; and wherein the fully synthetic, polyglycol-based
biocompatible hydrogel polymer matrix provides controlled release
of the at least one cell, when implanted at a target site in an
animal's body, to the target site of the animal's body.
2. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the at least one first monomeric unit is PEG-based
and fully synthetic, and wherein the at least one second monomeric
unit is PEG-based and fully synthetic.
3. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the cell is selected from a mammalian cell, insect
cell, protozoal cell, bacterial cell, viral cell, or fungal
cell.
4. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 3, wherein the mammalian cell is a stem cell.
5. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the culture medium comprises a growth factor.
6. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the first monomeric unit is derived from a
MULTIARM-(5-50k)-SH, a MULTIARM-(5-50k)-NH2 or a
MULTIARM-(5-50k)-AA monomer and the second monomeric unit is
derived from a MULTIARM-(5-50k)-SG, a MULTIARM-(5-50k)-SGA, or a
MULTIARM-(5-50k)-SS monomer.
7. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 6, wherein the first monomeric unit is derived from a
4ARM-5k-SH, 4ARM-2k-NH2, 4ARM-5k-NH2, 8ARM-20k-NH2, 4ARM-20k-AA, or
8ARM-20k-AA monomer, and the second monomeric unit is derived from
a 4ARM-10k-SG, 8ARM-15k-SG, 4ARM-20k-SGA, or 4ARM-20k-SS
monomer.
8. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the animal is a human.
9. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the polyglycol-based biocompatible hydrogel
polymer matrix is bioabsorbed within about 14 to 180 days.
10. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the controlled release of the at least one cell to
the target site of the animal's body comprises diffusion of the at
least one cell from the polyglycol-based biocompatible hydrogel
polymer matrix.
11. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 1, wherein the controlled release of the at least one cell to
the target site of the animal's body is at least partially through
degradation and bioabsorption of the polyglycol-based biocompatible
hydrogel polymer matrix.
12. A fully synthetic polyglycol-based biocompatible
pre-formulation, comprising: (a) at least one fully synthetic
polyglycol-based first compound comprising more than one
nucleophilic group; (b) at least one fully synthetic
polyglycol-based second compound comprising more than one
electrophilic group; (c) at least one cell; and (d) a culture
medium that supports growth of the at least one cell; wherein the
polyglycol-based biocompatible pre-formulation at least in part
polymerizes and/or gels to form a polyglycol-based biocompatible
hydrogel polymer matrix encapsulating the cell in the presence of
water.
13. The polyglycol-based biocompatible pre-formulation of claim 12,
wherein the cell is a mammalian stem cell.
14. The polyglycol-based biocompatible pre-formulation of claim 12,
wherein the culture medium comprises a buffer.
15. The polyglycol-based biocompatible pre-formulation of claim 12,
wherein the first compound is a MULTIARM-(5-50k)-SH, a
MULTIARM-(5-50k)-NH2, a MULTIARM-(5-50k)-AA, or a combination
thereof, and the second compound is a MULTIARM-(5-50k)-SG, a
MULTIARM-(5-50k)-SGA, a MULTIARM-(5-50k)-SS, or a combination
thereof.
16. The polyglycol-based biocompatible pre-formulation of claim 15,
wherein the first compound is 4ARM-5k-SH, 4ARM-2k-NH2, 4ARM-5k-NH2,
8ARM-20k-NH2, 4ARM-20k-AA, 8ARM-20k-AA, and a combination thereof,
and the second compound is 4ARM-10k-SG, 8ARM-15k-SG, 4ARM-20k-SGA,
4ARM-20k-SS, or a combination thereof.
17. The polyglycol-based biocompatible pre-formulation of preceding
claim 16, wherein the first compound is 8ARM-20k-NH2 and/or
8ARM-20k-AA, and the second compound is 4ARM-20k-SGA.
18. The polyglycol-based biocompatible pre-formulation of claim 12,
wherein the polyglycol-based biocompatible pre-formulation gels to
form a polyglycol-based biocompatible hydrogel polymer matrix in
between about 20 seconds and 10 minutes.
19. The polyglycol-based biocompatible hydrogel polymer matrix of
claim 12.
20. The method of treating a disease by administering the
polyglycol-based biocompatible hydrogel polymer matrix of claim 1.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/785,477, filed Mar. 14, 2013, which application
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Cell based therapies are important options for the treatment
of clinical indications including diseases, tissue damage,
neurological disorders, blood disorders, cancers, developmental
defects, wounds and orthopedic impediments. Many cell based
therapies are target specific, with cells being administered
directly to a target site. When cells are not suitably retained at
a target site after administration, there is both a loss of cells
available for the intended treatment as well as an increased risk
of cell differentiation at an alternative site. When cells are
administered to a target site without sufficient protection, the
cells may go through physiochemical changes such as hypertrophy,
necrosis, apoptosis, or senescence. Treatment efficacy is
attenuated when the administered cells are physiochemically altered
or not retained at the desired target site.
SUMMARY OF THE INVENTION
[0003] In one aspect, provided herein is a fully synthetic,
polyglycol-based biocompatible hydrogel polymer matrix comprising a
fully synthetic, polyglycol-based biocompatible hydrogel polymer
comprising at least one first monomeric unit bound through at least
one amide, thioester, or thioether linkage to at least one second
monomeric unit, wherein the polymer forms the matrix that
encapsulates at least one cell; and a culture medium which supports
the growth of the at least one cell. In certain embodiments, the
fully synthetic, polyglycol-based biocompatible hydrogel polymer
matrix provides controlled release of the at least one cell, when
implanted at a target site in an animal's body, to the target site
of the animal's body. In some embodiments, the at least one first
monomeric unit is PEG-based and fully synthetic, and the at least
one second monomeric unit is PEG-based and fully synthetic. In
certain embodiments, the cell is selected from a mammalian cell,
insect cell, protozoal cell, bacterial cell, viral cell, or fungal
cell. In some embodiments, the mammalian cell is a stem cell. In
certain embodiments, the culture medium comprises a growth
factor.
[0004] In another aspect, provided herein is a polyglycol-based
biocompatible hydrogel polymer matrix comprising at least one first
monomeric unit bound through at least one amide, thioester, or
thioether linkage to at least one second monomeric unit; a culture
medium; and at least one cell. In certain embodiments, the
polyglycol-based biocompatible hydrogel polymer matrix is fully
synthetic. In certain embodiments, the polyglycol-based
biocompatible hydrogel polymer matrix is based on polyethylene
glycol (PEG), polypropylene glycol (PPG), polybutylene glycol
(PBG), or a co-polymer thereof. In some embodiments, the
polyglycol-based biocompatible hydrogel polymer matrix is fully
synthetic and PEG-based. In certain embodiments, the at least one
first monomeric unit is PEG-based and fully synthetic, and wherein
the at least one second monomeric unit is PEG-based and fully
synthetic. In certain embodiments of the polyglycol-based
biocompatible hydrogel polymer, the polyglycol-based biocompatible
hydrogel polymer matrix encapsulates the cell. In some embodiments
of the polyglycol-based biocompatible hydrogel polymer, the
polyglycol-based biocompatible hydrogel polymer supports the
viability and growth of cells on the surface of the polymer. In
certain embodiments of the polyglycol-based biocompatible hydrogel
polymer, the polyglycol-based biocompatible hydrogel polymer
supports the viability and growth of cells within the
polyglycol-based biocompatible hydrogel polymer matrix. In some
embodiments, the cell is microorganism. In certain embodiments, the
cell is selected from a mammalian cell, insect cell, protozoal
cell, bacterial cell, viral cell, or fungal cell. In one
embodiment, the mammalian cell is a stem cell.
[0005] In some embodiments of the polyglycol-based biocompatible
hydrogel polymer matrix, the culture medium comprises a growth
medium. In certain embodiments, the culture medium comprises a
growth factor. In some embodiments, the polyglycol-based
biocompatible hydrogel polymer matrix releases the cell at a target
site of a human body. In certain embodiments, the at least one cell
is viable in the polyglycol-based biocompatible hydrogel polymer
matrix for at least one hour. In some embodiments, the at least one
cell is viable in the polyglycol-based biocompatible hydrogel
polymer matrix for at least 5 days. In certain embodiments, the at
least one cell is proliferates and grows in the polyglycol-based
biocompatible hydrogel polymer matrix. In certain embodiments, the
controlled release of the at least one cell to the target site of
the animal's body comprises diffusion of the at least one cell from
the polyglycol-based biocompatible hydrogel polymer matrix. In some
embodiments, the controlled release of the at least one cell to the
target site of the animal's body is at least partially through
degradation and bioabsorption of the polyglycol-based biocompatible
hydrogel polymer matrix.
[0006] In certain embodiments of the polyglycol-based biocompatible
hydrogel polymer matrix, the first monomeric unit is derived from a
MULTIARM-(5-50k)-SH, a MULTIARM-(5-50k)-NH2 or a
MULTIARM-(5-50k)-AA monomer. In some embodiments, the first
monomeric unit is a glycol, trimethylolpropane, glycerol,
diglycerol, pentaerythritol, sorbitol, hexaglycerol,
tripentaerythritol, or polyglycerol derivative. In certain
embodiments, MULTIARM is selected from 2ARM, 3ARM, 4ARM, 6ARM, and
8ARM. In some embodiments, the first monomeric unit comprises one
or more polyethylene glycol sections. In certain embodiments, the
first monomeric unit is derived from a 4ARM-5k-SH, 4ARM-2k-NH2,
4ARM-5k-NH2, 8ARM-20k-NH2, 4ARM-20k-AA, or 8ARM-20k-AA monomer.
[0007] In some embodiments of the polyglycol-based biocompatible
hydrogel polymer matrix, the second monomeric unit is derived from
a MULTIARM-(5-50k)-SG, a MULTIARM-(5-50k)-SGA, or a
MULTIARM-(5-50k)-SS monomer. In certain embodiments, the second
monomeric unit is a glycol, trimethylolpropane, glycerol,
diglycerol, pentaerythritol, sorbitol, hexaglycerol,
tripentaerythritol, or polyglycerol derivative. In some
embodiments, MULTIARM is selected from 2ARM, 3ARM, 4ARM, 6ARM, and
8ARM. In certain embodiments, the second monomeric unit comprises
one or more polyethylene glycol sections. In some embodiments, the
second monomeric unit is derived from a 4ARM-10k-SG, 8ARM-15k-SG,
4ARM-20k-SGA, or 4ARM-20k-SS monomer.
[0008] In another aspect, provided herein is a polyglycol-based
biocompatible pre-formulation, comprising at least one first
compound comprising more than one nucleophilic group, at least one
second compound comprising more than one electrophilic group, at
least one cell, and a culture medium, wherein the polyglycol-based
biocompatible pre-formulation at least in part polymerizes and/or
gels to form a polyglycol-based biocompatible hydrogel polymer
matrix encapsulating the cell in the presence of water. In certain
embodiments, the biocompatible pre-formulation is fully synthetic.
In some embodiments, the culture medium supports the growth of the
at least one cell. In certain embodiments, the biocompatible
pre-formulation is based on polyethylene glycol (PEG),
polypropylene glycol (PPG), polybutylene glycol (PBG), or a
co-polymer thereof. In some embodiments, the biocompatible
pre-formulation is PEG-based. In certain embodiments, the
biocompatible pre-formulation is fully synthetic and PEG-based.
[0009] In certain embodiments of the polyglycol-based biocompatible
pre-formulation, the cell is a microorganism. In some embodiments,
the cell is a mammalian cell, insect cell, protozoal cell,
bacterial cell, viral cell, or fungal cell. In certain embodiments,
the mammalian cell is a stem cell.
[0010] In some embodiments of the polyglycol-based biocompatible
pre-formulation, the culture medium comprises a growth medium. In
certain embodiments, the culture medium comprises a growth
factor.
[0011] In some embodiments of the polyglycol-based biocompatible
pre-formulation, the nucleophilic group comprises a thiol or amino
group. In certain embodiments, the electrophilic group comprises an
epoxide, N-succinimidyl succinate, N-succinimidyl glutarate,
N-succinimidyl succinamide or N-succinimidyl glutaramide. In
certain embodiments, the first compound and the second compound
comprise one or more polyglycol sections. In some embodiments, the
first compound is selected from a MULTIARM-(5-50k)-SH, a
MULTIARM-(5-50k)-NH2 and a MULTIARM-(5-50k)-AA and the second
compound is selected from a MULTIARM-(5-50k)-SG, a
MULTIARM-(5-50k)-SGA and a MULTIARM-(5-50k)-SS. In certain
embodiments, the first compound and the second compound are
independently a glycol, trimethylolpropane, glycerol, diglycerol,
pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol, or
polyglycerol derivative. In some embodiments, the MULTIARM is
independently selected from 2ARM, 3ARM, 4ARM, 6ARM and 8ARM.
[0012] In certain embodiments of the polyglycol-based biocompatible
pre-formulation, the first compound is selected from 4ARM-5k-SH,
4ARM-2k-NH2, 4ARM-5k-NH2, 8ARM-20k-NH2, 4ARM-20k-AA and
8ARM-20k-AA, and the second compound is selected from 4ARM-10k-SG,
8ARM-15k-SG, 4ARM-20k-SGA, and 4ARM-20k-SS. In a specific
embodiment, the first compound is 8ARM-20k-NH2 and/or 8ARM-20k-AA,
and the second compound is 4ARM-20k-SGA.
[0013] In some embodiments of the polyglycol-based biocompatible
pre-formulation, the at least one first compound is a
polyglycol-based, fully synthetic, biocompatible compound
comprising one or more nucleophilic groups, and the at least one
second compound is a polyglycol-based, fully synthetic,
biocompatible compound comprising one or more electrophilic groups.
In certain embodiments of the polyglycol-based biocompatible
pre-formulation, the at least one first compound is a PEG-based,
fully synthetic, biocompatible compound comprising one or more
nucleophilic groups, and the at least one second compound is a
PEG-based, fully synthetic, biocompatible compound comprising one
or more electrophilic groups.
[0014] In certain embodiments of the polyglycol-based
biocompatible, the polyglycol-based biocompatible pre-formulation
gels to form a polyglycol-based biocompatible hydrogel polymer
matrix in between about 20 seconds and 10 minutes. In some
embodiments, the polyglycol-based biocompatible hydrogel polymer
matrix gels at a predetermined time.
[0015] In certain embodiments of the polyglycol-based biocompatible
pre-formulation, the first compound and the second compound do not
react with the cell during formation of the polyglycol-based
biocompatible hydrogel polymer matrix. In some embodiments, the
cell remains unchanged after formation of the polyglycol-based
biocompatible hydrogel polymer matrix. In certain embodiments, the
viability of the cell is not affected by the formation of the
polyglycol-based biocompatible hydrogel polymer matrix. In some
embodiments, the viability of the cell is not affected by the
polyglycol-based biocompatible hydrogel polymer matrix. In certain
embodiments, the physiochemical properties of a wall of the cell
are not affected by the formation of the polyglycol-based
biocompatible hydrogel polymer matrix.
[0016] In certain embodiments, the polyglycol-based biocompatible
hydrogel polymer matrix is bioabsorbable. In some embodiments, the
polyglycol-based biocompatible hydrogel polymer matrix is
bioabsorbed within about 1 to 70 days. In other embodiments, the
polyglycol-based biocompatible hydrogel polymer matrix is
bioabsorbed within about 14 to 180 days. In certain embodiments,
the polyglycol-based biocompatible hydrogel polymer matrix is
substantially non-bioabsorbable.
[0017] In some embodiments, the cell is released from the
polyglycol-based biocompatible hydrogel polymer matrix through
diffusion, degradation of the polyglycol-based biocompatible
hydrogel polymer matrix, or any combination thereof. In certain
embodiments, the cell is initially released from the
polyglycol-based biocompatible hydrogel polymer matrix through
diffusion and later released through degradation of the
polyglycol-based biocompatible hydrogel polymer matrix. In some
embodiments, the cell is substantially released from the
polyglycol-based biocompatible hydrogel polymer matrix within 180
days. In certain embodiments, the cell is substantially released
from the polyglycol-based biocompatible hydrogel polymer matrix
within 14 days. In some embodiments, the cell is substantially
released from the polyglycol-based biocompatible hydrogel polymer
matrix within 24 hours. In other embodiments, the cell is
substantially released from the polyglycol-based biocompatible
hydrogel polymer matrix within one hour. In certain embodiments,
the release of the cell is essentially inhibited until a time that
the polyglycol-based biocompatible hydrogel polymer matrix starts
to degrade. In some embodiments, the polyglycol-based biocompatible
hydrogel polymer matrix has a pore size, wherein the pore size is
small enough to essentially inhibit the release of the cell before
the time that the polyglycol-based biocompatible hydrogel polymer
matrix starts to degrade. In certain embodiments, at least a
portion of the cell is released before the time that the
polyglycol-based biocompatible hydrogel polymer matrix starts to
degrade. In other embodiments, the polyglycol-based biocompatible
hydrogel polymer matrix has a pore size, wherein the pore size is
large enough to allow at least a partial release of the cell before
the time that the polyglycol-based biocompatible hydrogel polymer
matrix starts to degrade.
[0018] In certain embodiments, the polyglycol-based biocompatible
hydrogel polymer matrix minimizes the degradation or denaturing of
the cell. In some embodiments, the cell is protected from the
enzymes and pH conditions of the gastrointestinal tract. In certain
embodiments, the cell remains viable after release from the
polyglycol-based biocompatible hydrogel polymer matrix.
[0019] In a further aspect provided herein are methods of treating
a disease or condition by administering a polyglycol-based
biocompatible pre-formulation comprising more than one nucleophilic
group, at least one second compound comprising more than one
electrophilic group, at least one cell, and a culture medium,
wherein the polyglycol-based biocompatible pre-formulation at least
in part polymerizes and/or gels to form a polyglycol-based
biocompatible hydrogel polymer matrix encapsulating the cell in the
presence of water. In another aspect provided herein are method of
treating a disease or condition by administering a polyglycol-based
biocompatible hydrogel polymer matrix comprising at least one first
monomeric unit bound through at least one amide, thioester, or
thioether linkage to at least one second monomeric unit; a culture
medium; and at least one cell.
[0020] In another aspect provided herein is a polyglycol-based
biocompatible hydrogel polymer matrix comprising at least one first
monomeric unit bound through at least one amide, thioester, or
thioether linkage to at least one second monomeric unit, and a
culture medium. In a further aspect, provided herein is a
polyglycol-based biocompatible hydrogel polymer matrix comprising
at least one first monomeric unit bound through at least one amide,
thioester, or thioether linkage to at least one second monomeric
unit, and at least one cell.
[0021] In a further aspect, provided herein is a polyglycol-based
biocompatible pre-formulation, comprising at least one first
compound comprising more than one nucleophilic group, at least one
second compound comprising more than one electrophilic group, and
at least one cell, wherein the polyglycol-based biocompatible
pre-formulation at least in part polymerizes and/or gels to form a
polyglycol-based biocompatible hydrogel polymer matrix
encapsulating the cell in the presence of water. In another aspect,
provided herein is a polyglycol-based biocompatible
pre-formulation, comprising at least one first compound comprising
more than one nucleophilic group, at least one second compound
comprising more than one electrophilic group, and a culture medium,
wherein the polyglycol-based biocompatible pre-formulation at least
in part polymerizes and/or gels to form a polyglycol-based
biocompatible hydrogel polymer matrix in the presence of water.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0023] FIG. 1 shows the effect of addition of degradable acetate
amine 8ARM-20k-AA or 4ARM-20k-AA on degradation times. Degradations
occurred in phosphate buffered saline (PBS) at 37.degree. C.
[0024] FIG. 2 shows the effect of polymer concentration on
degradation time for 75% Acetate Amine formulation and 100% Acetate
Amine formulation.
[0025] FIG. 3 shows the effect of a polymer left in the air as the
percent of water weight loss over time.
[0026] FIG. 4 shows a sample plot generated by the Texture Analyzer
Exponent software running the firmness test. The peak force was
recorded as the polymer firmness, which represents the point where
the target penetration depth of 4 mm has been reached by the
probe.
[0027] FIG. 5 shows a sample plot generated by the Texture Analyzer
Exponent software running the elastic modulus test under
compression. The modulus was calculated from the initial slope of
the curve up to 10% of the maximum compression stress.
[0028] FIG. 6 shows an exemplary plot generated by the Texture
Analyzer Exponent software running the adhesion test. A contact
force of 100.0 g was applied for 10 seconds. The tack was measured
as the peak force after lifting the probe from the sample. The
adhesion energy or the work of adhesion was calculated as the area
under the curve representing the tack force (points 1 to 2). The
stringiness was defined as the distance traveled by the probe while
influencing the tack force (points 1 and 2).
[0029] FIG. 7 shows the firmness vs. degradation time plotted as
percentages for the polymer formulation: 8ARM-20k-AA/8ARM-20k-NH2
(70/30) & 4ARM-20k-SGA at 4.8% solution with 0.3% HPMC. The
error bars represent the standard deviations of 3 samples. The
degradation time for the polymer was 18 days.
[0030] FIG. 8 shows the chlorhexidine cumulative % elution.
[0031] FIG. 9 shows that for a polymer, the triamcinolone
cumulative % elution for 60, 90 and 240 day polymers.
[0032] FIG. 10 shows that for short degradation time version of the
hydrogel polymer loaded with Depo-Medrol, the methylprednisolone
cumulative % elution.
[0033] FIG. 11 shows that for long degradation time version of a
polymer loaded with Depo-Medrol, the methylprednisolone cumulative
% elution.
[0034] FIG. 12 shows the effect of solid phosphate powder
concentration on polymer gel time (A) and solution pH (B).
[0035] FIG. 13 shows the effect of sterilization on gel times for
polymers of various concentrations (A) and (B).
[0036] FIG. 14 shows the storage stability of kits at 5.degree. C.,
20.degree. C. and 37.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Cell therapy is used for many clinical indications in
multiple target sites and by several modes of cell delivery. Cells
are directed to a target site via local or systemic administration.
An important example of cell therapy comprises the steps of stem
cell engraftment, cell differentiation, and replacement of damaged
tissue, whereby the target tissue has improved function. Cell based
therapies can be administered to damaged tissue following events
such as myocardial infarction, infection or cancer treatment.
During and after administration of a cell based therapy to a target
site, the therapeutic cells may not be sufficiently protected.
Cells which are not protected may undergo apoptosis, necrosis,
hypertrophy, or senescence; thereby diminishing the efficacy of
treatment. Cell based therapies which are delivered in a suitable
environment allow for cell survival. In some instances, cells
delivered in a suitable environment proliferate, differentiate, and
integrate with target tissue. Cell based therapies which are
retained in a suitable environment at the target site allow for
proper cell functionality at the target site.
[0038] Cell based treatments administered systemically expose many
regions in the body to the administered cells in addition to the
target site. Diffusion of stem cells away from the target site
increases the risk of undesired cell differentiation and subsequent
complications. Importantly, the loss of cell retention at the
target site increases the amount of administered cells necessary
for therapeutic efficacy. Localized cell delivery directly to a
target site limits exposure of the administered cells to the areas
surrounding the target site. Localized cell delivery and cell
retention enables the administration of a controlled therapeutic
dose. In some instances, a therapeutic dose is controlled by
extended release of the cells. In some instances, localized cell
delivery treatments are more effective because dosages can be
increased with less concern for adverse side effects. In further
instances, extended release of the cells also reduces the number of
doses necessary in the course of treatment.
[0039] A biocompatible pre-formulation to form a biocompatible
hydrogel polymer matrix enables the administration and retention of
cells directly to target sites. The biocompatible pre-formulation
at least in part polymerizes and/or gels to form the biocompatible
hydrogel polymer matrix. The biocompatible hydrogel polymer matrix
comprises at least one cell. In some embodiments, the biocompatible
hydrogel matrix comprises a biocompatible hydrogel scaffold. The
biocompatible hydrogel polymer matrix provides structural and
nutritional support for the cells after administration of the
polymer matrix or pre-formulation to a target site. In some
embodiments, the biocompatible hydrogel scaffold provides
structural and nutritional support for the cells after
administration of the polymer matrix or pre-formulation to a target
site. In certain embodiments, the biocompatible hydrogel polymer
matrix provides structural and nutritional support for the cells
after administration of the polymer matrix or pre-formulation to a
target site. The biocompatible hydrogel polymer matrix enables the
cells to be retained at a target site for a pre-determined amount
of time. In certain embodiments, the biocompatible hydrogel polymer
matrix provides a protective and nutrient rich environment suitable
for cell survival, growth or proliferation. In certain embodiments,
the biocompatible hydrogel polymer matrix provides a protective and
nutrient rich environment suitable for cell survival,
proliferation, differentiation, and tissue integration. A
biocompatible pre-formulation to form a biocompatible hydrogel
polymer matrix further enables the controlled release of cells at
target sites. In certain embodiments, the controlled release of
cells at target sites is through diffusion, degradation of the
biocompatible hydrogel polymer matrix, or any combination thereof.
In some instances, the hydrogel polymer matrix is biodegradable. In
certain instances, delivery, retention, and controlled release of
the cells using a biocompatible hydrogel polymer matrix minimizes
cell hypertrophy, senescence, apoptosis, and necrosis. In some
instances, the biocompatible hydrogel polymer matrix protects the
cells from the enzymes and pH conditions of the gastrointestinal
tract. In some instances, the polymer matrix is configured to
maintain the physiochemical properties of the cells during
administration, retention, biocompatible hydrogel polymer matrix
degradation, or release of the cells to the target site. In some
instances, the cells remain viable during and after polymerization
of the biocompatible hydrogel polymer matrix. In some instances,
the cells remain viable when added to an already polymerized and/or
gelled biocompatible hydrogel polymer matrix. In some instances,
the cells remain viable when administered. In some instances, the
cells remain viable after delivery to a target site. In some
instances, the cells remain viable during release from the
biocompatible hydrogel polymer matrix. In some instances, the cells
remain viable during degradation of the biocompatible hydrogel
polymer matrix.
[0040] A biocompatible hydrogel polymer matrix enables the delivery
of cells to a target site where the cells will eventually be
released from the polymer matrix by diffusion, polymer matrix
degradation or any combination thereof. In some instances, polymer
matrix degradation times are controlled by varying the composition
of the biocompatible pre-formulation components allowing for the
appropriate application and placement of the biocompatible hydrogel
polymer matrix. In some instances, polymer matrix degradation times
are controlled by varying the pH of the pre-formulation allowing
for the appropriate application and placement of the biocompatible
hydrogel polymer matrix. In some instances, polymer matrix
degradation times are controlled by varying the concentrations of
the biocompatible pre-formulation components allowing for the
appropriate application and placement of the biocompatible hydrogel
polymer matrix. In some embodiments, the cells are released from
the biocompatible hydrogel polymer matrix in a precise and
consistent manner. In certain instances, the biocompatible hydrogel
polymer matrix is bioabsorbed over a defined period of time. In
some embodiments, the biocompatible hydrogel polymer matrix
provides the sustained release of cells at a target site. In
certain embodiments, the sustained and controlled release reduces
the systemic exposure to the cells. In certain embodiments, the
controlled release allows for cell retention at a target site. In
some instances, the cells are released from the biocompatible
hydrogel polymer matrix over an extended period of time. In certain
instances, delivery of the cells in a biocompatible hydrogel
polymer matrix provides a depot of the cells (e.g., under the
skin), wherein the depot releases the cells over an extended period
of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 10, days, 14 days, 3 weeks, 4 weeks). In some instances, the
biocompatible hydrogel polymer matrix releases the cells after a
delay as a delayed burst.
[0041] The biocompatible hydrogel polymer matrix comprising at
least one cell may start out as a liquid biocompatible
pre-formulation which is delivered to a target site using minimally
invasive techniques. The initial liquid state allows the
formulation to be delivered through small catheters directed by
endoscopes or other image guided techniques to the target site
(e.g., bronchoscope for lung, thoracoscope for the chest cavity,
laparoscope for the abdominal cavity, cystoscope for the bladder,
arthroscope for joint space, etc.). Once in the body, the liquid
formulation polymerizes into a biocompatible hydrogel polymer
matrix. In some instances, the biocompatible hydrogel polymer
matrix adheres to the tissue and the at least one cell is
maintained at the target site. In some instances, the biocompatible
hydrogel polymer matrix is delivered to a target site after
polymerization. In some instances, polymerization times are
controlled by varying the composition of the biocompatible
pre-formulation components allowing for the appropriate application
and placement of the biocompatible hydrogel polymer matrix. The
controlled gelling allows the use of the biocompatible hydrogel
polymer matrix to deliver at least one cell directly to the
affected target tissue, thereby minimizing systemic exposure. In
some embodiments, the biocompatible hydrogel polymer matrix may
polymerize outside the body. In certain embodiments, exposure to
the cells is limited to the tissue around the target site. In some
embodiments, the patient is not exposed systemically to the cell
therapy. In certain embodiments, the biocompatible pre-formulation
allows the cells to remain viable during and after polymerization.
In some embodiments, the cells are combined with a biocompatible
hydrogel polymer matrix after polymerization and/or gel formation.
In some embodiments, the biocompatible hydrogel polymer matrix
further polymerizes and/or gels after delivery to a target
site.
[0042] Cells may also be administered via a biocompatible hydrogel
polymer matrix directly on a wound or surgical site. Biocompatible
pre-formulations may form a biocompatible hydrogel polymer matrix
that is easily applied on the wound or surgical site and the
surrounding skin. The biocompatible hydrogel polymer matrix enables
the administration of cells directly to the wound or surgical site.
Biocompatible pre-formulations may polymerize and/or gel prior to
or after application to the wound or surgical site. In some
instances, once the biocompatible pre-formulation is applied, e.g.,
sprayed over the wound or surgical site, in the liquid form, the
biocompatible pre-formulation gels quickly and forms a solid
biocompatible hydrogel polymer matrix layer over the wound or
surgical site. The biocompatible hydrogel polymer matrix seals the
wound or surgical site and it also sticks to the surrounding skin.
The biocompatible hydrogel polymer matrix layer over the wound or
surgical site acts as a barrier to keep the wound or surgical site
from getting infected. In some instances, the biocompatible
hydrogel polymer matrix layer in contact with the skin makes the
skin surface sticky and thus allows a bandage to stick to the skin
more effectively. Most importantly, the biocompatible hydrogel
polymer matrix is non-toxic. After healing has taken place, the
biocompatible hydrogel polymer matrix dissolves and is absorbed
without producing toxic by-products. In some embodiments, the wound
or surgical site is healed by the formation of a graft after the
administration of stem cells with a biocompatible hydrogel polymer
matrix. In certain embodiments, the biocompatible pre-formulation
is applied to a wound or surgical site without the cells losing
viability. In certain embodiments, the biocompatible hydrogel
polymer matrix keeps the wound or surgical site sealed for 24-48
hours and protects it from infection, which avoids repeat visits to
the hospital and thus saving costs. In certain embodiments,
exposure to the cells is limited to the tissue around the target
site. In some embodiments, the patient is not exposed systemically
to the cell therapy.
[0043] In some embodiments, the biocompatible hydrogel polymer
matrix is also loaded with one or more additional components, such
as a buffer or a therapeutic agent. The physical and chemical
nature of the biocompatible hydrogel polymer matrix is such that a
large variety of cell types and additional components may be used
with the biocompatible pre-formulation that forms the biocompatible
hydrogel polymer matrix. In some embodiments, the additional
components enhance the viability and functionality of the cells. In
some embodiments, the additional components comprise activation
factors. In some embodiments the activation factors include growth
factors for cell growth stimulation and proliferation.
Exemplary Biocompatible Hydrogel Components
[0044] Provided herein are biocompatible pre-formulations,
comprising at least one first compound comprising more than one
nucleophilic group, at least one second compound comprising more
than one electrophilic group, at least one cell, and optionally
additional components. An exemplary additional component is a
culture medium. In certain embodiments, the culture medium is a
buffer. In certain embodiments the culture medium contains
nutrients for the at least one cell. In certain embodiments the at
least one cell is a stem cell. In certain embodiments, the at least
one first compound is formulated in a buffer. In certain
embodiments, the at least one second compound is formulated in a
buffer. In certain embodiments, the at least one cell is formulated
in a buffer. In certain embodiments, at least one biocompatible
pre-formulation component is a solid. In certain embodiments, all
components of the biocompatible pre-formulations are solids. In
certain embodiments, at least one biocompatible pre-formulation
component is a liquid. In certain embodiments, all biocompatible
pre-formulation components are liquids. In certain embodiments, the
biocompatible pre-formulation components form a biocompatible
hydrogel polymer matrix at a target site by mixing the at least one
first compound, the at least one second compound, the at least one
cell, and the optional additional component in the presence of
water and delivering the mixture to the target site such that the
biocompatible hydrogel polymer matrix at least in part polymerizes
and/or gels at the target site. In certain embodiments, the
biocompatible pre-formulation forms a biocompatible hydrogel
polymer matrix at a target site by mixing the at least one first
compound, the at least one second compound, and the at least one
cell in the presence of water and delivering the mixture to the
target site such that the biocompatible hydrogel polymer matrix at
least in part polymerizes and/or gels at the target site. In
certain embodiments, the optional additional component, e.g.
buffer, is added after the formulation is combined. In certain
embodiments, the biocompatible pre-formulation forms a
biocompatible hydrogel polymer matrix prior to application at a
target site by mixing the at least one first compound, the at least
one second compound, the at least one cell, and the optional
additional component in the presence of water and delivering the
mixture to the target site such that the biocompatible hydrogel
polymer matrix at least in part polymerizes and/or gels prior to
application at a target site. In certain embodiments, the
biocompatible pre-formulation forms a biocompatible hydrogel
polymer matrix prior to application at a target site by mixing the
at least one first compound, the at least one second compound, and
the at least one cell in the presence of water and delivering the
mixture to the target site such that the biocompatible hydrogel
polymer matrix at least in part polymerizes and/or gels prior to
application at a target site. In certain embodiments, the optional
additional component, e.g. buffer, is added after the formulation
is combined. In certain embodiments, the biocompatible
pre-formulations are biodegradable. In certain embodiments, the
biocompatible hydrogel polymer matrix comprises a biocompatible
hydrogel scaffold. In certain embodiments, the biocompatible
hydrogel scaffold comprises the at least one first compound and the
at least one second compound. In certain embodiments, the
biocompatible hydrogel scaffold comprises the at least one first
compound, the at least one second compound and a buffer. In certain
embodiments, the biocompatible hydrogel scaffold is fully
synthetic.
[0045] Provided herein are biocompatible pre-formulations,
comprising at least one first compound comprising more than one
nucleophilic group, at least one second compound comprising more
than one electrophilic group, a buffer, and optionally additional
components. An exemplary additional component is at least one cell.
In certain embodiments the cell is a stem cell. In certain
embodiments, the buffer is a culture medium. In certain embodiments
the culture medium provides nutrients to a cell. In certain
embodiments, the at least one first compound is formulated in a
buffer. In certain embodiments, the at least one second compound is
formulated in a buffer. In certain embodiments, at least one
biocompatible pre-formulation component is a solid. In certain
embodiments, all biocompatible pre-formulations are solids. In
certain embodiments, at least one biocompatible pre-formulation
component is a liquid. In certain embodiments, all biocompatible
pre-formulation components are liquids. In certain embodiments, the
biocompatible pre-formulation forms a biocompatible hydrogel
polymer matrix at a target site by mixing the at least one first
compound, the at least one second compound, the buffer, and the
optional additional component in the presence of water and
delivering the mixture to the target site such that the
biocompatible hydrogel polymer matrix at least in part polymerizes
and/or gels at the target site. In certain embodiments, the
biocompatible pre-formulation forms a biocompatible hydrogel
polymer matrix at a target site by mixing the at least one first
compound, the at least one second compound, and the buffer in the
presence of water and delivering the mixture to the target site
such that the biocompatible hydrogel polymer matrix at least in
part polymerizes and/or gels at the target site. In certain
embodiments, the optional additional component, e.g. cell, is added
after the formulation is combined. In certain embodiments, the
biocompatible pre-formulation forms a biocompatible hydrogel
polymer matrix prior to application at a target site by mixing the
at least one first compound, the at least one second compound, the
buffer, and the optional additional component in the presence of
water and delivering the mixture to the target site such that the
biocompatible hydrogel polymer matrix at least in part polymerizes
and/or gels prior to application at a target site. In certain
embodiments, the biocompatible pre-formulation forms a
biocompatible hydrogel polymer matrix prior to application at a
target site by mixing the at least one first compound, the at least
one second compound, and the buffer in the presence of water and
delivering the mixture to the target site such that the
biocompatible hydrogel polymer matrix at least in part polymerizes
and/or gels prior to application at a target site. In certain
embodiments, the optional additional component, e.g. cell, is added
after the formulation is combined. In certain embodiments, the
biocompatible pre-formulations are biodegradable. In certain
embodiments, the biocompatible hydrogel polymer matrix comprises a
biocompatible hydrogel scaffold. In certain embodiments, the
biocompatible hydrogel scaffold comprises the at least one first
compound, the at least one second compound and a buffer. In certain
embodiments, the biocompatible hydrogel scaffold is fully
synthetic.
[0046] In some embodiments, the biocompatible pre-formulation
compounds comprise monomers which polymerize into polymers. In some
embodiments, the biocompatible pre-formulation monomers polymerize
to form a biocompatible hydrogel polymer matrix. In some
embodiments, a polymer is a biocompatible hydrogel polymer matrix.
In some embodiments, a polymer is a biocompatible hydrogel
scaffold. In some embodiments, the biocompatible pre-formulation
compounds gel to form a biocompatible hydrogel polymer matrix. In
some embodiments, the biocompatible pre-formulation compounds gel
to form a biocompatible hydrogel scaffold. In some embodiments, the
biocompatible pre-formulation compounds polymerize and gel to form
a biocompatible hydrogel polymer matrix. In some embodiments, the
biocompatible pre-formulation compounds polymerize and gel to form
a biocompatible hydrogel polymer scaffold. In some embodiments, the
biocompatible hydrogel polymer matrix further polymerizes after
hydrogel polymer matrix formation. In some embodiments, the
biocompatible hydrogel polymer matrix gels after hydrogel polymer
matrix formation. In some embodiments, the biocompatible hydrogel
polymer matrix further polymerizes and gels after hydrogel polymer
matrix formation.
[0047] In some embodiments, the first or second compound comprising
more than one nucleophilic or electrophilic group are glycol-based.
In some embodiments, glycol-based compounds include ethylene
glycol, propylene glycol, butylene glycol, alkyl glycols of various
chain lengths, and any combination or copolymers thereof. In some
embodiments, the glycol-based compounds are polyglycol-based
compounds. In some embodiments, the polyglycol-based compounds
include, but are not limited to, polyethylene glycols (PEGs),
polypropylene glycols (PPGs), polybutylene glycols (PBGs), and
polyglycol copolymers. In some embodiments, glycol-based compounds
include polyethylene glycol, polypropylene glycol, polybutylene
glycol, polyalkyl glycols of various chain lengths, and any
combination or copolymers thereof. In some embodiments, the
glycol-based compounds are fully synthetic. In some embodiments,
the polyglycol-based compounds are fully synthetic.
[0048] In some embodiments, the first or second compound comprising
more than one nucleophilic or electrophilic group are polyol
derivatives. In certain embodiments, the first or second compound
is a dendritic polyol derivative. In some embodiments, the first or
second compound is a glycol, trimethylolpropane, glycerol,
diglycerol, pentaerythritiol, sorbitol, hexaglycerol,
tripentaerythritol, or polyglycerol derivative. In certain
embodiments, the first or second compound is a glycol,
trimethylolpropane, pentaerythritol, hexaglycerol, or
tripentaerythritol derivative. In some embodiments, the first or
second compound is a trimethylolpropane, glycerol, diglycerol,
pentaerythritiol, sorbitol, hexaglycerol, tripentaerythritol, or
polyglycerol derivative. In some embodiments, the first or second
compound is a pentaerythritol, di-pentaerythritol, or
tripentaerythritol derivative. In certain embodiments, the first or
second compound is a hexaglycerol
(2-ethyl-2-(hydroxymethyl)-1,3-propanediol, trimethylolpropane)
derivative. In some embodiments, the first or second compound is a
sorbitol derivative. In certain embodiments, the first or second
compound is a glycol, propyleneglycol, glycerin, diglycerin, or
polyglycerin derivative.
[0049] In some embodiments, the first and/or second compound
comprise polyethylene glycol (PEG) chains comprising one to 200
ethylene glycol subunits. In certain embodiments, the first and/or
second compound may further comprise polypropylene glycol (PPG)
chains comprising one to 200 propylene glycol subunits. The PEG or
PPG chains extending from the polyols are the "arms" linking the
polyol core to the nucleophilic or electrophilic groups.
Exemplary Nucleophilic Monomers
[0050] The biocompatible pre-formulation comprises at least one
first compound comprising more than one nucleophilic group. In some
embodiments, the first compound is a monomer configured to form a
polymer matrix through the reaction of a nucleophilic group in the
first compound with an electrophilic group of a second compound. In
some embodiments, the first compound monomer is fully synthetic. In
some embodiments, the nucleophilic group is a hydroxyl, thiol, or
amino group. In preferred embodiments, the nucleophilic group is a
thiol or amino group. In some embodiments, the at least one first
compound is glycol-based. In some embodiments, glycol-based
compounds include ethylene glycol, propylene glycol, butylene
glycol, alkyl glycols of various chain lengths, and any combination
or copolymers thereof. In some embodiments, glycol-based compounds
are polyglycol-based compounds. In some embodiments, the
polyglycol-based compounds include, but are not limited to,
polyethylene glycols (PEGs), polypropylene glycols (PPGs),
polybutylene glycols (PBGs), and polyglycol copolymers. In some
embodiments, glycol-based compounds include polyethylene glycol,
polypropylene glycol, polybutylene glycol, polyalkyl glycols of
various chain lengths, and any combination or copolymers thereof.
In some embodiments, the glycol-based compounds are fully
synthetic. In some embodiments, the polyglycol-based compounds are
fully synthetic.
[0051] In certain embodiments, the nucleophilic group is connected
to the polyol derivative through a suitable linker. Suitable
linkers include, but are not limited to, esters (e.g., acetates) or
ethers. In some instances, monomers comprising ester linkers are
more susceptible to biodegradation. Examples of linkers comprising
a nucleophilic group include, but are not limited to,
mercaptoacetate, aminoacetate (glycin) and other amino acid esters
(e.g., alanine, .beta.-alanine, lysine, ornithine),
3-mercaptopropionate, ethylamine ether, or propylamine ether. In
some embodiments, the polyol core derivative is bound to a
polyethylene glycol or polypropylene glycol subunit, which is
connected to the linker comprising the nucleophilic group. The
molecular weight of the first compound (the nucleophilic monomer)
is about 500 to 40000. In certain embodiments, the molecular weight
of a first compound (a nucleophilic monomer) is about 100, about
500, about 1000, about 2000, about 3000, about 4000, about 5000,
about 6000, about 7000, about 8000, about 9000, about 10000, about
12000, about 15000, about 20000, about 25000, about 30000, about
35000, about 40000, about 50000, about 60000, about 70000, about
80000, about 90000, or about 100000. In some embodiments, the
molecular weight of a first compound is about 500 to 2000. In
certain embodiments, the molecular weight of a first compound is
about 15000 to about 40000. In some embodiments, the first compound
is water soluble.
[0052] In some embodiments, the first compound is a
MULTIARM-(5k-50k)-polyol derivative comprising polyglycol subunits
and more than two nucleophilic groups. MULTIARM refers to number of
polyglycol subunits that are attached to the polyol core and these
polyglycol subunits link the nucleophilic groups to the polyol
core. In some embodiments, MULTIARM is 3ARM, 4ARM, 6ARM, 8ARM,
10ARM, 12ARM. In some embodiments, MULTIARM is 4ARM or 8ARM. In
some embodiments, the first compound is MULTIARM-(5k-50k)-NH2,
MULTIARM-(5k-50k)-AA, or a combination thereof. In certain
embodiments, the first compound is 4ARM-(5k-50k)-NH2,
4ARM-(5k-50k)-AA, 8ARM-(5k-50k)-NH2, and 8ARM-(5k-50k)-AA, or a
combination thereof. In some embodiments, the polyol derivative is
a glycol, trimethylolpropane, glycerol, diglycerol,
pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol, or
polyglycerol derivative.
[0053] Examples of the construction of monomers comprising more
than one nucleophilic group are shown below with a
trimethylolpropane or pentaerythritol core polyol. The compounds
shown have thiol or amine electrophilic groups that are connected
to variable lengths PEG subunit through acetate, propionate or
ethyl ether linkers (e.g., structures below of ETTMP (A; n=1),
4ARM-PEG-NH2 (B; n=1), and 4ARM-PEG-AA (C; n=1)). Monomers using
other polyol cores are constructed in a similar way.
##STR00001##
[0054] Suitable first compounds comprising a nucleophilic group
(used in the amine-ester chemistry) include, but are not limited
to, pentaerythritol polyethylene glycol amine (4ARM-PEG-NH2)
(molecular weight selected from about 5000 to about 40000, e.g.,
5000, 10000, or 20000), pentaerythritol polyethylene glycol amino
acetate (4ARM-PEG-AA) (molecular weight selected from about 5000 to
about 40000, e.g., 5000, 10000, or 20000), hexaglycerin
polyethylene glycol amine (8ARM-PEG-NH2) (molecular weight selected
from about 5000 to about 40000, e.g., 10000, 20000, or 40000), or
tripentaerythritol glycol amine (8ARM(TP)-PEG-NH2) (molecular
weight selected from about 5000 to about 40000, e.g., 10000, 20000,
or 40000). Within this class of compounds, 4 (or 8)ARM-PEG-AA
comprises ester (or acetate) groups while the 4 (or 8)ARM-PEG-NH2
monomers do not comprise ester (or acetate) groups.
[0055] Other suitable first compounds comprising a nucleophilic
group (used in the thiol-ester chemistry) include, but not limited
to, glycol dimercaptoacetate (THIOCURE.RTM. GDMA),
trimethylolpropane trimercaptoacetate (THIOCURE.RTM. TMPMA),
pentaerythritol tetramercaptoacetate (THIOCURE.RTM. PETMA), glycol
di-3-mercaptopropionate (THIOCURE.RTM. GDMP), trimethylolpropane
tri-3-mercaptopropionate (THIOCURE.RTM. TMPMP), pentaerythritol
tetra-3-mercaptopropionate (THIOCURE.RTM. PETMP),
polyol-3-mercaptopropionates, polyester-3-mercaptopropionates,
propyleneglycol 3-mercaptopropionate (THIOCURE.RTM. PPGMP 800),
propyleneglycol 3-mercaptopropionate (THIOCURE.RTM. PPGMP 2200),
ethoxylated trimethylolpropane tri-3-mercaptopropionate
(THIOCURE.RTM. ETTMP-700), and ethoxylated trimethylolpropane
tri-3-mercaptopropionate (THIOCURE.RTM. ETTMP-1300).
Exemplary Electrophilic Monomers
[0056] The biocompatible pre-formulation comprises at least one
second compound comprising more than one electrophilic group. In
some embodiments, the second compound is a monomer configured to
form a polymer matrix through the reaction of an electrophilic
group in the second compound with a nucleophilic group of a first
compound. In some embodiments, the second compound monomer is fully
synthetic. In some embodiments, the electrophilic group is an
epoxide, maleimide, succinimidyl, or an alpha-beta unsaturated
ester. In preferred embodiments, the electrophilic group is an
epoxide or succinimidyl. In some embodiments, the at least one
second compound is glycol-based. In some embodiments, glycol-based
compounds include ethylene glycol, propylene glycol, butylene
glycol, alkyl glycols of various chain lengths, and any combination
or copolymers thereof. In some embodiments, the glycol-based
compound is a polyglycol-based compound. In some embodiments, the
polyglycol-based compounds include, but are not limited to,
polyethylene glycols (PEGs), polypropylene glycols (PPGs),
polybutylene glycols (PBGs), and polyglycol copolymers. In some
embodiments, glycol-based compounds include polyethylene glycol,
polypropylene glycol, polybutylene glycol, polyalkyl glycols of
various chain lengths, and any combination or copolymers thereof.
In some embodiments, the glycol-based compounds are fully
synthetic. In some embodiments, the polyglycol-based polymer is
fully synthetic.
[0057] In certain embodiments, the electrophilic group is connected
to the polyol derivative through a suitable linker. Suitable
linkers include, but are not limited to, esters, amides, or ethers.
In some instances, monomers comprising ester linkers are more
susceptible to biodegradation. Examples of linkers comprising an
electrophilic group include, but are not limited to, succinimidyl
succinate, succinimidyl glutarate, succinimidyl succinamide,
succinimidyl glutaramide, or glycidyl ether. In some embodiments,
the polyol core derivative is bound to a polyethylene glycol or
polypropylene glycol subunit, which is connected to the linker
comprising the electrophilic group. The molecular weight of the
second compound (the electophilic monomer) is about 500 to 40000.
In certain embodiments, the molecular weight of a second compound
(an electophilic monomer) is about 100, about 500, about 1000,
about 2000, about 3000, about 4000, about 5000, about 6000, about
7000, about 8000, about 9000, about 10000, about 12000, about
15000, about 20000, about 25000, about 30000, about 35000, about
40000, about 50000, about 60000, about 70000, about 80000, about
90000, or about 100000. In some embodiments, the molecular weight
of a second compound is about 500 to 2000. In certain embodiments,
the molecular weight of a second compound is about 15000 to about
40000. In some embodiments, the second compound is water
soluble.
[0058] In some embodiments, the second compound is a
MULTIARM-(5k-50k)-polyol derivative comprising polyglycol subunits
and more than two electrophilic groups. MULTIARM refers to number
of polyglycol subunits that are attached to the polyol core and
these polyglycol subunits link the nucleophilic groups to the
polyol core. In some embodiments, MULTIARM is 3ARM, 4ARM, 6ARM,
8ARM, 10ARM, 12ARM or any combination thereof. In some embodiments,
MULTIARM is 4ARM or 8ARM. In certain embodiments, the second
compound is selected from MULTIARM-(5-50k)-SG,
MULTIARM-(5-50k)-SGA, MULTIARM-(5-50k)-SS, MULTIARM-(5-50k)-SSA,
and a combination thereof. In certain embodiments, the second
compound is selected from 4ARM-(5-50k)-SG, 4ARM-(5-50k)-SGA,
4ARM-(5-50k)-SS, 8ARM-(5-50k)-SG, 8ARM-(5-50k)-SGA and
8ARM-(5-50k)-SS, and a combination thereof. In some embodiments,
the polyol derivative is a glycol, trimethylolpropane, glycerol,
diglycerol, pentaerythritol, sorbitol, hexaglycerol,
tripentaerythritol, or polyglycerol derivative.
[0059] Examples of the construction of monomers comprising more
than one electrophilic group are shown below with a pentaerythritol
core polyol. The compounds shown have a succinimidyl electrophilic
group, a glutarate or glutaramide linker, and a variable lengths
PEG subunit (e.g., structures below of 4ARM-PEG-SG (D; n=3) and
4ARM-PEG-SGA (E; n=3)). Monomers using other polyol cores or
different linkers (e.g., succinate (SS) or succinamide (SSA) are
constructed in a similar way.
##STR00002##
[0060] Suitable second compounds comprising an electrophilic group
include, but are not limited to, pentaerythritol polyethylene
glycol maleimide (4ARM-PEG-MAL) (molecular weight selected from
about 5000 to about 40000, e.g., 10000 or 20000), pentaerythritol
polyethylene glycol succinimidyl succinate (4ARM-PEG-SS) (molecular
weight selected from about 5000 to about 40000, e.g., 10000 or
20000), pentaerythritol polyethylene glycol succinimidyl glutarate
(4ARM-PEG-SG) (molecular weight selected from about 5000 to about
40000, e.g., 10000 or 20000), pentaerythritol polyethylene glycol
succinimidyl glutaramide (4ARM-PEG-SGA) (molecular weight selected
from about 5000 to about 40000, e.g., 10000 or 20000), hexaglycerin
polyethylene glycol succinimidyl succinate (8ARM-PEG-SS) (molecular
weight selected from about 5000 to about 40000, e.g., 10000 or
20000), hexaglycerin polyethylene glycol succinimidyl glutarate
(8ARM-PEG-SG) (molecular weight selected from about 5000 to about
40000, e.g., 10000, 15000, 20000, or 40000), hexaglycerin
polyethylene glycol succinimidyl glutaramide (8ARM-PEG-SGA)
(molecular weight selected from about 5000 to about 40000, e.g.,
10000, 15000, 20000, or 40000), tripentaerythritol polyethylene
glycol succinimidyl succinate (8ARM(TP)-PEG-SS) (molecular weight
selected from about 5000 to about 40000, e.g., 10000 or 20000),
tripentaerythritol polyethylene glycol succinimidyl glutarate
(8ARM(TP)-PEG-SG) (molecular weight selected from about 5000 to
about 40000, e.g., 10000, 15000, 20000, or 40000), or
tripentaerythritol polyethylene glycol succinimidyl glutaramide
(8ARM(TP)--PEG-SGA) (molecular weight selected from about 5000 to
about 40000, e.g., 10000, 15000, 20000, or 40000). The 4 (or
8)ARM-PEG-SG monomers comprise ester groups, while the 4 (or
8)ARM-PEG-SGA monomers do not comprise ester groups.
[0061] Other suitable second compounds comprising an electrophilic
group are sorbitol polyglycidyl ethers, including, but not limited
to, sorbitol polyglycidyl ether (DENACOL.RTM. EX-611), sorbitol
polyglycidyl ether (DENACOL.RTM. EX-612), sorbitol polyglycidyl
ether (DENACOL.RTM. EX-614), sorbitol polyglycidyl ether
(DENACOL.RTM. EX-614 B), polyglycerol polyglycidyl ether
(DENACOL.RTM. EX-512), polyglycerol polyglycidyl ether
(DENACOL.RTM. EX-521), diglycerol polyglycidyl ether (DENACOL.RTM.
EX-421), glycerol polyglycidyl ether (DENACOL.RTM. EX-313),
glycerol polyglycidyl ether (DENACOL.RTM. EX-313),
trimethylolpropane polyglycidyl ether (DENACOL.RTM. EX-321),
sorbitol polyglycidyl ether (DENACOL.RTM. EJ-190).
Formation of Biocompatible Hydrogel Polymer Matrices
[0062] Provided herein are biocompatible pre-formulations,
comprising at least one first compound comprising more than one
nucleophilic group, at least one second compound comprising more
than one electrophilic group, at least one cell, and optionally
additional components. An exemplary additional component is a
culture medium. In certain embodiments, the culture medium is a
buffer. In certain embodiments, the culture medium is a nutrient
rich medium. In certain embodiments the cell is a stem cell. The
biocompatible pre-formulation undergoes polymerization and/or
gelling to form a biocompatible hydrogel polymer matrix. In certain
embodiments, the biocompatible hydrogel polymer matrix is
biodegradable. In certain embodiments, the biocompatible hydrogel
polymer matrix comprises a biocompatible hydrogel scaffold.
[0063] Provided herein are biocompatible pre-formulations,
comprising at least one first compound comprising more than one
nucleophilic group, at least one second compound comprising more
than one electrophilic group, a culture medium, and optionally
additional components. An exemplary additional component is at
least one cell. In certain embodiments the cell is a stem cell. In
certain embodiments, the culture medium is a buffer. In certain
embodiments, the culture medium is a nutrient rich medium. The
biocompatible pre-formulation undergoes polymerization and/or
gelling to form a biocompatible hydrogel polymer matrix. In certain
embodiments, the biocompatible hydrogel polymer matrix is
biodegradable. In certain embodiments, the biocompatible hydrogel
polymer matrix comprises a biocompatible hydrogel scaffold.
[0064] In certain embodiments, the pre-formulation safely undergoes
polymerization at a target site inside or on a mammalian body, for
instance at the site of a wound, surgical site, or in a joint. In
certain embodiments, the biocompatible hydrogel polymer matrix
forms a wound patch, suture, or joint spacer. In some embodiments,
the first compound and the second compound are monomers forming a
polymer matrix through the reaction of a nucleophilic group in the
first compound with the electrophilic group in the second compound.
In certain embodiments, the monomers are polymerized at a
predetermined time. In some embodiments, the monomers are
polymerized under mild and nearly neutral pH conditions. In certain
embodiments, the biocompatible hydrogel polymer matrix does not
change volume after gelling.
[0065] In some embodiments, the first and second compounds react to
form amide, thioester, or thioether bonds. When a thiol nucleophile
reacts with a succinimidyl electrophile, a thioester is formed.
When an amino nucleophile reacts with a succinimidyl electrophile,
an amide is formed.
[0066] In some embodiments, one or more first compounds comprising
an amino group react with one or more second compounds comprising a
succinimidyl ester group to form amide linked first and second
monomer units. In certain embodiments, one or more first compounds
comprising a thiol group react with one or more second compounds
comprising a succinimidyl ester group to form thioester linked
first and second monomer units. In some embodiments, one or more
first compounds comprising an amino group react with one or more
second compounds comprising an epoxide group to from amine linked
first and second monomer units. In certain embodiments, one or more
first compounds comprising a thiol group react with one or more
second compounds comprising an epoxide group to form thioether
linked first and second monomer units.
[0067] In some embodiments, a first compound is mixed with a
different first compound before addition to one or more second
compounds. In other embodiments, a second compound is mixed with a
different second compound before addition to one or more first
compounds. In certain embodiments, the properties of the
biocompatible pre-formulation and the biocompatible hydrogel
polymer matrix are controlled by the properties of the at least one
first and at least one second monomer mixture.
[0068] In some embodiments, one first compound is used in the
biocompatible hydrogel polymer matrix. In certain embodiments, two
different first compounds are mixed and used in the biocompatible
hydrogel polymer matrix. In some embodiments, three different first
compounds are mixed and used in the biocompatible hydrogel polymer
matrix. In certain embodiments, four or more different first
compounds are mixed and used in the biocompatible hydrogel polymer
matrix.
[0069] In some embodiments, one second compound is used in the
biocompatible hydrogel polymer matrix. In certain embodiments, two
different second compounds are mixed and used in the biocompatible
hydrogel polymer matrix. In some embodiments, three different
second compounds are mixed and used in the biocompatible hydrogel
polymer matrix. In certain embodiments, four or more different
second compounds are mixed and used in the biocompatible hydrogel
polymer matrix.
[0070] In some embodiments, a first compound comprising ether
linkages to the nucleophilic group are mixed with a different first
compound comprising ester linkages to the nucleophilic group. This
allows the control of the concentration of ester groups in the
resulting biocompatible hydrogel polymer matrix. In certain
embodiments, a second compound comprising ester linkages to the
electrophilic group are mixed with a different second compound
comprising ether linkages to the electrophilic group. In some
embodiments, a second compound comprising ester linkages to the
electrophilic group are mixed with a different second compound
comprising amide linkages to the electrophilic group. In certain
embodiments, a second compound comprising amide linkages to the
electrophilic group are mixed with a different second compound
comprising ether linkages to the electrophilic group.
[0071] In some embodiments, a first compound comprising an
aminoacetate (e.g., glycine derived) nucleophile is mixed with a
different first compound comprising an amine nucleophile (e.g., an
ethylamine ether) at a specified molar ratio (x/y). In certain
embodiments, the molar ratio (x/y) is 5/95, 10/90, 15/85, 20/80,
25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35,
70/30, 75/25, 80/20, 85/15, 90/10, or 95/5. In certain embodiments,
a first compound comprising an aminoacetate (e.g., glycine derived)
nucleophile is mixed with a different first compound comprising an
amine nucleophile (e.g., an ethylamine ether) at a specified weight
ratio (x/y). In certain embodiments, the weight ratio (x/y) is
5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55,
50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or
95/5. In certain embodiments, the mixture of two first compounds is
mixed with one or more second compounds at a molar amount
equivalent to the sum of x and y.
[0072] In some embodiments, the first compound comprising more than
one nucleophilic group and the at least one cell are pre-mixed in
the presence of water. In some embodiments, the first compound
comprising more than one nucleophilic group and the cell are
pre-mixed without the presence of water. Once pre-mixing is
complete, the second compound comprising more than one
electrophilic group is added to the pre-mixture in the presence of
water to form a biocompatible hydrogel polymer matrix. Shortly
after final mixing, the biocompatible hydrogel polymer matrix
mixture is delivered to the target site. In certain embodiments, an
optional additional component is added to the pre-mix, the second
compound, or to the mixture just before delivery of the
biocompatible hydrogel polymer matrix mixture to the target site.
In certain embodiments, an optional additional component is added
to the pre-mix, the second compound, or to the mixture after
delivery of the biocompatible hydrogel polymer matrix mixture to
the target site. In some embodiments, the additional component is a
buffer. In some embodiments, the biocompatible hydrogel polymer
matrix polymerizes and/or gels prior to delivery to the target
site. In some embodiments, the biocompatible hydrogel polymer
matrix polymerizes and/or gels at the target site.
[0073] In some embodiments, the first compound comprising more than
one nucleophilic group and the buffer are pre-mixed in the presence
of water. In some embodiments, the first compound comprising more
than one nucleophilic group and the buffer are pre-mixed without
the presence of water. Once pre-mixing is complete, the second
compound comprising more than one electrophilic group is added to
the pre-mixture in the presence of water, forming a biocompatible
hydrogel polymer matrix. Shortly after final mixing, the
biocompatible hydrogel polymer matrix mixture is delivered to the
target site. In certain embodiments, an optional additional
component is added to the pre-mix, the second compound, or to the
mixture just before delivery of the biocompatible hydrogel polymer
matrix mixture to the target site. In certain embodiments, an
optional additional component is added to the pre-mix, the second
compound, or to the mixture after delivery of the biocompatible
hydrogel polymer matrix mixture to the target site. In some
embodiments, the additional component is at least one cell. In some
embodiments, the biocompatible hydrogel polymer matrix polymerizes
and/or gels prior to delivery to the target site. In some
embodiments, the biocompatible hydrogel polymer matrix polymerizes
and/or gels at the target site.
[0074] In other embodiments, the second compound comprising more
than one electrophilic group and the at least one cell are
pre-mixed in the presence of water. In other embodiments, the
second compound comprising more than one electrophilic group and
the cell are pre-mixed without the presence of water. Once
pre-mixing is complete, the first compound comprising more than one
nucleophilic group is added to the pre-mixture, forming a
biocompatible hydrogel polymer matrix. Shortly after final mixing,
the biocompatible hydrogel polymer matrix mixture is delivered to
the target site. In certain embodiments, an optional component is
added to the pre-mix, the first compound, or to the mixture just
before delivery of the biocompatible hydrogel polymer matrix
mixture to the target site. In certain embodiments, an optional
additional component is added to the pre-mix, the first compound,
or to the mixture after delivery of the biocompatible hydrogel
polymer matrix mixture to the target site. In some embodiments, the
additional component is a buffer. In some embodiments, the
biocompatible hydrogel polymer matrix polymerizes and/or gels prior
to delivery to the target site. In some embodiments, the
biocompatible hydrogel polymer matrix polymerizes and/or gels at
the target site.
[0075] In other embodiments, the second compound comprising more
than one electrophilic group and the buffer are pre-mixed in the
presence of water. In other embodiments, the second compound
comprising more than one electrophilic group and the buffer are
pre-mixed without the presence of water. Once pre-mixing is
complete, the first compound comprising more than one nucleophilic
group is added to the pre-mixture, forming a biocompatible hydrogel
polymer matrix. Shortly after final mixing, the biocompatible
hydrogel polymer matrix mixture is delivered to the target site. In
certain embodiments, an optional component is added to the pre-mix,
the first compound, or to the mixture just before delivery of the
biocompatible hydrogel polymer matrix mixture to the target site.
In certain embodiments, an optional additional component is added
to the pre-mix, the first compound, or to the mixture after
delivery of the biocompatible hydrogel polymer matrix mixture to
the target site. In some embodiments, the additional component is
at least one cell. In some embodiments, the biocompatible hydrogel
polymer matrix polymerizes and/or gels prior to delivery to the
target site. In some embodiments, the biocompatible hydrogel
polymer matrix polymerizes and/or gels at the target site.
[0076] In some embodiments, a first compound comprising more than
one nucleophilic group, a second compound comprising more than one
electrophilic group, and at least one cell are mixed together in
the presence of water, whereby a biocompatible hydrogel polymer
matrix is formed. In some embodiments, a first compound comprising
more than one nucleophilic group, a second compound comprising more
than one electrophilic group, and a buffer are mixed together in
the presence of water, whereby a biocompatible hydrogel polymer
matrix is formed. In some embodiments, a first compound comprising
more than one nucleophilic group, a second compound comprising more
than one electrophilic group, at least one cell, and a buffer are
mixed together in the presence of water, whereby a biocompatible
hydrogel polymer matrix is formed. In certain embodiments, the
first compound comprising more than one nucleophilic group, the
second compound comprising more than one electrophilic group,
and/or the cell are individually diluted in an aqueous buffer in
the pH range of about 5.0 to about 9.5, wherein the individual
dilutions or neat monomers are mixed and a biocompatible hydrogel
polymer matrix is formed. In some embodiments, the aqueous buffer
is in the pH range of about 6.0 to about 8.5. In certain
embodiments, the aqueous buffer is in the pH range of about 8. In
certain embodiments, the aqueous buffer is a culture medium. In
certain embodiments, the culture medium is a nutrient rich
medium.
[0077] In certain embodiments, the concentration of the monomers in
the aqueous is from about 1% to about 100%. In some embodiments,
the dilution is used to adjust the viscosity of the monomer
dilution. In certain embodiments, the concentration of a monomer in
the aqueous buffer is about 1%, about 2%, about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 95%, or about
100%.
[0078] In some embodiments, the electrophilic and nucleophilic
monomers are mixed in such ratio that there is a slight excess of
electrophilic groups present in the mixture. In certain
embodiments, this excess is about 10%, about 5%, about 2%, about
1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%,
about 0.4%, about 0.3%, about 0.2%, about 0.1%, or less than
0.1%.
[0079] In certain embodiments, the gelling time or curing time of
the biocompatible hydrogel polymer matrix is controlled by the
selection of the first and second compounds. In some embodiments,
the concentration of nucleophilic or electrophilic groups in the
first or second compound influences the gelling time of the
biocompatible pre-formulation. In certain embodiments, temperature
influences the gelling time of the biocompatible pre-formulation.
In some embodiments, the type of aqueous buffer influences the
gelling time of the biocompatible pre-formulation. In some
embodiments, the aqueous buffer is a culture medium. In certain
embodiments, the concentration of the aqueous buffer influences the
gelling time of the biocompatible pre-formulation. In some
embodiments, the nucleophilicity and/or electrophilicity of the
nucleophilic and electrophilic groups of the monomers influences
the gelling time of the biocompatible pre-formulation. In some
embodiments, the cell type influences the gelling time of the
biocompatible pre-formulation. In some embodiments, the cell
concentration influences the gelling time of the biocompatible
pre-formulation.
[0080] In some embodiments, the gelling time or curing time of the
biocompatible hydrogel polymer matrix is controlled by the pH of
the aqueous buffer. In certain embodiments, the gelling time is
between about 20 seconds and 10 minutes. In some embodiments, the
gelling time is less than 30 minutes, less than 20 minutes, less
than 10 minutes, less than 5 minutes, less than 4.8 minutes, less
than 4.6 minutes, less than 4.4 minutes, less than 4.2 minutes,
less than 4.0 minutes, less than 3.8 minutes, less than 3.6
minutes, less than 3.4 minutes, less than 3.2 minutes, less than
3.0 minutes, less than 2.8 minutes, less than 2.6 minutes, less
than 2.4 minutes, less than 2.2 minutes, less than 2.0 minutes,
less than 1.8 minutes, less than 1.6 minutes, less than 1.4
minutes, less than 1.2 minutes, less than 1.0 minutes, less than
0.8 minutes, less than 0.6 minutes, or less than 0.4 minutes. In
certain embodiments, the pH of the aqueous buffer is from about 5
to about 9.5. In some embodiments, the pH of the aqueous buffer is
from about 7.0 to about 9.5. In specific embodiments, the pH of the
aqueous buffer is about 8. In some embodiments, the pH of the
aqueous buffer is about 5, about 5.5, about 6.0, about 6.5, about
6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about
7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.8, about
7.9, about 8.0, about 8.1 about 8.2 about 8.3, about 8.4, about
8.5, about 9.0, or about 9.5.
[0081] In certain embodiments, the gelling time or curing time of
the biocompatible pre-formulation is controlled by the type of
aqueous buffer. In some embodiments, the aqueous buffer is a
physiologically acceptable buffer. In certain embodiments, aqueous
buffers include, but are not limited to, aqueous saline solutions,
phosphate buffered saline, borate buffered saline, a combination of
borate and phosphate buffers wherein each component is dissolved in
separate buffers,
N-2-Hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid
(HEPES), 3-(N-Morpholino) propanesulfonic acid (MOPS),
2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid
(TES), 3-[N-tris(Hydroxy-methyl)
ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid
(EPPS), Tris[hydroxymethyl]-aminomethane (THAM), and
Tris[hydroxymethyl]methyl aminomethane (TRIS). In some embodiments,
the thiol-ester chemistry (e.g., ETTMP nucleophile with SGA or SG
electrophile) is performed in borate buffer. In certain
embodiments, the amine-ester chemistry (NH2 or AA nucleophile with
SGA or SG electrophile) is performed in phosphate buffer. In some
embodiments the aqueous buffer is a culture medium. In certain
embodiments, culture media include, but are not limited to, DMEM,
IMDM, OptiMEM.RTM., AlgiMatrix.TM., Fetal Bovine Serum, GS1-R.RTM.,
GS2-Mt, iSTEM.RTM., NDiff.RTM. N2,NDiff.RTM. N2-AF, RHB-A.RTM.,
RHB-Basal.RTM., RPMI, SensiCell.TM., GlutaMAX.TM., FluoroBrite.TM.,
Gibco.RTM. TAP, Gibco.RTM. BG-11, LB, M9 Minimal, Terrific Broth,
2YXT, MagicMedia.TM., ImMedia.TM., SOC, YPD, CSM, YNB, Grace's
Insect Media, 199/109 and HamF10/HamF12. In certain embodiments,
the cell culture medium may be serum free. In certain embodiments,
the culture media may include additives. In some embodiments,
culture media additives include, but are not limited to,
antibiotics, vitamins, proteins, inhibitors, small molecules,
minerals, inorganic salts, nitrogen, growth factors, amino acids,
serum, carbohydrates, lipids, hormones and glucose. In some
embodiments, growth factors include, but are not limited to, EGF,
bFGF, FGF, ECGF, IGF-1, PDGF, NGF, TGF-.alpha. and TGF-.beta.. In
certain embodiments, the culture medium may not be aqueous. In
certain embodiments, the non-aqueous culture media include, but are
not limited to, frozen cell stocks, lyophilized medium, and
agar.
[0082] In certain embodiments, the biocompatible hydrogel polymer
matrix comprises a biocompatible hydrogel scaffold. In certain
embodiments, the biocompatible hydrogel scaffold comprises the
pre-formulation at least one first compound and the pre-formulation
at least one second compound. In certain embodiments, the
biocompatible hydrogel scaffold comprises a buffer. In certain
embodiments, the biocompatible hydrogel scaffold is fully
synthetic. In certain embodiments, the biocompatible hydrogel
scaffold provides an environment suitable for sustained cell
viability and/or growth.
[0083] In certain embodiments, the first compound and the second
compound do not react with the cell during formation of the
biocompatible hydrogel polymer matrix. In some embodiments, the
cell remains unchanged after polymerization of the first and second
compounds (i.e., monomers). In certain embodiments, the cell does
not change the properties of the biocompatible hydrogel polymer
matrix. In some embodiments, the physiochemical properties of the
cell and the biocompatible hydrogel polymer matrix formulation are
not affected by the polymerization of the monomers. In certain
embodiments, delivery of the cell using a biocompatible hydrogel
polymer matrix minimizes the degradation or denaturing of the cell.
In some instances, the physiochemical properties of the cell are
not affected by the delivery or release of the cell to the target
site.
[0084] In some embodiments, the biocompatible hydrogel polymer
matrix formulations further comprise a contrast agent for
visualizing the biocompatible hydrogel polymer matrix formulation
and locating a tumor using e.g., X-ray, fluoroscopy, or computed
tomography (CT) imaging. In certain embodiments, the contrast agent
enables the visualization of the bioabsorption of the biocompatible
hydrogel polymer matrix. In some embodiments, the contrast agent is
a radiopaque material. In certain embodiments, the radiopaque
material is selected from, but not limited to, sodium iodide,
potassium iodide, and barium sulfate, VISIPAQUE.RTM.,
OMNIPAQUE.RTM., or HYPAQUE.RTM., tantalum, and similar commercially
available compounds, or combinations thereof. In other embodiments,
the biocompatible hydrogel polymer matrix further comprises a
pharmaceutically acceptable dye.
[0085] In some embodiments, the biocompatible hydrogel polymer
matrix formulations further comprise a viscosity enhancer. Examples
of viscosity enhancer include, but are not limited to,
hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose,
polyvinylcellulose, polyvinylpyrrolidone.
Area of for Treatment--Target Sites
[0086] In certain embodiments, the target site is inside a mammal.
In some embodiments, the target site is inside a human being. In
certain embodiments, the target site is on the human body. In some
embodiments, the target site is accessible through surgery. In
certain embodiments, the target site is accessible through
minimally invasive surgery. In some embodiments, the target site is
accessible through an endoscopic device. In certain embodiments,
the target site is a wound on the skin of a mammal. In other
embodiments, the target site is in a joint or on a bone of a
mammal. In some embodiments, the target site is a surgical site in
a mammal
[0087] In some embodiments, a biocompatible pre-formulation or a
biocompatible hydrogel polymer matrix is used as a sealant or
adhesive. In certain embodiments, the biocompatible pre-formulation
or biocompatible hydrogel polymer matrix is used to seal a wound on
a mammal. In other embodiments, the biocompatible pre-formulation
or biocompatible hydrogel polymer matrix is used to fill cavities,
e.g., in a joint space to form a gel cushion. In other embodiments,
the biocompatible pre-formulation or biocompatible hydrogel polymer
matrix is used as a carrier for delivery of cells to target
sites.
[0088] In some embodiments, the biocompatible hydrogel polymer
matrix formulation is polymerized ex vivo. In certain embodiments,
the ex vivo polymerized biocompatible hydrogel polymer matrix
formulation is delivered through traditional routes of
administration (e.g., oral, implantation, or rectal). In other
embodiments, the ex vivo polymerized biocompatible hydrogel polymer
matrix formulation is delivered during surgery to a target
site.
Delivery of the Biocompatible Hydrogel Formulation to a Target
Site
[0089] In some embodiments, the biocompatible pre-formulation is
delivered as a biocompatible pre-formulation to a target site
through a catheter or a needle to form a biocompatible hydrogel
polymer matrix at the target site. In other embodiments, the
biocompatible pre-formulation is delivered to the target site in or
on the mammal using syringe and needle. In some embodiments, a
delivery device is used to deliver the biocompatible
pre-formulation to the target site. In some embodiments, the
biocompatible pre-formulation is delivered to the target site so
that the biocompatible pre-formulation mostly covers the target
site. In certain embodiments, the biocompatible pre-formulation
substantially covers an exposed portion of diseased tissue. In some
embodiments, the biocompatible pre-formulation does not spread to
any other location intentionally. In some embodiments, the
biocompatible pre-formulation substantially covers diseased tissue
and does not significantly cover healthy tissue. In certain
embodiments, the biocompatible hydrogel polymer matrix does not
significantly cover healthy tissue. In some embodiments, the
biocompatible pre-formulation gels over the target site and
thoroughly covers diseased tissue. In some embodiments, the
biocompatible hydrogel polymer matrix adheres to tissue. In some
embodiments, the biocompatible hydrogel polymer matrix mixture gels
after delivery at the target site, covering the target site. In
some embodiments, the biocompatible hydrogel polymer matrix mixture
gels prior to delivery at the target site.
[0090] In some embodiments, the gelling time of the biocompatible
pre-formulation is set according to the preference of the doctor
delivering the biocompatible pre-formulation mixture to a target
site. In most instances, a physician delivers the biocompatible
pre-formulation mixture to the target within 15 to 30 seconds. In
certain embodiments, the gelling time is between about 20 seconds
and 10 minutes. In some embodiments, the gelling time or curing
time of the biocompatible pre-formulation is controlled by the pH
of the aqueous buffer. In certain embodiments, the gelling time or
curing time of the biocompatible pre-formulation is controlled by
the selection of the first and second compounds. In some
embodiments, the concentration of nucleophilic or electrophilic
groups in the first or second compound influences the gelling time
of the biocompatible pre-formulation. In some embodiments, cell
concentration influences the gelling time of the biocompatible
pre-formulation. In some embodiments, cell type influences the
gelling time of the biocompatible pre-formulation. In some
embodiments, optional addition components influence the gelling
time of the biocompatible pre-formulation.
[0091] In some embodiments, curing of the biocompatible hydrogel
polymer matrix is verified post-administration. In certain
embodiments, the verification is performed in vivo at the delivery
site. In other embodiments, the verification is performed ex vivo.
In some embodiments, curing of the biocompatible hydrogel polymer
matrix is verified visually through the fiber-optics of an
endoscopic device. In certain embodiments, curing of biocompatible
hydrogel polymer matrices comprising radiopaque materials is
verified using X-ray, fluoroscopy, or computed tomography (CT)
imaging. A lack of flow of the biocompatible hydrogel polymer
matrix indicates that the biocompatible hydrogel polymer matrix has
gelled and the biocompatible hydrogel is sufficiently cured. In
further embodiments, curing of the biocompatible hydrogel polymer
matrix is verified by evaluation of the residue in the delivery
device, for instance the residue in the catheter of the
bronchoscope or other endoscopic device, or the residue in the
syringe used to deliver the biocompatible hydrogel polymer matrix.
In other embodiments, curing of the biocompatible hydrogel polymer
matrix is verified by depositing a small sample (e.g., .about.1 mL)
on a piece of paper or in a small vessel and subsequent evaluation
of the flow characteristics after the gelling time has passed.
[0092] In some embodiments, the biocompatible pre-formulation
delivers at least one cell to a target site. In some embodiments,
the biocompatible pre-formulation delivers nutrients to at least
one cell located at a target site. In some embodiments, the
biocompatible pre-formulation delivers structural support to at
least one cell located at a target site. In some embodiments, the
biocompatible pre-formulation delivers at least one cell and at
least one buffer to a target site. In some embodiments, the
biocompatible hydrogel polymer matrix delivers at least one cell to
a target site. In some embodiments, the biocompatible hydrogel
polymer matrix delivers nutrients to at least one cell located at a
target site. In some embodiments, the biocompatible hydrogel
polymer matrix delivers structural support to at least one cell
located at a target site. In some embodiments, the biocompatible
hydrogel polymer matrix delivers at least one cell to a target
site.
Bioabsorbance of the Biocompatible Hydrogel Polymer Matrix
[0093] In some embodiments, the biocompatible hydrogel polymer
matrix is a bioabsorbable polymer. In certain embodiments, the
biocompatible hydrogel polymer matrix is bioabsorbed within about 5
to 30 days. In some embodiments, the biocompatible hydrogel polymer
matrix is bioabsorbed within about 30 to 180 days. In some
embodiments, the biocompatible hydrogel polymer matrix is
bioabsorbed within about 1 to 70 days. In preferred embodiments,
the biocompatible hydrogel polymer matrix is bioabsorbed within
about 14 to 180 days. In some embodiments the biocompatible
hydrogel polymer matrix is bioabsorbed within about 365 days, 180
days, about 150 days, about 120 days, about 90 days, about 80 days,
about 70 days, about 60 days, about 50 days, about 40 days, about
35 days, about 30 days, about 28 days, about 21 days, about 14
days, about 10 days, about 7 days, about 6 days, about 5 days,
about 4 days, about 3 days, about 2 days, or about 1 day. In
certain embodiments the biocompatible hydrogel polymer matrix is
bioabsorbed within less than 365 days, 180 days, less than 150
days, less than 120 days, less than 90 days, less than 80 days,
less than 70 days, less than 60 days, less than 50 days, less than
40 days, less than 35 days, less than 30 days, less than 28 days,
less than 21 days, less than 14 days, less than 10 days, less than
7 days, less than 6 days, less than 5 days, less than 4 days, less
than 3 days, less than 2 days, or less than 1 day. In some
embodiments the biocompatible hydrogel polymer matrix is
bioabsorbed within more than 365 days, 180 days, more than 150
days, more than 120 days, more than 90 days, more than 80 days,
more than 70 days, more than 60 days, more than 50 days, more than
40 days, more than 35 days, more than 30 days, more than 28 days,
more than 21 days, more than 14 days, more than 10 days, more than
7 days, more than 6 days, more than 5 days, more than 4 days, more
than 3 days, more than 2 days, or more than 1 day. In some
embodiments, the biocompatible hydrogel polymer matrix is
substantially non-bioabsorbable.
[0094] The biocompatible hydrogel polymer matrix is slowly
bioabsorbed, dissolved, and or excreted. In some instances, the
rate of bioabsorption is controlled by the number of ester groups
in the biocompatible and/or biodegradable hydrogel polymer matrix.
In other instances, the higher the concentration of ester units is
in the biocompatible hydrogel polymer matrix, the longer is its
lifetime in the body. In further instances, the electron density at
the carbonyl of the ester unit controls the lifetime of the
biocompatible hydrogel polymer matrix in the body. In certain
instances, biocompatible hydrogel polymer matrices without ester
groups are essentially not biodegradable. In additional instances,
the molecular weight of the first and second compounds controls the
lifetime of the biocompatible hydrogel polymer matrix in the body.
In further instances, the number of ester groups per gram of
polymer matrix controls the lifetime of the biocompatible hydrogel
polymer matrix in the body.
[0095] In some instances, the lifetime of the biocompatible
hydrogel polymer matrix can be estimated using a model, which
controls the temperature and pH at physiological levels while
exposing the biocompatible hydrogel polymer matrix to a buffer
solution. In certain instances, the biodegradation of the
biocompatible hydrogel polymer matrix is substantially
non-enzymatic degradation.
[0096] In some embodiments, the selection of reaction conditions
determines the degradation time of the biocompatible hydrogel
polymer matrix. In certain embodiments, the concentration of the
first compound and second compound monomers determines the
degradation time of the resulting biocompatible hydrogel polymer
matrix. In some instances, a higher monomer concentration leads to
a higher degree of cross-linking in the resulting biocompatible
hydrogel polymer matrix. In certain instances, more cross-linking
leads to a later degradation of the biocompatible hydrogel polymer
matrix. In certain embodiments, temperature determines the
degradation time of the resulting biocompatible hydrogel polymer
matrix. In some instances, a higher monomer concentration leads to
a higher degree of cross-linking in the resulting biocompatible
hydrogel polymer matrix.
[0097] In certain embodiments, the composition of the linker in the
first and/or second compound influences the speed of degradation of
the resulting biocompatible hydrogel polymer matrix. In some
embodiments, the more ester groups are present in the biocompatible
hydrogel polymer matrix, the faster the degradation of the
biocompatible hydrogel polymer matrix. In certain embodiments, the
higher the concentration of mercaptopropionate (ETTMP), acetate
amine (AA), glutarate or succinate (SG or SS) monomers, the faster
the rate of degradation.
[0098] In certain embodiments, the composition of the cell
influences the speed of degradation of the resulting biocompatible
hydrogel polymer matrix. In certain embodiments, the concentration
of the cell influences the speed of degradation of the resulting
biocompatible hydrogel polymer matrix. In certain embodiments, the
composition of a buffer influences the speed of degradation of the
resulting biocompatible hydrogel polymer matrix. In certain
embodiments, the concentration of a buffer influences the speed of
degradation of the resulting biocompatible hydrogel polymer matrix.
In certain embodiments, the pH of a buffer influences the speed of
degradation of the resulting biocompatible hydrogel polymer matrix.
In certain embodiments, the composition of the optional additional
components influences the speed of degradation of the resulting
biocompatible hydrogel polymer matrix.
Pre-Formulations and Hydrogel Matrices for Cell Delivery in the
Treatment of Disease
[0099] The treatment of tendon injuries by stem cells necessitates
controlled delivery and release of cells at the target area. For
example, bone marrow mesenchymal stem cells (MSCs) have a
beneficial effect on the healing of tendon injuries in a horse.
Current methodologies inject MSCs in autologous bone marrow
aspirate in large numbers (10-20 million cells), however less than
25% of the MSCs remain in the injury area after 24 hours due to
systemic clearance. Retention of cells at the delivery site may
encourage the cells to engraft into the tissue resulting in
increased amounts of MSCs available to contribute to tissue
healing. The biocompatible pre-formulation and hydrogel polymer
matrix described herein are configured to deliver cells such as
MSCs within a pliable, injectable and absorbable gel that is
tolerated clinically and is compatible with cell survival and
growth. In some embodiments, the biocompatible pre-formulation and
hydrogel polymer matrix described herein provide improved cell
viability over cells injected without the use of a biocompatible
pre-formulation. In certain embodiments, the biocompatible hydrogel
polymer matrix functions as a scaffold supporting the growth of
cells loaded on or within the hydrogel polymer matrix.
[0100] In some embodiments, the biocompatible pre-formulation or
hydrogel polymer matrix described herein is delivered to a target
site on or in a mammal. In certain embodiments, the biocompatible
pre-formulation or hydrogel polymer matrix is delivered to a target
site in a joint. In some embodiments, the biocompatible
pre-formulation forms a biocompatible hydrogel polymer matrix
inside a joint. In certain embodiments, the biocompatible
pre-formulation forms a sticky biocompatible polymer matrix to seal
a wound on or in an animal. In some embodiments, the biocompatible
pre-formulation forms a suture. In certain embodiments, the wound
patch, joint spacer, or suture gels at least in part at the target
site in or on the mammal. In some embodiments, the wound patch,
joint spacer, or suture polymerizes at least in part at a target
site. In some embodiments, the wound patch, joint spacer, or suture
adheres at least partially to the target site.
[0101] In certain embodiments, the biocompatible pre-formulation is
used as a "liquid suture" or as a drug delivery platform to
transport medications directly to the targeted site in or on the
mammal. In some embodiments the target site is a joint, a wound or
a surgical site. In some embodiments, the spreadability, viscosity,
optical clarity, and adhesive properties of the biocompatible
pre-formulation or hydrogel polymer matrix are optimized to create
materials ideal as liquid sutures for the treatment of diseases. In
certain embodiments, the gel time is controlled from 50 seconds to
15 minutes.
[0102] In some embodiments, a biocompatible pre-formulation or
hydrogel polymer matrix comprising at least one cell is delivered
to a target site in a mammal. In some embodiments, the
biocompatible pre-formulation or hydrogel polymer matrix is
configured to deliver cells into damaged tissue in order to treat
disease or injury. In some embodiments, the diseases include, but
are not limited to, cancer, diabetes, Alzheimer's disease,
Parkinson's disease, Huntington's disease, and Celiac disease. In
some embodiments, the injury is caused by cardiac failure, muscle
damage, brain damage, or neurological disorders. In some
embodiments, the injury is a spinal cord injury. In some
embodiments, the delivered cells are configured to treat orthopedic
diseases or injuries. In some embodiments, the delivered cells are
configured to repair tendons, joints, bone defects, muscle, or
nerves.
Control of Release Rate of a Cell
[0103] In some embodiments, the biocompatible hydrogel polymer
matrix slowly delivers at least one cell to a target site by
diffusion and/or osmosis over time ranging from hours to days. In
certain embodiments, the cell is delivered directly to the target
site. In some embodiments, the procedure of delivering a
biocompatible hydrogel polymer matrix comprising a cell to a target
site is repeated several times, if needed. In other embodiments,
the cell is released from the biocompatible hydrogel polymer matrix
through biodegradation of the biocompatible hydrogel polymer
matrix. In some embodiments, the cell is released through a
combination of diffusion, osmosis, and/or biocompatible hydrogel
degradation mechanisms. In certain embodiments, the release profile
of the cell from the biocompatible hydrogel polymer matrix is
unimodal. In some embodiments, the release profile of the cell from
the biocompatible hydrogel polymer matrix is bimodal. In certain
embodiments, the release profile of the cell from the biocompatible
hydrogel polymer matrix is multimodal.
[0104] In some embodiments, the cell is released from the
biocompatible hydrogel polymer matrix though diffusion or osmosis.
In certain embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within 180 days. In some
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within 14 days. In certain
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within 24 hours. In some
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within one hour. In certain
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within about 180 days, about
150 days, about 120 days, about 90 days, about 80 days, about 70
days, about 60 days, about 50 days, about 40 days, about 35 days,
about 30 days, about 28 days, about 21 days, about 14 days, about
10 days, about 7 days, about 6 days, about 5 days, about 4 days,
about 3 days, about 2 days, about 1 day, about 0.5 day, about 6
hours, about 4 hours, about 2 hours, about or 1 hour. In some
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within more than 180 days,
more than 150 days, more than 120 days, more than 90 days, more
than 80 days, more than 70 days, more than 60 days, more than 50
days, more than 40 days, more than 35 days, more than 30 days, more
than 28 days, more than 21 days, more than 14 days, more than 10
days, more than 7 days, more than 6 days, more than 5 days, more
than 4 days, more than 3 days, more than 2 days, more than 1 day,
more than 0.5 day, more than 6 hours, more than 4 hours, more than
2 hours, more than or 1 hour. In certain embodiments, the cell is
substantially released from the biocompatible hydrogel polymer
matrix within less than 180 days, less than 150 days, less than 120
days, less than 90 days, less than 80 days, less than 70 days, less
than 60 days, less than 50 days, less than 40 days, less than 35
days, less than 30 days, less than 28 days, less than 21 days, less
than 14 days, less than 10 days, less than 7 days, less than 6
days, less than 5 days, less than 4 days, less than 3 days, less
than 2 days, less than 1 day, less than 0.5 day, less than 6 hours,
less than 4 hours, less than 2 hours, less than or 1 hour. In some
embodiments, the cell is substantially released from the
biocompatible hydrogel polymer matrix within about one day to about
fourteen days. In certain embodiments, the cell is substantially
released from the biocompatible hydrogel polymer matrix within
about one day to about 70 days.
[0105] In some embodiments, release of the cell from the
biocompatible hydrogel polymer matrix is controlled by the
composition of the biocompatible hydrogel polymer matrix. In
certain embodiments, the cell is released when the biocompatible
hydrogel polymer matrix starts to degrade. In some embodiments, the
pore size of the biocompatible hydrogel polymer matrix is small
enough to prevent the early phase release of the cell (i.e.,
release before the degradation of the biocompatible hydrogel
polymer matrix). In certain embodiments, the pore size of the
biocompatible hydrogel polymer matrix is large enough to allow the
early phase release of the cell.
[0106] In some embodiments, large PEG groups in the monomers leads
to large pore sizes in the resulting biocompatible hydrogel polymer
matrix allowing the elution of large cells. In certain embodiments,
large molecular weights of the monomers lead to biocompatible
hydrogel polymer matrices with large pore sizes. In some
embodiments, large monomer molecular weights of about 10 kDa lead
to biocompatible hydrogel polymer matrices with large pore sizes.
In certain embodiments, large monomer molecular weights of about 20
kDa lead to biocompatible hydrogel polymer matrices with large pore
sizes.
[0107] In some embodiments, small PEG groups in the monomers leads
to small pore sizes in the resulting biocompatible hydrogel polymer
matrix restricting the elution of small (and large) cells. In
certain embodiments, small molecular weights of the monomers lead
to biocompatible hydrogel polymer matrices with small pore sizes.
In some embodiments, small monomer molecular weights of about 5 kDa
lead to biocompatible hydrogel polymer matrices with small pore
sizes. In certain embodiments, small monomer molecular weights of
about 10 kDa in an 8-ARM monomer lead to biocompatible hydrogel
polymer matrices with small pore sizes. In some embodiments, the
small pore sizes restrict the elution of cells.
Exemplary Cells
[0108] In some embodiments, the biocompatible hydrogel polymer
matrix comprises at least one cell. In some embodiments, the
biocompatible hydrogel polymer matrix is delivered with a cell.
Examples of cells include, but are not limited to mammalian,
insect, protozoal, bacterial, viral, and fungal. In some
embodiments, the cells may be genetically engineered. In some
embodiments, the cells may be a vaccine.
[0109] In certain embodiments, the cell is a mammalian cell.
Examples of mammalian cells include, but are not limited to human,
murine, hamster, rat, canine and primate. Examples of human cells
include, but are not limited to embryonic, adult, bone marrow
stromal, embryonic germline, fetal, oligopotent progenitor, somatic
and induced pluripotent. Mammalian cells include, but are not
limited to, established or developed cell lines. Examples of cell
lines include, but are not limited to, HEK-293, CHO, 293-T, A2780,
BHK-21, BCP-1, DU145, H1299, HeLa, High-Five, HUVEC, MCF-7 and RBL.
Mammalian cells include, but are not limited to, stem cells.
Examples of stem cells include, but are not limited to adult,
embryonic, hematopoietic, embryonic, mesenchymal, multipotent,
neural, pluripotent, totipotent, umbilical cord and unipotent.
[0110] In certain embodiments, the cell is an insect cell. In
certain embodiments, the insect cell is genetically engineered. In
certain embodiments, the insect cell is non-infectious. Examples of
insect cells include, but are not limited to, Spodoptera
frugiperda, Drosophila and Trichoplusia ni.
[0111] In certain embodiments, the cell is a protozoa cell. In
certain embodiments, the protozoa cell is genetically engineered.
In certain embodiments, the protozoa cell is a vaccine. In certain
embodiments, the protozoa cell is non-infectious. Examples of
protozoa cells include, but are not limited to, Giardia lamblia,
Entamoeba histolytica, Plasmodium knowlesi and Balantidium
coli.
[0112] In certain embodiments, the cell is a bacterial cell. In
certain embodiments, the bacterial cell is genetically engineered.
In certain embodiments, the bacterial cell is a vaccine. In certain
embodiments, the bacterial cell is non-infectious. Examples of
bacterial cells include, but are not limited to, Acetobacter
aurantius, Agrobacterium radiobacter, Anaplasma phagocytophilum,
Azorhizobium caulinodans, Bacillus anthracis, Bacillus brevis,
Bacillus cereus, Bacillus subtilis, Bacteroides fragilis,
Bacteroides gingivalis, Bacteroides melaminogenicus, Bartonella
quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia
burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis,
Burkholderia mallei, Burkholderia pseudomallei, Burkholderia
cepacia, Calymmatobacterium granulomatis, Campylobacter coli,
Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori,
Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila
psittaci, Clostridium botulinum, Clostridium difficile,
Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella
burnetii, Enterobacter cloacae, Enterococcus faecalis, Enterococcus
faecium, Enterococcus galllinarum, Enterococcus maloratus,
Escherichia coli, Francisella tularensis, Fusobacterium nucleatum,
Gardnerella vaginalis, Haemophilus influenzae, Haemophilus
parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis,
Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus
acidophilus, Lactococcus lactis, Legionella pneumophila, Listeria
monocytogenes, Methanobacterium extroquens, Microbacterium
multiforme, Micrococcus luteus, Moraxella catarrhalis,
Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma
pneumonic, Neisseria gonorrhoeae, Neisseria meningitidis,
Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus,
Porphyromonas gingivalis, Prevotella melaminogenica, Pseudomonas
aeruginosa, Rhizobium radiobacter, Rickettsia rickettsii, Rothia
dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella
typhimurium, Shigella dysenteriae, Staphylococcus aureus,
Staphylococcus epidermidis, Stenotrophomonas maltophilia,
Streptococcus pneumoniae, Streptococcus pyogenes, Treponema
pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma,
Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica
and Yersinia pseudotuberculosis.
[0113] In certain embodiments, the cell is a viral cell. In certain
embodiments, the viral cell is genetically engineered. In certain
embodiments, the cell is a vaccine. In certain embodiments, the
cell is non-infectious. In certain embodiments, the viral cell is a
bacteriophage. Examples of viral cells include, but are not limited
to, Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses,
Reoviruses, Picornaviruses, Togaviruses, Orthomyxoviruses,
Rhabdoviruses, Retroviruses and Hepadnaviruses.
[0114] In certain embodiments, the cell is a fungal cell. In
certain embodiments, the fungal cell is genetically engineered. In
certain embodiments, the fungal cell is non-infectious. Examples of
fungal cells include, but are not limited to, Cryptococcus
neoformans, Cryptococcus gattii, Candida albicans, Candida
tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei,
Candida parapsilosis, Candida guilliermondii, Candida viswanathii,
Candida lusitaniae, Rhodotorula mucilaginosa, Schizosaccharomyces
pombe, Saccharomyces cerevisiae, Brettanomyces bruxellensis,
Candida stellata, Schizosaccharomyces pombe, Torulaspora
delbrueckii, Zygosaccharomyces bailii, Yarrowia lipolytica,
Saccharomyces exiguus and Pichia pastoris.
Exemplary Culture Media
[0115] In some embodiments, the biocompatible hydrogel polymer
matrix comprises a buffer or culture medium. In some embodiments,
the biocompatible hydrogel polymer matrix comprises a buffer and at
least one cell. In some embodiments, the culture medium is a
buffer. In some embodiments, the culture medium comprises a growth
medium. In some embodiments, the culture medium is nutrient rich.
In certain embodiments, the culture medium provides nutrients
sufficient for cell viability, growth, and/or proliferation. In
certain embodiments, culture media include, but are not limited to,
DMEM, IMDM, OptiMEM.RTM., AlgiMatrix.TM., Fetal Bovine Serum,
GS1-R.RTM., GS2-M.RTM., iSTEM.RTM., NDiff.RTM. N2,NDiff.RTM. N2-AF,
RHB-A.RTM., RHB-Basal.RTM., RPMI, SensiCell.TM., GlutaMAX.TM.,
FluoroBrite.TM., Gibco.RTM. TAP, Gibco.RTM. BG-11, LB, M9 Minimal,
Terrific Broth, 2YXT, MagicMedia.TM., ImMedia.TM., SOC, YPD, CSM,
YNB, Grace's Insect Media, 199/109 and HamF10/HamF12. In certain
embodiments, the cell culture medium may be serum free. In certain
embodiments, the culture medium includes additives. In some
embodiments, culture medium additives include, but are not limited
to, antibiotics, vitamins, proteins, inhibitors, small molecules,
minerals, inorganic salts, nitrogen, growth factors, amino acids,
serum, carbohydrates, lipids, hormones and glucose. In some
embodiments, growth factors include, but are not limited to, EGF,
bFGF, FGF, ECGF, IGF-1, PDGF, NGF, TGF-.alpha. and TGF-.beta.. In
certain embodiments, the culture medium may not be aqueous. In
certain embodiments, the non-aqueous culture medium include, but
are not limited to, frozen cell stocks, lyophilized medium and
agar.
Exemplary Combinations
[0116] In some embodiments, one or more optional additional
components can be incorporated into the biocompatible hydrogel
polymer matrix formulation. Provided herein are biocompatible
pre-formulations, comprising at least one first compound comprising
more than one nucleophilic group, at least one second compound
comprising more than one electrophilic group, at least one cell,
and optionally additional components. An exemplary additional
component is a buffer. In certain embodiments, the cell is a stem
cell. In certain embodiments, the additional component is a culture
medium. In certain embodiments, the culture medium is nutrient
rich. A biocompatible hydrogel polymer matrix is formed following
mixing the first compound, the second compound, and the at least
one cell in the presence of water; wherein the biocompatible
hydrogel polymer matrix gels at a target site. In some embodiments
a buffer or other additional components may be added to the
pre-formulation mix prior to or after biocompatible hydrogel
polymer matrix formation. In some embodiments, the first compound
and the second compound do not react with the at least one cell
during formation of the biocompatible hydrogel polymer matrix. In
certain embodiments, the biocompatible hydrogel polymer matrix
comprises a biocompatible hydrogel scaffold. In certain
embodiments, the biocompatible hydrogel scaffold comprises the at
least one first compound and the at least one second compound. In
certain embodiments, the biocompatible hydrogel scaffold comprises
a buffer. In certain embodiments, the biocompatible hydrogel
scaffold is fully synthetic.
[0117] Provided herein are biocompatible pre-formulations,
comprising at least one first compound comprising more than one
nucleophilic group, at least one second compound comprising more
than one electrophilic group, a buffer, and optionally additional
components. An exemplary additional component is at least one cell.
In certain embodiments the cell is a stem cell. In certain
embodiments, the buffer is a culture medium. In certain
embodiments, the culture medium is nutrient rich. A biocompatible
hydrogel polymer matrix is formed following mixing the first
compound, the second compound, and the buffer in the presence of
water; wherein the biocompatible hydrogel polymer matrix gels at a
target site. In some embodiments at least one cell or other
additional components may be added to the mix prior to or after
biocompatible hydrogel polymer matrix formation. In some
embodiments, the first compound and the second compound do not
react with the at least one cell during formation of the
biocompatible hydrogel polymer matrix. In certain embodiments, the
biocompatible hydrogel polymer matrix comprises a biocompatible
hydrogel scaffold. In certain embodiments, the biocompatible
hydrogel scaffold comprises the at least one first compound, the at
least one second compound and a buffer. In certain embodiments, the
biocompatible hydrogel scaffold is fully synthetic.
[0118] In certain embodiments, the biocompatible pre-formulation or
biocompatible hydrogel polymer matrix comprises at least one
additional component. Additional components include, but are not
limited to, proteins, biomolecules, growth factors, anesthetics,
antibacterials, antivirals, immunosuppressants, anti-inflammatory
agents, anti-proliferative agents, anti-angiogenesis agents and
hormones.
[0119] In some embodiments, the biocompatible hydrogel polymer
matrix or biocompatible pre-formulation further comprise a
visualization agent for visualizing the placement of the
biocompatible hydrogel polymer matrix at a target site The
visualization agent assists in visualizing the placement using
minimally invasive delivery, e.g., using an endoscopic device. In
certain embodiments, the visualization agent is a dye. In specific
embodiments, the visualization agent is a colorant.
[0120] In some embodiments, the biocompatible hydrogel polymer
matrix formulations further comprise a contrast agent for
visualizing the biocompatible hydrogel formulation and locating a
tumor using e.g., X-ray, fluoroscopy, or computed tomography (CT)
imaging. In certain embodiments, the contrast agent is radiopaque.
In some embodiments, the radiopaque material is selected from
sodium iodide, potassium iodide, barium sulfate, VISIPAQUE.RTM.,
OMNIPAQUE.RTM., or HYPAQUE.RTM., tantalum, and similar commercially
available compounds, or combinations thereof.
EXAMPLES
[0121] The following specific examples are to be construed as
merely illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever.
[0122] The following are general characteristics of the
biocompatible pre-formulations and biocompatible hydrogel polymer
matrices consistent with biocompatibility.
TABLE-US-00001 Pre-formulations Property Characteristics 1 In vivo
polymerizable Could be polymerized inside mammalian cavity or over
the skin 2 Reaction mixture pH Physiological to 8.0 pH range 3
Reaction temperature Ambient to body temperature 4 Formulation Two
or three component system; Mixed physical form immediately prior to
use, may contain radiopaque agent such as barium sulphate or iodine
containing organic compounds or other known radiopaque agents 5
Mixing time for the Few seconds (~10 sec) reaction to start 6 Gel
formation time Gel formation time ranges from 10 seconds to 120
seconds, or could be as long as 30 minutes depending on the
application 7 Solution viscosity Solution viscosity ranges from 1
to 800 cps 8 Sterilization ETO to E-beam sterilizable capability 9
Localized delivery Ideal for localized delivery for small
molecules, large molecules and cells 10 Stability of drugs in All
small molecule drugs and formulation mixture proteins studied so
far have been found to be stable
[0123] The following are some characteristics of adhesive
biocompatible hydrogel polymer matrices.
TABLE-US-00002 Hydrogel Property Characteristics 1 Tissue adhesion
Sticky formulations, physicochemical characteristics ideal for
bonding to skin, bones, or other mammalian tissues 2 Polymer
hardness Can be controlled from soft tissues to harder cartilage
like materials 3 Bioabsorption Time About 2 weeks up to 10 years,
or totally non-bioabsorbable 4 Biocompatibility Highly
biocompatible; passed all the subjected ISO 10993 tests 5 Polymer
cytotoxicity Non-cytotoxic formulations 6 Small molecule elution
Small drug molecules elution can be controlled and thus
pharmaceutical drugs could also be delivered using the
formulations, if needed 7 Compatibility with Highly compatible due
to physiological pH of the polymers proteins and Cells
[0124] Biocompatible pre-formulation chemical components used to
form biocompatible hydrogel polymer matrices are listed in Table 1.
These biocompatible pre-formulation components will be referred to
by their abbreviations. Several USP grade viscosity enhancing
agents were purchased from Sigma-Aldrich and were stored at
25.degree. C. They include methylcellulose (Methocel.RTM. MC,
10-25MPA.S) abbreviated as MC; hypromellose
(hydroxypropylmethylcellulose 2910) abbreviated as HPMC; and
povidone K-30 (polyvinylpyrrolidone) abbreviated as PVP.
[0125] The biocompatible pre-formulation components were stored at
5.degree. C. and allowed to warm to room temperature before use,
which typically took 30 minutes. After use the contents were purged
with N.sub.2 for approximately 30 seconds before sealing with
parafilm and returning to 5.degree. C. Alternately, the
biocompatible pre-formulation components were stored at -20.degree.
C. and allowed to warm to room temperature before use under the
flow of inert gas, which typically took 30 minutes. The
biocompatible pre-formulation components were purged with inert gas
for at least 30 seconds before returning to -20.degree. C.
[0126] A 0.15 M phosphate buffer was made by dissolving 9.00 g
(0.075 mol) NaH.sub.2PO.sub.4 in 500 mL of distilled water at
25.degree. C. with magnetic stirring. The pH was then adjusted to
7.99 with the dropwise addition of 50% aqueous NaOH. Several other
phosphate buffers were prepared in a similar fashion: 0.10 M
phosphate at pH 9, 0.10 M phosphate at pH 7.80, 0.10 M phosphate at
7.72, 0.10 M phosphate at pH 7.46, 0.15 M phosphate at pH 7.94,
0.15 M phosphate at pH 7.90, 0.4 M phosphate at pH 9, and 0.05 M
phosphate at pH 7.40.
[0127] A sterile 0.10 M phosphate buffer at pH 7.58 with 0.30% HPMC
was prepared for use in kits. First, 1.417 g HPMC was dissolved in
471 mL of 0.10 M phosphate buffer at pH 7.58 by vigorous shaking.
The viscous solution was allowed to clarify overnight. The solution
was filtered through a 0.22 .mu.m filter (Corning #431097) with
application of light vacuum. The viscosity of the resulting
solution was measured to be 8.48 cSt+/-0.06 at 20.degree. C.
[0128] A sterile 0.10 M phosphate buffer at pH 7.58 with 0.3% HPMC
was prepared. First, a 0.10 M phosphate buffer was made by
dissolving 5.999 g (0.05 mol) of NaH.sub.2PO.sub.4 in 500 mL of
distilled water at 20.degree. C. with magnetic stirring. The pH was
then adjusted to 7.58 with the dropwise addition of 50% aqueous
NaOH. Then, 1.5 g of HPMC was dissolved in 500 mL of the above
buffer solution by vigorous shaking. The viscous solution was
allowed to clarify overnight. The solution was filtered through a
0.22 .mu.m filter (Corning #431097) with application of light
vacuum. The viscosity of the resulting solution was measured via
the procedure as described in the Viscosity Measurements section
and was found to be 8.48 cSt+/-0.06 at 20.degree. C.
[0129] Phosphate buffered saline (PBS) was prepared by dissolving
two PBS tablets (Sigma Chemical, P4417) in 400 mL of distilled
water at 25.degree. C. with vigorous shaking. The solution has the
following composition and pH: 0.01 M phosphate, 0.0027 M potassium
chloride, 0.137 M sodium chloride, pH 7.46.
[0130] A 0.058 M phosphate buffer was made by dissolving 3.45 g
(0.029 mol) of NaH.sub.2PO.sub.4 in 500 mL of distilled water at
25.degree. C. with magnetic stirring. The pH was then adjusted to
7.97 with the dropwise addition of 50% aqueous NaOH.
[0131] A 0.05 M borate buffer was made by dissolving 9.53 g (0.025
mol) of Na.sub.2B.sub.4O.sub.7.10H2O in 500 mL of distilled water
at 25.degree. C. with magnetic stirring. The pH was then adjusted
to 7.93 or 8.35 with the dropwise addition of 6.0 N HCl.
[0132] An antiseptic liquid component was prepared in a similar
fashion with a commercial 2% chlorhexidine solution. To 100 mL of
2% chlorhexidine solution was dissolved 0.3 g of HPMC. The viscous
solution was allowed to clarify overnight at 5.degree. C. The
resulting clear blue solution has the following composition: 2%
chlorhexidine, 0.3% HPMC and an unknown quantity of nontoxic blue
dye and detergent.
[0133] Other liquid components were prepared in a similar fashion
by simply dissolving the appropriate amount of the desired additive
to the solution. For example, an antiseptic liquid component with
1% denatonium benzoate, a bittering agent, was prepared by
dissolving 2 g of denatonium benzoate in 200 mL of 2% chlorhexidine
solution.
[0134] Alternatively, commercially available drug solutions were
used as the liquid component. For example, saline solution,
Kenalog-10 (10 mg/mL solution of triamcinolone acetonide) and
Depo-Medrol (40 mg/mL of methylprednisolone acetate) were used.
[0135] The amine or thiol component (typically in the range of 0.1
mmol arms equivalents) was added to a 50 mL centrifuge tube. A
volume of reaction buffer was added to the tube via a pipette such
that the final concentration of solids in solution was about 5
percent. The mixture was gently swirled to dissolve the solids
before adding the appropriate amount of ester or epoxide.
Immediately after adding the ester or epoxide, the entire solution
was shaken for 10 seconds before letting it rest.
TABLE-US-00003 TABLE 1 Components used in biocompatible
pre-formulations. Pre-formulation Components Technical Name
ETTMP-1300 Ethoxylated trimethylolpropane tri(3-mercaptopropionate)
4ARM-5k-SH 4ARM PEG Thiol (pentaerythritol) 4ARM-2k-NH2 4ARM PEG
Amine (pentaerythritol), HCl Salt, MW 2000 4ARM-5k-NH2 4ARM PEG
Amine (pentaerythritol), HCl Salt, MW 5000 8ARM-20k-NH2 8ARM PEG
Amine (hexaglycerol), HCl Salt, MW 20000 4ARM-20k-AA 4ARM PEG
Acetate Amine HCl Salt, MW 20000 8ARM-20k-AA 8ARM PEG Acetate Amine
(hexaglycerol) HCl Salt, MW 20000 8ARM-20k-AA 8ARM PEG Acetate
Amine (hexaglycerol) TFA Salt, MW 20000 4ARM-10k-SG 4ARM PEG
Succinimidyl Glutarate (pentaerythritol), MW 10000 8ARM-15k-SG 8ARM
PEG Succinimidyl Glutarate (hexaglycerol), MW 15000 4ARM-20k-SGA
4ARM PEG Succinimidyl Glutaramide (pentaerythritol), MW 20000
4ARM-10k-SS 4ARM PEG Succinimidyl Succinate (pentaerythritol), MW
10000 EJ-190 Sorbitol polyglycidyl ether MC Methyl Cellulose
(Methocel .RTM. MC) HPMC Hypromellose
(Hydroxypropylmethylcellulose) PVP Povidone
(polyvinylpyrrolidone)
[0136] The gel time for all cases was measured starting from the
addition of the ester or epoxide until the gelation of the
solution. The gel point was noted by pipetting 1 mL of the reaction
mixture and observing the dropwise increase in viscosity.
Degradation of the polymers was performed by the addition of 5 to
10 mL of phosphate buffered saline to ca. 5 g of the material in a
50 mL centrifuge tube and incubating the mixture at 37.degree. C.
The degradation time was measured starting from the day of addition
of the phosphate buffer to complete dissolution of the polymer into
solution.
Example 1
Manufacture of a Biocompatible Hydrogel Polymer Matrix (Amine-Ester
Chemistry)
[0137] A solution of 8ARM-20K-NH2 was prepared in a Falcon tube by
dissolving about 0.13 g solid monomer in about 2.5 mL of sodium
phosphate buffer (buffer pH 7.36). The mixture was shaken for about
10 seconds at ambient temperature until complete dissolution was
obtained. The Falcon tube was allowed to stand at ambient
temperature. In another Falcon tube, 0.10 g of 8ARM-15K-SG was
dissolved in the same phosphate buffer as above. The mixture was
shaken for about 10 seconds and at this point all the powder
dissolved. The 8ARM-15K-SG solution was poured immediately into the
8ARM-20K-NH2 solution and a timer was started. The mixture was
shaken and mixed for about 10 seconds and a 1 mL solution of the
mixture was pipetted out using a mechanical high precision pipette.
The gel time of 1 mL liquid was collected and then verified with
the lack of flow for the remaining liquids. The gel time data of
the formulation was recorded and was about 90 seconds.
Example 2
Manufacture of a Biocompatible Hydrogel Polymer Matrix (Amine-Ester
Chemistry)
[0138] A solution of amines was prepared in a Falcon tube by
dissolving about 0.4 g solid 4ARM-20k-AA and about 0.2 g solid
8ARM-20k-NH2 in about 18 mL of sodium phosphate buffer (buffer pH
7.36). The mixture was shaken for about 10 seconds at ambient
temperature until complete dissolution was obtained. The Falcon
tube was allowed to stand at ambient temperature. To this solution,
0.3 g of 8ARM-15K-SG was added. The mixture was shaken to mix for
about 10 seconds until all the powder dissolved. 1 mL of the
mixture was pipetted out using a mechanical high precision pipette.
The gel time of the formulation was collected using the process
described above. The gel time was about 90 seconds.
Example 3
Manufacture of a Biocompatible Hydrogel Polymer Matrix (Thiol-Ester
Chemistry)
[0139] A solution of ETTMP-1300 was prepared in a Falcon tube by
dissolving about 0.04 g monomer in about 5 mL of sodium borate
buffer (buffer pH 8.35). The mixture was shaken for about 10
seconds at ambient temperature until complete dissolution was
obtained. The Falcon tube was allowed to stand at ambient
temperature. To this solution, 0.20 g of 8ARM-15K-SG was added. The
mixture was shaken for about 10 seconds until the powder dissolved.
1 mL of the mixture was pipetted out using a mechanical high
precision pipette. The gel time was found to be about 70
seconds.
Example 4
Manufacture of a Biocompatible Hydrogel Polymer Matrix
(Thiol-Epoxide Chemistry)
[0140] A solution of ETTMP-1300 was prepared in a Falcon tube by
dissolving about 0.04 g monomer in about 5 mL of sodium borate
buffer (buffer pH 8.35). The mixture was shaken for about 10
seconds at ambient temperature until complete dissolution was
obtained. The Falcon tube was allowed to stand at ambient
temperature. To this solution, 0.10 g of EJ-190 was added. The
mixture was shaken for about 10 seconds until complete dissolution
is obtained. 1 mL of the mixture was pipetted out using a
mechanical high precision pipette. The gel time was found to be
about 6 minutes.
Example 5
In Vitro Bioabsorbance Testing
[0141] A 0.10 molar buffer solution of pH 7.40 was prepared with
deionized water. A 50 mL portion of this solution was transferred
to a Falcon tube. A sample polymer was prepared in a 20 cc syringe.
After curing, a 2-4 mm thick slice was cut from the polymer slug
and was placed in the Falcon tube. A circulating water bath was
prepared and maintained at 37.degree. C. The Falcon tube with
polymer was placed inside the water bath and time was started. The
dissolution of the polymer was monitored and recorded. The
dissolution time ranged from 1-90 days depending on the type of
sample polymer.
Example 6
Gelling and Degradation Times of Amine-Ester Polymers
[0142] Amines studied were 8ARM-20k-NH2 and 4ARM-5k-NH2. The
formulation details and material properties are given in Table 2.
With 8ARM-20k-NH2, it was found that a phosphate buffer with 0.058
M phosphate and pH of 7.97 was necessary to obtain acceptable gel
times of around 100 seconds. Using a 0.05 M phosphate buffer with a
pH of 7.41 resulted in a more than two-fold increase in gel time
(270 seconds).
[0143] With the 8ARM-20k-NH2, the ratio of 4ARM-10k-SS to
4ARM-20k-SGA was varied from 50:50 to 90:10. The gel time remained
consistent, but there was a marked shift in degradation time around
a ratio of 80:20. For formulations with ratios of 75:25 and 50:50,
degradation times spiked to one month and beyond. Using lower
amounts of 4ARM-20k-SGA (80:20, 85:15, 90:10) resulted in
degradation times of less than 7 days.
[0144] As a comparison, the 4ARM-5k-NH2 was used in a formulation
with a ratio of 4ARM-10k-SS to 4ARM-20k-SGA of 80:20. As was
expected, the degradation time remained consistent, which suggests
that the mechanism of degradation was unaffected by the change in
amine. However, the gel time increased by 60 seconds, which may
reflect the relative accessibility of reactive groups in a high
molecular weight 8ARM amine and a low molecular weight 4ARM
amine.
TABLE-US-00004 TABLE 2 Gel and degradation times for varying
4ARM-10k- SS/4ARM-20k-SGA ratios with 8ARM-15k-SG ester. Phosphate
Ratio of Reaction Buffer Gel Degradation Pre-formulation
4ARM-10k-SS/ Concentration and Time Time Components 4ARM-20k-SGA pH
(s) (days) 8ARM-20k-NH2 50/50 0.05M 270 N/A 4ARM-10k-SS,
4ARM-20k-SGA pH 7.41 8ARM-20k-NH2 50/50 0.058M 100 >41
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97 8ARM-20k-NH2 75/25 0.058M 90 29
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97 8ARM-20k-NH2 80/20 0.058M 100 7
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97 4ARM-5k-NH2 80/20 0.058M 160 6
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97 8ARM-20k-NH2 85/15 0.058M 100 5
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97 8ARM-20k-NH2 90/10 0.058M 90 6
4ARM-10k-SS, 4ARM-20k-SGA pH 7.97
Example 7
Gelling and Degradation Times of Thiol-Ester Polymers
[0145] Thiols studied were 4ARM-5k-SH and ETTMP-1300. The
formulation details and material properties are given in Table 3.
It was found that a 0.05 M borate buffer with a pH of 7.93 produced
gel times of around 120 seconds. Increasing the amount of
4ARM-20k-SGA in the formulation increased the gel time to 190
seconds (25:75 ratio of 4ARM-10k-SS to 4ARM-20k-SGA) up to 390
seconds (0:100 ratio of 4ARM-10k-SS to 4ARM-20k-SGA). Using a 0.05
M borate buffer with a pH of 8.35 resulted in a gel time of 65
seconds, about a two-fold decrease in gel time. Thus, the gel time
may be tailored by simply adjusting the pH of the reaction
buffer.
[0146] The ratio of 4ARM-10k-SS to 4ARM-20k-SGA was varied from
0:100 to 100:0. In all cases, the degradation time did not vary
significantly and was typically between 3 and 5 days. It is likely
that degradation is occurring via alternate pathways.
TABLE-US-00005 TABLE 3 Gel and degradation times for varying
4ARM-10k-SS/4ARM- 20k-SGA ratios with 4ARM-5k-SH and ETTMP-1300
thiols. Phosphate Ratio of Reaction Buffer Gel Degradation
Pre-formulation 4ARM-10k-SS/ Concentration and Time Time Components
4ARM-20k-SGA pH (s) (days) 4ARM-5k-SH 50/50 0.05M 65 N/A
4ARM-10k-SS, 4ARM-20k-SGA pH 8.35 4ARM-5k-SH 50/50 0.05M 120 4
4ARM-10k-SS, 4ARM-20k-SGA pH 7.93 4ARM-5k-SH 75/25 0.05M 125 4
4ARM-10k-SS, 4ARM-20k-SGA pH 7.93 4ARM-5k-SH 90/10 0.05M 115 4
4ARM-10k-SS, 4ARM-20k-SGA pH 7.93 4ARM-5k-SH 25/75 0.05M 190 4
4ARM-10k-SS, 4ARM-20k-SGA pH 7.93 4ARM-5k-SH 10/90 0.05M 200 4
4ARM-10k-SS, 4ARM-20k-SGA pH 7.93 ETTMP-1300 0/100 0.05M 390 3
4ARM-20k-SGA 4ARM-5k-SH 100/0 0.05M 120 4 4ARM-10k-SS pH 7.93
Example 8
Gelling and Degradation Times of Amine-Ester and Thiol-Ester
Polymers
[0147] An amine (4ARM-5k-NH2) and a thiol (4ARM-5k-SH) were studied
with the ester 4ARM-10k-SG. The formulation details and material
properties are given in Table 4. A 0.058 M phosphate buffer with a
pH of 7.97 yielded a gel time of 150 seconds with the amine. A 0.05
M borate buffer with a pH of 8.35 produced a gel time of 75 seconds
with the thiol.
[0148] The amine-based polymer appeared to show no signs of
degradation, as was expected from the lack of degradable groups.
However, the thiol-based polymer degraded in 5 days. This suggests
that degradation is occurring through alternate pathways, as was
observed in the thiol formulations with 4ARM-10k-SS and
4ARM-20k-SGA (vida supra).
TABLE-US-00006 TABLE 4 Gel and degradation times for amines and
thiols with 4ARM-10k-SG biocompatible pre-formulations. Reaction
Buffer Type, Gel Degradation Pre-formulation Concentration, and
Time Time Components pH (s) (days) 4ARM-5k-NH2 & Phosphate 150
Indefinite 4ARM-10k-SG (0.058M, pH 7.97) 4ARM-5k-SH & Borate 75
5 4ARM-10k-SG (0.05M, pH 8.35)
Example 9
Gelling and Degradation Times of Thiol-Sorbitol Polyglycidyl Ether
Polymers
[0149] With ETTMP-1300 conditions such as high pH (10), high
solution concentration (50%), or high borate concentration (0.16 M)
were necessary for the mixture to gel. Gel times ranged from around
30 minutes to many hours. The conditions that were explored
include: pH from 7 to 12; solution concentration from 5% to 50%;
borate concentration from 0.05 M to 0.16 M; and thiol to epoxide
ratios from 1:2 to 2:1.
[0150] The high pH necessary for the reaction to occur could result
in degradation of the thiol. Thus, a polymer with EJ-190 and
4ARM-5k-SH was prepared. A 13% solution formulation exhibited a gel
time of 230 seconds at a pH of between 9 and 10. The degradation
time was 32 days. At a lower pH of around 8, the mixture exhibited
gel times in the range of 1 to 2 hours.
Example 10
General Procedure for the Preparation of Polymerizable
Biocompatible Pre-Formulations
[0151] Several representative sticky formulations are listed in
Table 5 along with specific reaction details for the preparation of
polymerizable biocompatible pre-formulations. The biocompatible
hydrogel polymers were prepared by first dissolving the amine
component in phosphate buffer or the thiol component in borate
buffer. The appropriate amount of the ester component was then
added and the entire solution was mixed vigorously for 10 to 20
seconds. The gel time was measured starting from the addition of
the ester until the gelation of the solution.
TABLE-US-00007 TABLE 5 (A) Summary of the reaction details for
several representative sticky formulations without viscosity
enhancer; (B) more detailed tabulation of a selection of the
reaction details including moles (degradation times were measured
in phosphate buffered saline (PBS) at 37.degree. C.). (A) Amine or
Pre-formulation Thiol/Ester % Gel Degradation Components Molar
Ratio Buffer Solution Time (s) Time (days) 8ARM-20k-NH2 3 0.15 M
phosphate, 3 130 N/A 4ARM-20K-SGA pH 7.99 8ARM-20k-NH2 1/3 0.15 M
phosphate, 3 300 N/A 4ARM-20K-SGA pH 7.99 8ARM-20k-NH2 3 0.15 M
phosphate, 8 50 N/A 4ARM-10K-SS pH 7.99 8ARM-20k-NH2 1/3 0.15 M
phosphate, 8 80 N/A 4ARM-10K-SS pH 7.99 4ARM-20K-AA/ 3 0.15 M
phosphate, 5 210 1 to 3 8ARM-20k-NH2 pH 7.99 (75/25) 4ARM-20K-SGA
4ARM-20K-AA/ 5 0.15 M phosphate, 10 180 1 to 3 8ARM-20k-NH2 pH 7.99
(75/25) 4ARM-20K-SGA 4ARM-5K-NH2 5 0.10 M phosphate, 10 160 7
4ARM-10K-SG pH 7.80 4ARM-5K-NH2 5 0.10 M phosphate, 20 160 1 to 3
4ARM-10K-SS pH 7.80 4ARM-5K-NH2 3 0.10 M phosphate, 5 160 13
4ARM-10K-SG pH 7.80 4ARM-5K-NH2 5 0.15 M phosphate, 20 80 7
4ARM-10K-SG pH 7.99 4ARM-5K-NH2 5 0.15 M phosphate, 30 70 10
4ARM-10K-SG pH 7.99 4ARM-5K-NH2 5 0.15 M phosphate, 19 60 53
4ARM-20K-SGA pH 7.99 4ARM-5K-NH2 5 0.15 M phosphate, 12 70 53
4ARM-20K-SGA pH 7.99 4ARM-5K-NH2 1/5 0.15 M phosphate, 19 160 15
4ARM-10K-SG pH 7.99 4ARM-SH-5K 5 0.05 M borate, 20 120 2 to 4
4ARM-10K-SG pH 7.93 4ARM-NH2-2K 5 0.10 M phosphate, 10 120 15
8ARM-15K-SG pH 7.46 4ARM-NH2-2K 7 0.10 M phosphate, 30 150 N/A
4ARM-20K-SGA pH 7.80 (B) Polymer % Pre-formulation Arms Solution
Components MW Mmoles Wt (g) Arm mmoles Eq (w/v) 8ARM-20k-NH2 20000
1000 0.075 8 0.00375 0.03 4ARM-20k-SGA 20000 1000 0.05 4 0.0025
0.01 Buffer Volume (phosphate) 4.1 3.0 8ARM-20k-NH2 20000 1000
0.025 8 0.00125 0.01 4ARM-20k-SGA 20000 1000 0.15 4 0.0075 0.03
Buffer Volume (phosphate) 5.8 3.0 8ARM-20k-NH2 20000 1000 0.3 8
0.015 0.12 4ARM-10k-SS 10000 1000 0.1 4 0.01 0.04 Buffer Volume
(phosphate) 5 8.0 8ARM-20k-NH2 20000 1000 0.1 8 0.005 0.04
4ARM-10k-SS 10000 1000 0.3 4 0.03 0.12 Buffer Volume (phosphate) 5
8.0
TABLE-US-00008 TABLE 6 Gel times for the
8ARM-20k-NH2/4ARM-20k-SGA(1/1) sticky polymers including HPMC as
viscosity enhancer with varying buffers and concentrations.
Pre-formulation Amine/Ester % Gel Time Components Molar Ratio
Buffer Solution (min) 8ARM-20k-NH2 1 0.10M 4.8 1.5 4ARM-20K-SGA
phosphate, 0.3% HPMC pH 7.80 8ARM-20k-NH2 1 0.10M 4.8 3.5
4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.46 8ARM-20k-NH2 1 0.05M 4.8
4.5 4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.42 8ARM-20k-NH2 1 0.05M
4 5.5 4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.42 8ARM-20k-NH2 1
0.05M 3 8.5 4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.42 8ARM-20k-NH2
1 0.05M 4.8 6.75 4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.24
8ARM-20k-NH2 1 0.05M 3 12 4ARM-20K-SGA phosphate, 0.3% HPMC pH 7.24
8ARM-20k-NH2 1 0.05M 2.5 15.5 4ARM-20K-SGA phosphate, 0.3% HPMC pH
7.24
[0152] Gel times ranged from 60 to 300 seconds and were found to be
easily tuned by adjusting the reaction buffer pH, buffer
concentration, or polymer concentration. An example of gel time
control for a single formulation is shown in Table 6, where the gel
time for the 8ARM-20k-NH2/4ARM-20k-SGA (1/1) polymer was varied
from 1.5 to 15.5 minutes.
[0153] In some instances, the stickiness of the polymers originates
from a mismatching in the molar equivalents of the components. A
variety of sticky materials using combinations of 4 or 8 armed
amines of molecular weights between 2 and 20 thousand and 4 or 8
armed esters of molecular weights between 10 and 20 thousand were
created. It was found that in comparison with the 8 armed esters,
the 4 armed esters resulted in stickier materials. For the amine
component, it was found that smaller molecular weights led to
stickier materials and higher amine to ester molar ratios.
[0154] A mismatch (amine to ester molar ratio) of at least 3 was
required to qualitatively sense stickiness. More preferably, a
ratio of around 5 produced a desirable level of stickiness combined
with polymer strength. Polymers with amine to ester molar ratios
higher than 5 may be formed as well, but some reaction conditions,
such as the polymer concentration, may need to be adjusted to
obtain a reasonable gel time. Furthermore, it was found that the
use of a viscosity enhanced solution improves the polymers by
increasing their strength and elasticity, allowing for higher amine
to ester molar ratios (Example 11; Table 9).
[0155] The materials formed were typically transparent and elastic.
Stickiness was tested for qualitatively by touch. Thus, a sticky
material adhered to a human finger or other surface and remained in
place until removed. Degradation times varied from 1 to 53 days. In
certain instances, the polymer properties, such as gel and
degradation times, pore sizes, swelling, etc. may be optimized for
different applications without losing the stickiness.
Example 11
General Procedure for the Preparation of Solutions with Enhanced
Viscosity
[0156] Polymer solutions with enhanced viscosities were prepared by
the addition of a viscosity enhancing agent to the reaction buffer.
Table 9B lists the viscosity enhancing agents studied, including
observations on the properties of the formed polymers. Stock
solutions of reaction buffers were prepared with varying
concentrations of methylcellulose (MC), hypromellose (HPMC) or
polyvinylpyrrolidone (PVP). As an example, a 2% (w/w) HPMC solution
in buffer was made by adding 0.2 g of HPMC to 9.8 mL of 0.10 M
phosphate buffer at pH 7.80, followed by vigorous shaking. The
solution was allowed to stand overnight. Buffer solutions with HPMC
concentrations ranging from 0.01% to 2.0% were prepared in a
similar fashion. Buffer solutions with PVP concentrations ranging
from 5% to 20% and buffer solutions with MC concentrations ranging
from 1.0 to 2.0% were also prepared by a similar method.
[0157] The polymers were formed in the same method as described
above in the general procedures for the preparation of the sticky
materials (Example 10). A typical procedure involved first
dissolving the amine component in the phosphate buffer containing
the desired concentration of viscosity enhancing agent. The
appropriate amount of the ester component was then added and the
entire solution was mixed vigorously for 10 to 20 seconds. The gel
time was measured starting from the addition of the ester until the
gelation of the solution.
[0158] Several representative formulations are listed in Table 7
and Table 8 along with specific reaction details. The percent of
degradable acetate amine component by mole equivalents is
represented by a ratio designated in parenthesis. For example, a
formulation with 75% degradable amine will be written as
8ARM-20k-AA/8ARM-20k-NH2 (75/25). The polymer was prepared by first
dissolving the formulation amine component in phosphate buffer. The
appropriate amount of the formulation ester component was then
added and the entire solution was mixed vigorously for 10 to 20
seconds. The gel time was measured starting from the addition of
the ester until the gelation of the solution.
[0159] The gel time is dependent on several factors: pH, buffer
concentration, polymer concentration, temperature and the
biocompatible pre-formulation monomers used. Previous experiments
have shown that the extent of mixing has little effect on the gel
time once the components are in solution, which typically takes up
to 10 seconds. The effect of biocompatible pre-formulation monomer
addition on buffer pH was measured. For the 8ARM-20k-NH2 &
4ARM-20k-SGA formulation, the buffer pH drops slightly from 7.42 to
7.36 upon addition of the biocompatible pre-formulation monomers.
For the 8ARM-20k-AA/8ARM-20k-NH2 (70/30) & 4ARM-20k-SGA
formulation, the buffer pH drops from 7.4 to 7.29 upon addition of
the biocompatible pre-formulation monomers. The additional decrease
in the pH was found to originate from acidic residues in the
degradable acetate amine. The same pH drop phenomenon was observed
for the 4ARM-20k-AA amine. In certain instances, a quality control
specification on the acetate amine solution pH may be required to
improve the consistency of degradable formulations.
[0160] The effect of reaction buffer pH on gel times was measured.
The gel times increase with an increase in the concentration of
hydronium ions in an approximately linear fashion. More generally,
the gel times decrease with an increase in the buffer pH. In
addition, the effect of reaction buffer phosphate concentration on
gel times was determined. The gel times decrease with an increase
in the phosphate concentration. Furthermore, the effect of polymer
concentration on gel times was investigated. The gel times decrease
significantly with an increase in the polymer concentration. At low
polymer concentrations where the gel time is greater than 5
minutes, hydrolysis reactions of the ester begin to compete with
the formation of the polymer. The effect of temperature on gel
times appears to follow the Arrhenius equation. The gel time is
directly related to the extent of reaction of the polymer solution
and so this behavior is not unusual.
[0161] The rheology of the polymers during the gelation process as
a function of the percent time to the gel point was determined.
When 100% represents the gel point and 50% represents half the time
before the gel point, the viscosity of the reacting solution
remains relatively constant until about 80% of the gel point. After
that point, the viscosity increases dramatically, representing the
formation of the solid gel.
[0162] The gel time stability of a single formulation using the
same lot of biocompatible pre-formulation monomers over the course
of about a year was measured. The biocompatible pre-formulation
monomers were handled according to the standard protocol outlined
above. The gel times remained relatively stable; some variations in
the reaction buffer may account for differences in the gel
times.
TABLE-US-00009 TABLE 7 (A) Summary of the reaction details for
several representative sticky formulations; (B) more detailed
tabulation of a selection of the reaction details including moles
(degradation times were measured in phosphate buffered saline (PBS)
at 37.degree. C.). (A) % Gel Degradation Pre-formulation Components
Buffer Solution Time (s) Time (days) 4ARM-20k-AA/8ARM-20k-NH2 0.10
M phosphate, 5 150 21 (60/40) pH 7.80 4ARM-20k-SGA
4ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 5 150 21 (60/40) pH 7.80
4ARM-20k-SGA 0.3% HPMC 8ARM-20k-NH2 0.10 M phosphate, 4.8 100 N/A
4ARM-20k-SGA pH 7.80 0.3% HPMC 8ARM-20k-NH2 0.10 M phosphate, 4.8
70 48 8ARM-15k-SG pH 7.80 0.3% HPMC 4ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 110 12 (60/40) pH 7.80 8ARM-15k-SG 0.3% HPMC
4ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 20 160 21 (60/40) pH
7.80 4ARM-20k-SGA 0.3% HPMC 8ARM-20k-NH2 0.10 M phosphate, 4.8 90
N/A 4ARM-20k-SGA pH 7.80 8ARM-20k-NH2 0.10 M phosphate, 4.8 80 N/A
4ARM-20k-SGA pH 7.80 1.0% HPMC 8ARM-20k-NH2 0.10 M phosphate, 4.8
210 N/A 4ARM-20k-SGA pH 7.46 0.3% HPMC 8ARM-20k-NH2 0.05 M
phosphate, 4.8 270 N/A 4ARM-20k-SGA pH 7.42 0.3% HPMC 8ARM-20k-NH2
0.05 M phosphate, 4 330 N/A 4ARM-20k-SGA pH 7.42 0.3% HPMC
8ARM-20k-NH2 0.05 M phosphate, 3 510 N/A 4ARM-20k-SGA pH 7.42 0.3%
HPMC 8ARM-20k-NH2 0.05 M phosphate, 4.8 405 N/A 4ARM-20k-SGA pH
7.24 0.3% HPMC 8ARM-20k-NH2 0.05 M phosphate, 3 720 N/A
4ARM-20k-SGA pH 7.24 0.3% HPMC 8ARM-20k-NH2 0.05 M phosphate, 2.5
930 N/A 4ARM-20k-SGA pH 7.24 0.3% HPMC 8ARM-20k-AA 0.10 M
phosphate, 4.8 90 6 4ARM-20k-SGA 7.46 pH HPMC (0.3%)
8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 100 16 (75/25) pH
7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 95 256 (60/40) pH 7.46 (estimated) 4ARM-20k-SGA HPMC
(0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 120 N/A
(50/50) pH 7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2
0.10 M phosphate, 4.8 100 21 (70/30) pH 7.46 4ARM-20k-SGA HPMC
(0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 100 28
(65/35) pH 7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-NH2 0.10 M
phosphate, 4.8 90 N/A 4ARM-20k-SGA pH 7.80 1.5% HPMC
8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 90 16 (75/25) pH
7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 105 21 (70/30) pH 7.46 4ARM-20k-SGA HPMC (0.3%)
8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 120 N/A (50/50) pH
7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 70 7 (70/30) pH 7.46 8ARM-15k-SG HPMC (0.3%)
4ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 260 10 (70/30) pH
7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 70 17 (60/40) pH 7.46 8ARM-15k-SG HPMC (0.3%)
8ARM-20k-AA 0.10 M phosphate, 4.8 85 7 4ARM-20k-SGA pH 7.46 HPMC
(0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4.8 95 13 (70/30)
pH 7.46 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M
phosphate, 4.8 95 10 (75/25) pH 7.46 4ARM-20k-SGA HPMC (0.3%)
8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 4 110 In Progress
(75/25) pH 7.58 4ARM-20k-SGA HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2
0.10 M phosphate, 3.5 150 In Progress (75/25) pH 7.58 4ARM-20k-SGA
HPMC (0.3%) 8ARM-20k-AA/8ARM-20k-NH2 0.10 M phosphate, 3 190 In
Progress (75/25) pH 7.58 4ARM-20k-SGA HPMC (0.3%) (B) Polymer %
Pre-formulation Arms Solution Components MW Mmoles Wt (g) Arm
mmoles Eq (w/v) 8ARM-20k-NH2 20000 1000 0.04 8 0.002 0.016
4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016 Buffer Volume
(phosphate) 2.5 4.8 Viscosity Enhancer 0.3% HPMC 8ARM-20k-NH2 20000
1000 0.08 8 0.004 0.032 8ARM-15k-SG 15000 1000 0.06 8 0.004 0.032
Buffer Volume (phosphate) 2.9 4.8 Viscosity Enhancer 0.3% HPMC
8ARM-20k-AA 20000 1000 0.04 8 0.002 0.016 4ARM-20k-SGA 20000 1000
0.08 4 0.004 0.016 Buffer Volume (phosphate) 2.5 4.8 Viscosity
Enhancer 0.3% HPMC 4ARM-20k-AA 20000 1000 0.06 4 0.003 0.012
8ARM-20k-NH2 20000 1000 0.02 8 0.001 0.008 4ARM-20k-SGA 20000 1000
0.1 4 0.005 0.02 Buffer Volume (phosphate) 3.6 5.0 Viscosity
Enhancer 0.3% HPMC 4ARM-20k-AA 20000 1000 0.12 4 0.006 0.024
8ARM-20k-NH2 20000 1000 0.04 8 0.002 0.016 8ARM-15k-SG 15000 1000
0.075 4 0.005 0.02 Buffer Volume (phosphate) 4.9 4.8 Viscosity
Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.06 8 0.003 0.024
8ARM-20k-NH2 20000 1000 0.02 8 0.001 0.008 4ARM-20k-SGA 20000 1000
0.16 4 0.008 0.032 Buffer Volume (phosphate) 5 4.8 Viscosity
Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.03 8 0.0015 0.012
8ARM-20k-NH2 20000 1000 0.02 8 0.001 0.008 4ARM-20k-SGA 20000 1000
0.1 4 0.005 0.02 Buffer Volume (phosphate) 3.1 4.8 Viscosity
Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.02 8 0.001 0.008
8ARM-20k-NH2 20000 1000 0.02 8 0.001 0.008 4ARM-20k-SGA 20000 1000
0.08 4 0.004 0.016 Buffer Volume (phosphate) 2.5 4.8 Viscosity
Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.035 8 0.00175 0.014
8ARM-20k-NH2 20000 1000 0.015 8 0.00075 0.006 4ARM-20k-SGA 20000
1000 0.1 4 0.005 0.02 Buffer Volume (phosphate) 3.1 4.8 Viscosity
Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.039 8 0.00195 0.0156
8ARM-20k-NH2 20000 1000 0.021 8 0.00105 0.0084 4ARM-20k-SGA 20000
1000 0.12 4 0.006 0.024 Buffer Volume (phosphate) 3.75 4.8
Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.09 8 0.0045
0.036 8ARM-20k-NH2 20000 1000 0.03 8 0.0015 0.012 4ARM-20k-SGA
20000 1000 0.24 4 0.012 0.048 Buffer Volume (phosphate) 9 4.0
Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.075 8 0.00375
0.03 8ARM-20k-NH2 20000 1000 0.025 8 0.00125 0.01 4ARM-20k-SGA
20000 1000 0.2 4 0.01 0.04 Buffer Volume (phosphate) 8.55 3.5
Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.06 8 0.003
0.024 8ARM-20k-NH2 20000 1000 0.02 8 0.001 0.008 4ARM-20k-SGA 20000
1000 0.16 4 0.008 0.032 Buffer Volume (phosphate) 8 3.0 Viscosity
Enhancer 0.3% HPMC
TABLE-US-00010 TABLE 8 (A) Summary of the reaction details for
several representative sticky formulations; (B) more detailed
tabulation of a selection of the reaction details including moles
(degradation times were measured in phosphate buffered saline (PBS)
at 37.degree. C.). (A) Components Poly. Estim. Deg. Appr. (Arm
Equiv. Mol %) Conc. Buffer Type & Components Time Gel Time
4ARM-20k-SGA 100% 5% Liquid 0.10 M 2 to 4 weeks 125 s 8ARM-20k-AA
65% 2.5 mL Phosphate, 8ARM-20k-NH2 35% pH 7.58 HPMC 0.3%
4ARM-20k-SGA 100% 5% Liquid 0.10 M 2 weeks 115 s 8-ARM-20k-AA 75%
2.5 mL Phosphate, 8ARM-20k-NH2 25% pH 7.58 HPMC 0.3% 4ARM-20k-SGA
100% 5% Liquid 0.10 M 2 weeks 155 s 8ARM-20k-AA 70% 2.5 mL
Phosphate, 8ARM-20k-NH2 30% pH 7.58 HPMC 0.3% 4ARM-20k-SGA 100% 5%
Liquid 0.10 M 2 weeks 110 s to 125 s 8ARM-20k-AA 75% 2.5 mL
Phosphate, 8ARM-20k-NH2 25% pH 7.58 HPMC 0.3% 4ARM-20k-SGA 100% 5%
Liquid 0.10 M 2 weeks 122 s 8ARM-20k-AA 75% 2.5 mL Phosphate,
8ARM-20k-NH2 25% pH 7.58 HPMC 0.3% 4ARM-20k-SGA 100% 5% Liquid 0.10
M 2 weeks 90 s to 120 s 8ARM-20k-AA 75% 2.5 mL Phosphate,
8ARM-20k-NH2 25% pH 7.58 HPMC 0.3% 1000 ppm Denatonium benzoate
4ARM-20k-SGA 100% 5% Liquid 0.10 M 2 weeks 90 s to 120 s
8ARM-20k-AA 75% 2.5 mL Phosphate, 8ARM-20k-NH2 25% pH 7.58 HPMC
0.3% 500 ppm Denatonium benzoate 4ARM-20k-SGA 100% 5% Liquid 0.10 M
2 weeks 90 s to 120s 8ARM-20k-AA 75% 2.5 mL Phosphate, 8ARM-20k-NH2
25% pH 7.58 HPMC 0.3% 100 ppm Denatonium benzoate 4ARM-20k-SGA 100%
5% Liquid 0.10 M 2 weeks 130 s 8ARM-20k-AA 70% 2.5 mL Phosphate,
8ARM-20k-NH2 30% pH 7.58 HPMC 0.3% 4ARM-20k-SGA 100% 4% Liquid 0.10
M 2 weeks 205 s to 230 s 8ARM-20k-AA 60% 2.25 mL Phosphate,
8-ARM-20k-NH2 40% pH 7.46 HPMC 0.3% 4ARM-20k-SGA 100% 6% Solid 0.10
M 30-60 days 90 s 8ARM-20k-AA 65% Freeze-dried (Aldrich) Phosphate,
8ARM-20k-NH2 35% Suggested use w/ pH 7.4 2 mL drug solution
4ARM-20k-SGA 100% 5% Liquid 0.10 M 2 weeks 90 s to 120 s
8ARM-20k-AA 75% 2.5 mL Phosphate, 8ARM-20k-NH2 25% pH 7.58 HPMC
0.3% 10000 ppm Denatonium benzoate 4ARM-20k-SGA 100% 5% Liquid 0.10
M 2 weeks 115 s 8ARM-20k-AA 75% 2.5 mL Phosphate, 8ARM-20k-NH2 25%
pH 7.58 HPMC 0.3% 4ARM-20k-SGA 100% 5% Liquid 0.10 M 2 weeks 150 s
8ARM-20k-AA 75% 2.5 mL Phosphate, 8ARM-20k-NH2 25% Using
freeze-dried pH 7.4 phosphate 1% Denatonium benzoate, 2%
Chlorhexidine 4ARM-20k-SGA 100% 6% Solid 0.10 M 2 weeks 110 s
8ARM-20k-AA 75% Freeze-dried (Aldrich) Phosphate, 8ARM-20k-NH2 25%
Suggested use w/ pH 7.4 2 mL drug solution 4ARM-20k-SGA 100% 6%
Liquid 0.01 M 2 weeks 27 min to 8ARM-20k-AA 70% 2.0 mL Phosphate,
31 min 8ARM-20k-NH2 30% Phosphate Buffered 0.137 M HPMC 0.3% Saline
(PBS) NaCl, 0.0027 M KCl, pH 7.2 4ARM-20k-SGA 100% 5% Liquid 0.10 M
2 weeks 158 s 8ARM-20k-AA 70% 2.5 mL Phosphate, 8ARM-20k-NH2 30%
Nolvasan (2% pH 7.4 Chlorhexidine) (B) Arms Pol. % Components MW
Mmoles Wt (g) Arm mmoles Eq Sol. (w/v) 8ARM-20k-AA 20000 1000 0.03
8 0.0015 0.012 8ARM-20k-NH2 20000 1000 0.01 8 0.0005 0.004
4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016 Buffer Volume
(phosphate) 2.5 4.8 Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000
1000 0.03 8 0.0015 0.012 8ARM-20k-NH2 20000 1000 0.01 8 0.0005
0.004 4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016 Buffer Volume
(phosphate) 2.5 4.8 Denatonium benzoate 1000 ppm Viscosity Enhancer
0.3% HPMC 8ARM-20k-AA 20000 1000 0.03 8 0.0015 0.012 8ARM-20k-NH2
20000 1000 0.01 8 0.0005 0.004 4ARM-20k-SGA 20000 1000 0.08 4 0.004
0.016 Buffer Volume (phosphate) 2.5 4.8 Denatonium benzoate 500 ppm
Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.03 8 0.0015
0.012 8ARM-20k-NH2 20000 1000 0.01 8 0.0005 0.004 4ARM-20k-SGA
20000 1000 0.08 4 0.004 0.016 Buffer Volume (phosphate) 2.5 4.8
Denatonium benzoate 100 ppm Viscosity Enhancer 0.3% HPMC
8ARM-20k-AA 20000 1000 0.03 8 0.0015 0.012 8ARM-20k-NH2 20000 1000
0.01 8 0.0005 0.004 4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016
Buffer Volume (phosphate) 2.5 4.8 Denatonium benzoate 10000 ppm
Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA 20000 1000 0.03 8 0.0015
0.012 8ARM-20k-NH2 20000 1000 0.01 8 0.0005 0.004 4ARM-20k-SGA
20000 1000 0.08 4 0.004 0.016 Solid Phosphate 0.043 Nolvasan Volume
(2% 2.5 4.8 chlorhexidine) Denatonium benzoate 10000 ppm
8ARM-20k-AA 20000 1000 0.026 8 0.0013 0.0104 8ARM-20k-NH2 20000
1000 0.014 8 0.0007 0.0056 4ARM-20k-SGA 20000 1000 0.08 4 0.004
0.016 Buffer Volume (phosphate) 2.5 4.8 Viscosity Enhancer 0.3%
HPMC 8ARM-20k-AA 20000 1000 0.028 8 0.0014 0.0112 8ARM-20k-NH2
20000 1000 0.012 8 0.0006 0.0048 4ARM-20k-SGA 20000 1000 0.08 4
0.004 0.016 Buffer Volume (phosphate) 2.5 4.8 Viscosity Enhancer
0.3% HPMC 8ARM-20k-AA 20000 1000 0.018 8 0.0009 0.0072 8ARM-20k-NH2
20000 1000 0.012 8 0.0006 0.0048 4ARM-20k-SGA 20000 1000 0.06 4
0.003 0.012 Buffer Volume (phosphate) 2.25 4 Viscosity Enhancer
0.3% HPMC 8ARM-20k-AA 20000 1000 0.026 8 0.0013 0.0104 8ARM-20k-NH2
20000 1000 0.014 8 0.0007 0.0056 4ARM-20k-SGA 20000 1000 0.08 4
0.004 0.016 Solid Phosphate 0.035 6 Drug Solution 2.0 mL
8ARM-20k-AA 20000 1000 0.027 8 0.00135 0.0108 8ARM-20k-NH2 20000
1000 0.009 8 0.00045 0.0036 4ARM-20k-SGA 20000 1000 0.072 4 0.0036
0.0144 Solid Phosphate 0.035 5.4 Drug Solution 2.0 mL 8ARM-20k-AA
20000 1000 0.028 8 0.0014 0.0112 8ARM-20k-NH2 20000 1000 0.012 8
0.0006 0.0048 4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016 Buffer
Volume (phosphate) 2 6 Viscosity Enhancer 0.3% HPMC 8ARM-20k-AA
20000 1000 0.028 8 0.0014 0.0112 8ARM-20k-NH2 20000 1000 0.012 8
0.0006 0.0048 4ARM-20k-SGA 20000 1000 0.08 4 0.004 0.016 Solid
Phosphate 0.043 Nolvasan Volume (2% 2.5 4.8 chlorhexidine)
Denatonium benzoate 1%
Cytotoxicity & Hemolysis Evaluation
[0163] Several polymer samples were sent out to NAMSA for
cytotoxicity and hemolysis evaluation. Cytotoxic effects were
evaluated according to ISO 10993-5 guidelines. Hemolysis was
evaluated according to procedures based on ASTM F756 and ISO
10993-4.
[0164] The polymer 8ARM-20k-NH2 & 4ARM-20k-SGA at 4.8% solution
with 0.3% HPMC was found to be non-cytotoxic and non-hemolytic. The
polymer 8ARM-20k-AA/8ARM-20k-NH2 (70/30) & 4ARM-20k-SGA at 4.8%
solution with 0.3% HPMC was found to be non-cytotoxic and
non-hemolytic. In addition, formulations involving 4ARM-20kAA and
8ARM-15k-SG were also non-cytotoxic and non-hemolytic.
Gel and Degradation Time Measurements
[0165] The gel time for all cases was measured starting from the
addition of the ester until the gelation of the solution. The gel
point was noted by pipetting 1 mL of the reaction mixture and
observing the dropwise increase in viscosity until the mixture
ceased to flow. Degradation of the polymers was performed by the
addition of 1 to 10 mL of phosphate buffered saline per 1 g of the
material in a 50 mL centrifuge tube and incubating the mixture at
37.degree. C. A digital water bath was used to maintain the
temperature. The degradation time was measured starting from the
day of addition of the phosphate buffer to complete dissolution of
the polymer into solution.
[0166] The effect of reaction buffer pH, phosphate concentration,
polymer concentration and reaction temperature on the gel times
were characterized. The buffer pH was varied from 7.2 to 8.0 by the
dropwise addition of either 50% aqueous NaOH or 6.0 N HCl.
Phosphate concentrations of 0.01, 0.02 and 0.05 M were prepared and
adjusted to pH 7.4. Polymer concentrations from 2 to 20% solution
were studied. Reaction temperatures of 5, 20, and 37.degree. C.
were tested by keeping the monomers, buffers, and reaction mixture
at the appropriate temperature. The 5.degree. C. environment was
provided by a refrigerator and the 37.degree. C. temperature was
maintained via the water bath. Room temperature was found to be
20.degree. C.
[0167] The effect of degradation buffer pH and the proportion of
degradable amine in the polymer formulation on the degradation
times were explored. The degradation buffer pH was varied from 7.2
to 9.0 by the dropwise addition of either 50% aqueous NaOH or 6.0 N
HCl. The degradable amine components studied were either the
4ARM-20k-AA or the 8ARM-20k-AA, and the percent of degradable amine
relative to the non-degradable amine was varied from 50 to
100%.
[0168] The degradation time is largely dependent on the buffer pH,
temperature, and the biocompatible pre-formulation monomers used.
Degradation occurs primarily through ester bond hydrolysis; in
biological systems, enzymatic pathways may also play a role. FIG. 1
compares the degradation times of formulations with 4ARM-20k-AA and
8ARM-20k-AA in varying amounts. In general, increasing the amount
of degradable acetate amine in relation to the non-degradable amine
decreases the degradation times. Additionally, in some instances,
the 8ARM-20k-AA exhibits a longer degradation time than the
4ARM-20k-AA per mole equivalent, which becomes especially apparent
when the percent of acetate amine drops below 70%.
[0169] The effect of the buffer pH on the degradation time was
investigated. The pH range between 7.2 and 9.0 was studied. In
general, a high pH environment results in a greatly accelerated
degradation. For example, an increase in pH from approximately 7.4
to 7.7 decreases the degradation time by about half.
[0170] The degradation time of different Acetate Amine formulations
was evaluated. The formulation with 70% Acetate Amine has a
degradation time of approximately 14 days whereas the formulation
with 62.5% Acetate Amine has a degradation time of approximately
180 days.
[0171] FIG. 2 shows the effect of polymer concentration on
degradation time for different Acetate Amine formulations, where
increasing polymer concentration slightly increases the degradation
time (75% Acetate Amine formulation). This effect is less apparent
for 100% Acetate Amine formulation, where the rate of ester
hydrolysis is more significant.
[0172] The monomers used in the formulations have also been found
to play a role in the way the polymer degrades. For the
8ARM-20k-AA/8ARM-20k-NH2 (70/30) & 4ARM-20k-SGA polymer,
degradation occurred homogeneously throughout the material,
resulting in a "smooth" degradation process. The polymer absorbed
water and swelled slightly over the initial few days. Then, the
polymer became gradually softer yet maintained its shape. Finally,
the polymer lost its shape and became a highly viscous fluid.
[0173] Fragmenting degradation processes are observed when the
amount of degradable amine becomes low, non-degradable regions in
the polymer may occur. For instance a 4ARM-20k-AA/8ARM-20k-NH2
(70/30) & 4ARM-20k-SGA formulation degraded into several large
fragments. For applications where the polymers are subjected to
great forces, fragmentation may also occur as the polymer becomes
softer and weaker over time.
Polymer Concentration
[0174] More dilute polymer solutions may be employed with minimal
changes in the mechanical properties. For the formulation
8ARM-20k-AA-20K/8ARM-20k-NH2 (75/25) with 4ARM-20k-SGA and 0.3%
HPMC, polymer concentrations of 3.0, 3.5 and 4.0% were studied. The
gel times increased steadily as the polymer concentration was
lowered. The firmness decreased slightly as the polymer
concentration was lowered. There was essentially no change in the
polymer adhesive properties. The elastic modulus decreased slightly
as the polymer concentration was lowered.
TABLE-US-00011 TABLE 9 (A) Reaction details for specific sticky
formulation; (B) Formulation results for a specific sticky
formulation with a variety of viscosity enhancing agents (the
biocompatible hydrogel surface spread test is conducted on a
hydrophilic biocompatible hydrogel surface composed of 97.5% water
at an angle of approximately 30.degree.; one drop of the polymer
solution from a 22 gauge needle is applied to the surface before
gelation); (C) the clarity of solutions containing a variety of
viscosity enhancing agents, as measured by the % transmission at
650 nm. (A) Pre-formulation Components MW wt (g) Arm mmoles Arms Eq
% Solution 8ARM-20k-NH2 20000 0.04 8 0.002 0.016 4ARM-20k-SGA 20000
0.08 4 0.004 0.016 Phosphate buffer 2.5 mL 0.10 M, pH 7.80 4.8 (B)
Viscous Approx. Gel Hydrogel Agent % Viscosity Time Surface Spread
(w/w) (cP) (s) Test Category Notes 0 (Original 1.1 80 2 Rigid, has
"bounce". Slight elasticity. Formulation) 5% PVP 1 to 5 90 2 to 3
No change, except for a slight increase in elasticity. 10% PVP 3 to
5 90 2 to 3 Slightly opaque, moderate increase in elasticity.
Slippery. 15% PVP 5 to 10 100 2 to 3 Opaque, definite increase in
elasticity. Slippery when wet, slightly sticky when dry. 20% PVP 10
110 2 Opaque, definite increase in elasticity. Slippery when wet,
very sticky when dry. 0.3% HPMC 8.4 80 2 No change. 1.0% HPMC 340.6
90 1 No change. 1.25% HPMC 1,000 90 1 No change. 1.5% HPMC 2,000
100 1 Slightly softer, lacks "bounce". 2.0% HPMC 4,000 100 1
Slightly softer, lacks "bounce". Slippery. (C) Sample %
Transmission @ 650 nm 0.10 M phosphate 100.0% buffer, pH 7.80 10%
PVP 99.9% 1.5% HPMC 95.7% 1.0% HPMC 96.8% 0.5% HPMC 99.1% 0.1% HPMC
99.6% Hydrogel Surface Spread Test Categories: 1) No spreading,
tight drops that stay in place; 2) Mild spreading, drops drip
slowly down; 3) Severe spreading, drops completely wet surface.
Water is in category 3.
[0175] Methylcellulose (MC) was found to behave similarly to
hypromellose (HPMC) and provided workable viscous solutions in the
concentration range of 0 to 2% (w/w). However, the HPMC dissolved
more readily than the MC, and the HPMC solutions possessed greater
optical clarity; thus the use of HPMC was favored. Povidone (PVP)
dissolved easily in the buffer, but provided minimal viscosity
enhancement even at 20% (w/w). Higher molecular weight grades of
PVP are available, but have not yet been explored.
[0176] For the most part, the polymers remain unchanged by the
addition of low concentrations of HPMC or PVP. However, there was a
noticeable change in the polymer around 0.3% HPMC that was
characterized by an enhanced elasticity, as evidenced by the
ability of the material to elongate more than usual without
breakage. Above 1.5% HPMC, the polymer became slightly softer and
exhibited less bounce. The gel times also remained within 10
seconds of the gel time for the formulation with no viscous agent.
In the case of PVP, significant changes in the polymer occurred
above 10% PVP. The polymer became more opaque with a noticeable
increase in elasticity and stickiness. At 15% to 20% PVP, the
polymer became similar to the sticky materials, but with a better
mechanical strength. The gel times also increased by roughly 20
seconds relative to the formulation with no viscous agent. Thus,
the addition of lower concentrations of PVP or HPMC to the polymer
solutions may be beneficial in improving the polymer's elasticity
and lubricity.
[0177] The results of the biocompatible hydrogel surface spread
test show that most formulations belong in category 2.
[0178] Based on these observations, a formulation utilizing 0.3%
HPMC was chosen for further evaluation. Above 1.0% HPMC, the
solutions became significantly more difficult to mix and
dissolution of the monomers became an issue. At 0.5% HPMC and
above, the formation of air bubbles during mixing became
significant. Furthermore, the solutions were not easily filtered
through a 0.5 .mu.m syringe filter to remove the bubbles. However,
the 0.3% HPMC solution was easily filtered even after moderate
mixing, resulting in a bubble-free, optically clear polymer.
Viscosity Measurements
[0179] The viscosities of the resulting buffer solutions were
measured with the appropriately sized Cannon-Fenske viscometer tube
from Ace Glass. Viscometer sizes used ranged from 25 to 300.
Measurements of select solutions were performed in triplicate at
both 20.degree. C. and 37.degree. C. The results are shown in Table
9B. To calculate the approximate dynamic viscosities, it was
assumed that all the buffer solutions had the same density as
water.
[0180] To characterize the rheology of the polymers during the
gelation process, a size 300 viscometer was used with a formulation
that was designed to gel after approximately 15 minutes. The
formulation used involved the 8ARM-20k-NH2 with the 4ARM-20k-SGA
ester at 2.5% solution and 0.3% HPMC. The reaction occurred in a
0.05 M phosphate buffer at a pH of 7.2. Thus, one viscosity
measurement with the size 300 viscometer was obtained in about one
minute and subsequent measurements may be obtained in quick
succession up to the gel point.
Hydrogel Surface Spread Test
[0181] To model the performance of the polymer solutions on a
hydrophilic surface the extent of spreading and dripping of
droplets on a high water content biocompatible hydrogel polymer
matrix surface at an incline of about 30.degree. was recorded. The
biocompatible hydrogel polymer matrix was made by dissolving 0.10 g
(0.04 mol arm eq.) of 8ARM-20k-NH2 in 7 mL 0.05 M phosphate buffer
at pH 7.4 in a Petri-dish, followed by the addition of 0.075 g
(0.04 mol arm eq.) of 8ARM-15k-SG ester. The solution was stirred
with a spatula for 10 to 20 seconds and allowed to gel, which
typically took 5 to 10 minutes. The water content of the resulting
polymer was 97.5%.
[0182] The test was performed by first preparing the polymer
solution in the usual fashion. After thorough mixing, the polymer
solution was dispensed dropwise through a 22 gauge needle onto the
biocompatible biocompatible hydrogel polymer matrix surface. The
results are shown in Table 9B and were divided into three general
categories: 1) no spreading, tight drops that stay in place; 2)
mild spreading, drops drip slowly down; 3) severe spreading, drops
completely wet surface. Water is in category 3.
Swelling & Drying Measurements
[0183] The extent of swelling in the polymers during the
degradation process was quantified as the liquid uptake of the
polymers. A known mass of the polymer was placed in PBS at
37.degree. C. At specified time intervals, the polymer was isolated
from the buffer solution, patted dry with paper towels and weighed.
The percent increase in the mass was calculated from the initial
mass.
[0184] The fate of the polymers in air under ambient conditions was
quantified as the weight loss over time. A polymer film of about 1
cm thickness was placed on a surface at 20.degree. C. Mass
measurements were performed at set intervals. The percent weight
loss was calculated from the initial mass value.
[0185] The percent of water uptake by the 8ARM-20k-NH2/4ARM-20k-SGA
polymers with 0, 0.3 and 1.0% HPMC was investigated. The 1.0% HPMC
polymer absorbed up to 30% of its weight in water until day 20.
After day 20, the polymer returned to about 10% of its weight in
water. In comparison, the 0% HPMC polymer initially absorbed up to
10% of its weight in water, but began to lose water gradually,
hovering about 5% of its weight in water. The 0.3% HPMC polymer
behaved in an intermediate fashion. It initially absorbed up to 20%
of its weight in water, but returned to about 10% of its weight in
water after a week and continued to slowly lose water.
[0186] The percent of weight loss under ambient conditions over 24
hours by the 8ARM-20k-AA/8ARM-20k-NH2 (75/25) & 4ARM-20k-SGA
polymer with 0.3% HPMC and 1.0% HPMC is shown in FIG. 3. Ambient
conditions were roughly 20.degree. C. and 30 to 50% relative
humidity. The rate of water loss was fairly constant over 6 hours
at about 10% per hour. After 6 hours, the rate slowed significantly
as the polymer weight approached a constant value. The rate of
water loss is expected to vary based on the polymer shape and
thickness, as well as the temperature and humidity.
Specific Gravity Measurements
[0187] The specific gravity of the polymers was obtained by
preparing the polymer solution in the usual fashion and pipetting
1.00 mL of the thoroughly mixed solution onto an analytical
balance. The measurements were performed in triplicate at
20.degree. C. The specific gravity was calculated by using the
density of water at 4.degree. C. as the reference.
[0188] The specific gravity of the polymers did not differ
significantly from that of the buffer solution only, both of which
were essentially the same as the specific gravity of water.
Exceptions may occur when the polymer solution is not filtered and
air bubbles become embedded in the polymer matrix.
Barium Sulfate Suspensions
[0189] For imaging purposes, barium sulfate was added to several
polymer formulations as a radiocontrast agent. Barium sulfate
concentrations of 1.0, 2.0, 5.0 and 10.0% (w/v) were explored. The
viscosity of the resulting polymer solutions was measured and the
effect of barium sulfate addition on the polymer gel times and
syringability characteristics were also studied.
[0190] Barium sulfate concentrations of 1.0, 2.0, 5.0 and 10.0%
(w/v) were explored. The opaque, milky white suspensions formed
similarly opaque and white polymers. No changes in the gel times
were observed. Qualitatively, the polymers appeared to have similar
properties to that of polymers without barium sulfate. All
formulations were able to be readily dispensed through a 22 gauge
needle.
[0191] The viscosity measurements for barium sulfate concentrations
of 1.0, 2.0, 5.0 and 10.0% was measured. The viscosity remained
relatively stable up to 2.0%; at 5.0%, the viscosity increased
slightly to about 2.5 cP. There was a sharp increase in the
viscosity to nearly 10 cP as the concentration approached 10.0%.
Thus, a barium sulfate concentration of 5.0% was chosen as a
balance between high contrast strength and similarity to unmodified
polymer formulations.
Biocompatible Hydrogel Firmness, Elastic Modulus, and Adhesion
[0192] The firmness of the polymers was characterized by a Texture
Analyzer model TA.XT.plus with Exponent software version 6.0.6.0.
The method followed the industry standard "Bloom Test" for
measuring the firmness of gelatins. In this test, the TA-8 V ball
probe was used to penetrate the polymer sample to a defined depth
and then return out of the sample to the original position. The
peak force measured is defined as the "firmness" of the sample. For
the polymers studied, a test speed of 0.50 mm/sec, a penetration
depth of 4 mm, and a trigger force of 5.0 g were used. The polymers
were prepared on a 2.5 mL scale directly in a 5 mL size vial to
ensure consistent sample dimensions. The vials used were
ThermoScientific/Nalgene LDPE sample vials, product#6250-0005
(LOT#7163281060). Measurements were conducted at 20.degree. C. The
polymers were allowed to rest at room temperature for approximately
1 hour before measuring. Measurements were performed in triplicate
for at least three samples. A sample plot generated by the Exponent
software running the firmness test is given in FIG. 4. The peak of
the plot represents the point at which the target penetration depth
of 4 mm was reached.
[0193] The elastic modulus of the polymers was characterized by a
Texture Analyzer model TA.XT.plus with Exponent software version
6.0.6.0. In this test, the TA-19 Kobe probe was used to compress a
polymer cylinder of known dimensions until fracture of the polymer
occurs. The probe has a defined surface area of 1 cm.sup.2. The
modulus was calculated as the initial slope up to 10% of the
maximum compression stress. For the polymers studied, a test speed
of 5.0 mm/min and a trigger force of 5.0 g were used. The sample
height was auto-detected by the probe. The polymers were prepared
on a 2.5 mL scale directly in a 5 mL size vial cap to ensure
consistent sample dimensions. The vials used were
ThermoScientific/Nalgene LDPE sample vials, product#6250-0005
(LOT#7163281060). Measurements were conducted at 20.degree. C. The
polymers were allowed to rest at room temperature for approximately
1 hour before measuring. Measurements were performed for at least
three samples. A sample plot generated by the Exponent software
running the modulus test is given in FIG. 5. The polymers typically
behaved elastically for the initial compression, as evidenced by
the nearly linear plot.
[0194] The adhesive properties of the polymers were characterized
by a Texture Analyzer model TA.XT.plus with Exponent software
version 6.0.6.0. In the adhesive test, the TA-57R 7 mm diameter
punch probe was used to contact the polymer sample with a defined
force for a certain amount of time, and then return out of the
sample to the original position. An exemplary plot generated by the
Exponent software running the adhesive test is given in FIG. 6. The
plot begins when the probe hits the surface of the polymer. The
target force is applied on the sample for a defined unit of time,
represented by the constant force region in the plot. Then, the
probe returns out of the sample to the original position and the
adhesive force between the probe and the sample is measured as the
"tack", which is the peak force required to remove the probe from
the sample. Other properties that were measured include the
adhesion energy or the work of adhesion, and the material's
"stringiness." The adhesion energy is simply the area under the
curve representing the tack force. Thus, a sample with a high tack
and low adhesion energy will qualitatively feel very sticky, but
may be cleanly removed with a quick pull; a sample with a high tack
and high adhesion energy will also feel very sticky, but the
removal of the material will be more difficult and may be
accompanied by stretching of the polymer, fibril formation and
adhesive residues. The elasticity of the polymer is proportional to
the measured "stringiness", which is the distance the polymer
stretches while adhered to the probe before failure of the adhesive
bond. For the polymers studied, a test speed of 0.50 mm/sec, a
trigger force of 2.0 g, and a contact force of 100.0 g and contact
time of 10.0 sec were used. The polymers were prepared on a 1.0 to
2.5 mL scale directly in a 5 mL size vial to ensure consistent
sample surfaces. The vials used were Thermo Scientific/Nalgene LDPE
sample vials. Measurements were conducted at 20.degree. C. The
polymers were allowed to rest at room temperature for approximately
1 hour before measuring. As reference materials, the adhesive
properties of a standard Post-It Note.RTM. and Scotch Tape.RTM.
were measured. All measurements were performed in triplicate. The
averages and standard deviations were calculated.
[0195] The effect of HPMC addition to the mechanical properties of
the polymers was explored, along with the effect of adding
degradable 8ARM-20k-AA amine. Under the stated conditions of the
firmness test, it was found that the addition of 0.3% HPMC
decreased the firmness of the polymer by about half. This
corresponds to a slight decrease in the elastic modulus. The 1.0%
HPMC polymer had approximately the same firmness as the 0.3% HPMC
polymer, but a slight decrease in the elastic modulus. The
disparity between the firmness and modulus tests is likely due to
experimental error. The polymer solutions were not filtered, so the
presence of air bubbles likely increased the errors. The water
content of the polymers may also change as the polymers were
sitting in the air, essentially changing the physical properties of
the materials.
[0196] It was found that the addition of the degradable 8ARM-20k-AA
amine did not substantially change the measured values of the
firmness or the elastic modulus. The measured values for a standard
commercial Post-It.TM. Note are also included as a reference. The
polymer tack was found to be around 40 mN, which is about three
times less than that of a Post-It.TM. Note. The adhesive properties
of the polymer were not found to vary with the addition of the
degradable amine.
[0197] FIG. 7 shows the firmness vs. degradation time for the
8ARM-20k-AA/8ARM-20k-NH2 (70/30) & 4ARM-20k-SGA at 4.8%
solution with 0.3% HPMC. The error bars represent the standard
deviations of 3 samples. The degradation time for the polymer was
18 days. The firmness of the polymer strongly correlated with the
extent of degradation. Swelling may also play a role during the
early stages.
[0198] The effect of various additives to the formulation on the
polymer properties was explored. Gel gel time, degradation time,
firmness, adhesion and elastic modulus was measured for polymers
prepared with varying combinations of 1% HPMC, 2% chlorhexidine and
1% denatonium benzoate. Essentially no change in the polymer
properties were found except for formulations containing 2%
chlorhexidine, which exhibited decreased firmness and elastic
modulus. It was apparent from visual inspection of the polymer that
the change was due to the detergent present in the Nolvasan
solution used and not the chlorhexidine; the detergent caused heavy
foaming during mixing that gelled into an aerated polymer.
Optical Clarity
[0199] A Thermo Scientific GENESYS 10S UV-Vis spectrophotometer was
used to measure the optical clarity of the viscous solutions. To a
quartz cuvette, 1.5 mL of the sample solution was pipetted. The
buffer solution with no additives was used as the reference. The
stable % transmission of the sample was recorded at 650 nm.
[0200] To measure the light transmission of the polymers, 1 mL of
polymer solution was filtered with a 5 .mu.m filter into a cuvette
before gelation. The cuvette was then placed horizontally so that
the polymer gelled on the side of the cuvette as a film. The film
thickness was found to be 3 mm. The polymer was allowed to cure for
15 minutes at room temperature before measuring the % light
transmission at 400, 525 and 650 nm with air as the reference.
[0201] All of the viscous solutions under consideration were found
to have acceptable to excellent optical clarity under the
concentration ranges used (greater than 97% transmission). For the
highly viscous solutions, air bubble formation during mixing was
observed, which may be resolved by the addition of an anti-foaming
agent, or through the use of a syringe filter (See Table 96).
[0202] The polymers exhibited excellent optical clarifies over the
visible spectrum. The lowest % transmission relative to buffer only
was 97.2% and the highest was 99.7%. The drop in the % transmission
at lower wavelengths is likely due to some energy absorption as the
ultraviolet region is approached.
Drug Elution: General Procedures
[0203] A Thermo Scientific GENESYS 10S UV-Vis spectrophotometer was
used to quantify the release of various drugs from several
polymers. First, the reference drug or drug solution was dissolved
in an appropriate solvent. Typically, phosphate buffered saline
(PBS), ethanol or dimethylsulfoxide (DMSO) were used as the
solvent. Next, the optimal absorption peak for identifying and
quantifying the drug was determined by performing a scan of the
drug solution between 200 and 1000 nm. With the absorption peak
selected, a reference curve was established by measuring the peak
absorbance for various concentrations of the drug. The different
drug concentration solutions were prepared by standard dilution
techniques using analytical pipettes. A linear fit of the
absorbance vs. drug concentration resulted in a general equation
that was used to convert the measured absorbance of the elution
samples to the drug concentration.
[0204] The polymer was prepared with a known drug dosage in the
same fashion as a doctor administering the polymer in a clinical
setting. However, in this case the polymer was molded into a
cylinder with a diameter of approximately 18 mm. The polymer
cylinder was then placed in a 50 mL Falcon tube with a set amount
of PBS and placed at 37.degree. C. The temperature was maintained
by a digitally controlled water bath.
[0205] Elution samples were collected daily by decanting the PBS
solution from the polymer. The volume of sample collected was
recorded. The polymer was placed in a volume of fresh PBS
equivalent to the volume of sample that was collected and returned
to 37.degree. C. The elution sample was analyzed by first diluting
the sample in the appropriate solvent using analytical pipettes
such that the measured absorbance was in the range determined by
the reference curve. The dilution factor was recorded. The drug
concentration was calculated from the measured absorbance via the
reference curve and the dilution factor. The drug amount was
calculated by multiplying the drug concentration with the sample
volume. The percent elution for that day was calculated by dividing
the drug amount by the total amount of drug administered.
Drug Elution: Chlorhexidine
[0206] The peak found between 255 and 260 nm was chosen and a
reference curve was established by measuring the peak absorbance
for 0, 0.5, 1, 2.5, 5, 10, 20, 40, and 50 ppm of chlorhexidine.
Concentrations above 50 ppm did not exhibit linear behavior in peak
absorbance.
[0207] The polymer was prepared with a commercial Nolvasan
solution, which corresponds to a 2% chlorhexidine dose (50 mg). The
elution volume was 2 mL of PBS per 1 g of polymer. The elution
samples were stored at 20.degree. C. The elution samples were
analyzed by diluting the sample 1,000-fold with dimethyl sulfoxide
(DMSO) in a quartz cuvette.
[0208] The chlorhexidine elution behavior proceeded similarly to
previous experiments with other small molecules. Almost half of the
chlorhexidine was released within the first three days. Then, the
elution rate slowed dramatically for the next three to four days
followed by another large release of chlorhexidine as the polymer
degrades (FIG. 8).
[0209] The elution of the steroidal drugs, triamcinolone and
methylprednisolone, behaved similarly. The first few days typically
exhibit an elevated elution rate, presumably as weakly bound
surface drug is released. Then, the elution is relatively constant
at a rate that is related to the drug solubility. Finally, the
remaining drug in the polymer is released as degradation begins.
Several examples are given in FIG. 9, FIG. 10, and FIG. 11 of the
control over the elution behavior that was developed. Drugs may be
released over a short time (weeks) or long period (years,
projected).
Example 12
General Procedure for the Preparation of Polymerizable
Biocompatible Pre-Formulations
[0210] Several representative formulations for both sticky and
non-sticky films are listed in Table 10 along with specific
reaction details. The films had thicknesses ranging from 100 to 500
.mu.m, and may be layered with different formulations in a
composite film.
TABLE-US-00012 TABLE 10 (A) Summary of the reaction details for
several representative thin film formulations; (B) more detailed
tabulation of a selection of the reaction details including moles
(films ranged in thickness from 100 to 500 .mu.m). (A)
Pre-formulation Amine/Ester % Components Molar Ratio Buffer
Solution 4ARM-20k-AA & 1 0.15 M 19.6 8ARM-15k-SG phosphate, pH
7.99 4ARM-5k-NH2 & 4.5/1 0.05 M 39 4ARM-10k-SG phosphate, pH
7.40 4ARM-5k-NH2 & 1 0.05 M 36.4 4ARM-10k-SG phosphate, pH 7.40
4ARM-5k-NH2 & 4.5/1 0.10 M 39 4ARM-10k-SG & phosphate, HPMC
(1.25%) pH 7.80 4ARM-2k-NH2 & 8/1 0.10 M 30.6 4ARM-10k-SG &
phosphate, HPMC (1.5%) pH 7.80 4ARM-2k-NH2 & 8/1 0.15 M 30
4ARM-20k-SGA & phosphate, MC (2%) pH 7.94 4ARM-2k-NH2 &
10/1 0.15 M 30 4ARM-20k-SGA & phosphate, MC (2%) pH 7.94 (B)
Polymer % Pre-formulation Arms Solution Components MW Mmoles Wt (g)
Arm mmoles Eq (w/v) 4ARM-20k-AA 20000 1000 0.2 4 0.01 0.04
8ARM-15k-SG 15000 1000 0.075 8 0.01 0.04 Buffer Volume 1.4 19.6
(phosphate) 4ARM-5k-NH2 5000 1000 0.27 4 0.05 0.22 4ARM-10k-SG
10000 1000 0.12 4 0.01 0.05 Buffer Volume 1 39.0 (phosphate)
4ARM-5k-NH2 5000 1000 0.17 4 0.03 0.14 4ARM-10k-SG 10000 1000 0.34
4 0.03 0.14 Buffer Volume 1.4 36.4 (phosphate) 4ARM-5k-NH2 5000
1000 0.27 4 0.05 0.22 4ARM-10k-SG 10000 1000 0.12 4 0.01 0.05
Buffer Volume 1 39.0 (phosphate) Viscosity Enhancer 1.25% HPMC
Example 13
Preparation of Kits and their Use
[0211] Several kits were prepared with the polymer formulation
tested earlier. The materials used to assemble the kits are listed
in Table 11 and the formulations used are listed in Table 12. The
kits are typically composed of two syringes, one syringe containing
the solid components and the other syringe containing the liquid
buffer. The syringes are connected via a mixing tube and a one-way
valve. The contents of the syringes are mixed via opening the valve
and transferring the contents of one syringe into the other,
repeatedly, for 10 to 20 seconds. The spent syringe and mixing tube
are then removed and discarded, and the active syringe is fitted
with a dispensing unit, such as a needle or cannula, and the
polymer solution is expelled until the onset of gelation. In other
embodiments, the viscous solution impedes the dissolution of the
solid components and thus a third syringe is employed. The third
syringe contains a concentrated viscous buffer that enhances the
viscosity of the solution once all the components have dissolved.
In some embodiments, the optical clarity of the resulting polymer
is improved through the addition of a syringe filter.
[0212] All of the formulations tested were easily dispensed through
a 22 gauge needle. The mixing action between the two syringes was
turbulent and the introduction of a significant amount of air
bubbles was apparent. Gentle mixing results in a clear material
free of bubbles. Alternatively, the use of a syringe filter was
found to remove bubbles without any change in the polymer
properties.
TABLE-US-00013 TABLE 11 Materials used to fabricate kits including
vendor, part number and lot number. Description Vendor Vincon
Tubing, 1/8'' I.D. Ryan Herco 1/4'' O.D. 1/16'' wall, 100 Ft. Flow
Solutions 12 mL Luer-Lock Syringe Tyco Healthcare, Kendall Monoject
.TM. 3 mL Luer-Lock Syringe Tyco Healthcare, Kendall Monoject .TM.
One Way Stopcock, QOSINA Female Luer Lock to Male Luer Female Luer
Lock Barb for QOSINA 1/8'' I.D. tubing, RSPC Non-vented Luer
Dispenser Tip Cap, White QOSINA 32 mm Hydrophilic Syringe Filter, 5
micron PALL .RTM. Life Sciences
TABLE-US-00014 TABLE 12 The detailed contents for four different
kits; the solid components are in one syringe, while the liquid
components are in another syringe; a mixing tube connects the two
syringes. Pre-formulation Components MW wt (g) Arm mmoles Arms Eq %
Solution 8ARM-20k-NH2 20000 0.04 8 0.002 0.016 4ARM-20k-SGA 20000
0.08 4 0.004 0.016 Phosphate buffer 2.5 mL 0.10 M, pH 7.80 4.8
Viscosity Enhancer No viscosity enhancer 8ARM-20k-NH2 20000 0.04 8
0.002 0.016 4ARM-20k-SGA 20000 0.08 4 0.004 0.016 Phosphate buffer
2.5 mL 0.10 M, pH 7.80 4.8 Viscosity Enhancer 0.3% HPMC
8ARM-20k-NH2 20000 0.04 8 0.002 0.016 4ARM-20k-SGA 20000 0.08 4
0.004 0.016 Phosphate buffer 2.5 mL 0.10 M, pH 7.80 4.8 Viscosity
Enhancer 7.5% Povidone 8ARM-20k-NH2 20000 0.04 8 0.002 0.016
4ARM-20k-SGA 20000 0.08 4 0.004 0.016 Phosphate buffer 2.5 mL 0.10
M, pH 7.80 4.8 Viscosity Enhancer 1.0% HPMC
[0213] Several additional kits were prepared with the polymer
formulation that performed the best in initial trials. The
materials used to assemble the kits are listed in Table 13. The
kits are typically composed of two syringes, one syringe containing
the solid components and the other syringe containing the liquid
buffer. The syringes were loaded by removing the plungers, adding
the components, purging the syringe with a gentle flow of nitrogen
gas for 20 seconds, and then replacing the plunger. Finally, the
plungers were depressed as much as possible to reduce the internal
volume of the syringes. The specifications for the amounts of
chemical components in the kits are listed in Table 14A. A summary
describing the lots of kits prepared is listed in Table 14B.
[0214] The syringes were connected directly after uncapping, the
male part locking into the female part. The contents of the
syringes were mixed via transferring the contents of one syringe
into the other, repeatedly, for 10 to 20 seconds. The spent syringe
was then removed and discarded, and the active syringe was fitted
with a dispensing unit, such as a needle or cannula, and the
polymer solution was expelled until the onset of gelation. In other
embodiments, the viscous solution impeded the dissolution of the
solid components and thus a third syringe was employed. The third
syringe contained a concentrated viscous buffer that enhanced the
viscosity of the solution once all the components had
dissolved.
[0215] All the formulations tested were easily dispensed through a
22 gauge needle. The mixing action between the two syringes was
turbulent and the introduction of a significant amount of air
bubbles was apparent. The use of a syringe filter was found to
remove bubbles without any change in the polymer properties.
[0216] The prepared kits were placed into foil pouches along with
one oxygen absorbing packet per pouch. The pouches were heat sealed
with a CHTC-280 PROMAX tabletop chamber sealing unit. Two different
modes of sealing were explored: under nitrogen and under vacuum.
The settings for sealing under nitrogen were: 30 seconds of vacuum,
20 seconds of nitrogen, 1.5 seconds of heat sealing, and 3.0
seconds of cooling. The settings for sealing under vacuum were: 60
seconds of vacuum, 0 seconds of nitrogen, 1.5 seconds of heat
sealing, and 3.0 seconds of cooling.
TABLE-US-00015 TABLE 13 Materials used to fabricate kits including
vendor, part number and lot number. Description Vendor 12 mL Male
Luer-Lock Syringe Tyco Healthcare, Kendall Monoject .TM. 5 mL
Female Luer Lock Syringe, Purple QOSINA Male Luer Lock Cap,
Non-vented QOSINA Female Non-vented Luer Dispensor QOSINA Tip Cap,
White 100 cc oxygen absorbing packet IMPAK 6.25'' .times. 9'' OD
PAKVF4 IMPAK Mylar foil pouch
TABLE-US-00016 TABLE 14 Specifications for kit components for the
8ARM-20k-AA/8ARM- 20-NH2 & 4ARM-20k-SGA formulation with 60,
65, 70 and 75% degradable amine (A). LOT formulation summary (B).
(A) Pre-formulation Specifications Components 60/40 65/35 70/30
75/25 8ARM-20k-AA 0.024-0.026 g 0.026-0.027 g 0.028-0.029 g
0.030-0.031 g 8ARM-20k-NH2 0.014-0.016 g 0.013-0.014 g 0.011-0.012
g 0.009-0.010 g 4ARM-20k-SGA 0.080-0.082 g 0.080-0.082 g
0.080-0.082 g 0.080-0.082 g Phosphate 2.50 mL of 0.10M phosphate,
pH 7.58, 0.30% HPMC Buffer (8.48 cSt +/- 0.06 @ 20.degree. C.) (B)
Formulation Buffer pH Sealing Method Notes 60/40 7.46 nitrogen
60/40 7.58 nitrogen 60/40 7.72 nitrogen 70/30 7.58 vacuum 70/30
7.58 vacuum no nitrogen purging of syringe 65/35 7.58 vacuum 75/25
7.58 vacuum 75/25 7.58 vacuum 75/25 7.58 nitrogen 65/35 7.58 vacuum
65/35 7.58 nitrogen
[0217] Several kits were prepared for use in beta testing. The
materials used to assemble the kits are listed in Table 15. The
kits are typically composed of two syringes, one syringe containing
the solid components and the other syringe containing the liquid
buffer. The syringes were loaded by removing the plungers, adding
the components, purging the syringe with a gentle flow of inert gas
for 10 seconds, and then replacing the plunger. Finally, the
plungers were depressed as much as possible to reduce the internal
volume of the syringes.
[0218] Alternatively, a single syringe kit may be prepared by
loading the solid components into one female syringe along with a
solid form of the phosphate buffer. The kit is then utilized in a
similar fashion as the dual syringe kit, except the user may use a
specified amount of a variety of liquids in a male syringe.
Typically, any substance provided in a liquid solution for
injection may be used. Some examples of suitable liquids are water,
saline, Kenalog-10, Depo-Medrol and Nolvasan.
[0219] The kits are utilized in the following fashion. The syringes
are connected directly after uncapping, the male part locking into
the female part. The contents of the syringes are mixed via
transferring the contents of one syringe into the other,
repeatedly, for 10 to 20 seconds. The spent syringe is then removed
and discarded, and the active syringe is fitted with a dispensing
unit, such as a needle, a spray nozzle or a brush tip, and the
polymer solution is expelled until the onset of gelation.
[0220] The prepared kits were placed into foil pouches along with
one oxygen absorbing packet and one indicating silica gel packet
per pouch. Labels were affixed to the pouches that displayed the
product and company name, contact information, LOT and batch
numbers, expiration date, and recommended storage conditions. A
radiation sterilization indicator that changes color from yellow to
red upon exposure to sterilizing radiation was also affixed to the
upper left corner of the pouch. The pouches were heat sealed with a
CHTC-280 PROMAX tabletop chamber sealing unit. The settings for
sealing under vacuum were: 50 seconds of vacuum, 1.5 seconds of
heat sealing, and 5.0 seconds of cooling.
[0221] An example detailing the lots of sterile kits prepared is
listed in Table 15. A previous study found that if the loaded
syringe was not purged with nitrogen before replacing the plunger
during kit preparation, the sterile kits exhibited an increase in
gel time of about 30 seconds relative to kits that had syringes
flushed with nitrogen. No significant difference was found between
kits that had been sealed under vacuum and kits that had been
sealed under nitrogen. It was easily observable when the
vacuum-sealed kits lost their seal, so it was decided to
vacuum-seal all kits as standard procedure. The effects of
including the oxygen absorbing packet and silica gel packet to the
kits on the long term storage stability is currently under
investigation.
TABLE-US-00017 TABLE 15 Materials used to fabricate kits including
vendor, and part number. Description Vendor Part # 10 mL Luer-Lok
Syringe BD 309604 Non-Vented Luer Dispenser QOSINA 65119 Tip Cap,
White 5 mL Female Luer-Lock Syringe, QOSINA C3610 Purple PP Male
Luer Lock Cap, QOSINA 11166 Non-Vented, PP Brush tip Flumatic
BT01225R 5.25'' .times. 8'' PAKVF4D IMPAK 0525MFDFZ08TE Mylar foil
pouch 3.5'' .times. 6.5'' PAKVF4W IMPAK 035MFW065Z Mylar foil pouch
Radiation Sterilization Indicator QOSINA 13124 100 cc oxygen
absorbing packet IMPAK OAP100 Indicating silica gel IMPAK
40ISG37
TABLE-US-00018 TABLE 16 Example specifications for kit components
for the 8-arm- AA-20K/8-arm-NH2-20K & 4-arm-SGA-20K formulation
with 75% degradable amine (A). LOT formulation summary (B). (A)
Components LOT# & Specifications 8ARM-20k-AA 0.029-0.031 g
8ARM-20k-NH2 0.009-0.011 g 4ARM-20k-SGA 0.079-0.081 g Phosphate
Buffer 2.50 mL of 0.10M phosphate, pH 7.58, 0.30% HPMC (8.48 cSt
+/- 0.06 @ 20.degree. C.) LOT Size 3 30 34 48 Gel Time (s) 110-125
Degradation Time 10-12 (days) (B) Components LOT# &
Specifications 8ARM-20k-AA 0.029-0.031 g 8ARM-20k-NH2 0.009-0.011 g
4ARM-20k-SGA 0.079-0.081 g Phosphate Buffer Powder 0.03-0.06 g
Nolvasan (2% 2.50 mL, 1% denatonium chlorhexidine) benzoate LOT
Size 64 Gel Time (s) 150 Degradation Time (days) 11
[0222] The kit preparation time was recorded. Loading one buffer
syringe took an average of 1.5 minutes, while one solids syringe
took an average of 4 minutes. Vacuum sealing one kit took
approximately 1.5 minutes. Thus, the time estimate for the
preparation of one kit was 7 minutes, or approximately 8 kits per
hour. The kit preparation time may be improved by premixing all the
solids in the correct ratios such that only one mass of solids
needs to be measured, and by optimizing the vacuum sealing
procedure by reducing the vacuum cycle time.
[0223] All the formulations tested were easily dispensed through a
23 to 34 gauge needle. Higher gauges exhibit a lower flow rate as
expected. The mixing action between the two syringes was turbulent
and the introduction of a significant amount of air bubbles was
apparent. The use of a syringe filter was found to remove bubbles
without any change in the polymer properties.
[0224] For the single syringe system, the effect of phosphate
powder use was investigated. FIG. 12 shows the effect of varying
amounts or concentrations of the solid phosphate on polymer gel
times and solution pH. The system was found to be relatively
insensitive to the amount of phosphate, tolerating up to 2-fold
differences without significant variation.
Kit Sterilization & Testing
[0225] The sealed kits were packed into large sized FedEx boxes.
Each box was sterilized via electron-beam radiation at NUTEK
Corporation according to a standard procedure that was developed.
Included in this report is a copy of the standard sterilization
procedure document.
[0226] For each lot of sterilized kits, a gel time and degradation
time test was performed on a randomly selected kit to verify the
viability of the materials. A previous study included a runner or
control box of kits that was not sterilized, and concluded that
environmental conditions during transit of the kits did not play a
significant role in gel time changes.
[0227] Sterilized kits were sent to NAMSA for sterility
verification according to USP<71>. The kits were verified as
sterile.
[0228] No physical changes in the monomer and phosphate buffer
solutions were observed post-sterilization. Prior experiments have
shown that the polymer gel times consistently increase by
approximately 30 seconds after sterilization. For example, a
polymer with a 90 second gel time will exhibit a 120 second gel
time after sterilization. The pH of the sterile buffer was
unchanged, so it was suspected that some monomer degradation during
sterilization occurred. This was confirmed by preparing
unsterilized polymers at various concentrations and comparing the
gel times, degradation times and mechanical properties with
sterilized polymers (FIG. 13). The current data shows that the
monomers experience roughly 15 to 20% degradation upon
sterilization. Thus, a 5% polymer after sterilization will behave
similarly to a 4% polymer. Additional experiments are planned to
establish a detailed quality control calibration curve.
Storage Stability
[0229] The sterilized kits were stored at 5.degree. C. Some kits
were stored at 20.degree. C. or 37.degree. C. to explore the effect
of temperature on storage stability. The stability of the kits was
primarily quantified by recording changes in gel time, which is
directly proportional to the extent of monomer degradation. The
37.degree. C. temperature was maintained by submerging the kits
fully into the water bath and thus represents the worst case
scenario regarding humidity.
[0230] The storage stability of the kits was explored by placing
some kits at 5.degree. C., 20.degree. C. or 37.degree. C. and
measuring the change in gel times at defined intervals. The kits
were prepared and sealed according to the procedures detailed in a
previous section. The results are shown in FIG. 14. Over 16 weeks,
no significant change in gel times were observed for kits stored at
5.degree. C. and 20.degree. C. At 37.degree. C., the gel time
begins to increase after roughly 1 week at a constant rate. The
foil pouch proved to be an effective moisture barrier. The
indicating silica gel packet exhibited only mild signs of moisture
absorption as evidenced by the color. Longer term data is still in
the process of being collected.
Example of Syringe Kit Preparation
[0231] One syringe kit was developed where the components are
stored in two syringes, a male and a female syringe. The female
syringe contains a mixture of white powders. The male syringe
contains a buffer solution. The two syringes are connected and the
contents mixed to produce a liquid polymer. The liquid polymer is
then sprayed or applied over the suture wound where it covers the
entire suture line. During the process, the polymer enters the
voids left by sutures and protects the wound from infections. At
the wound site, the liquid polymer turns into a solid gel and stays
at the site for over two weeks. During this time, the wound is
healed and infection free.
[0232] The components necessary to prepare the kit are disclosed in
Table 17 and Table 18. To prepare the powder components of the kit
to fill into the female syringe, the plunger of the 5 mL female
Luer-lock syringe was removed, and the syringe was capped with the
appropriate cap. 8ARM-20k-AA (0.028 g, the acceptable weight range
is 0.0270 g to 0.0300 g), 8ARM-20k-NH2 (0.012 g, the acceptable
weight range is 0.0100 g to 0.0130 g), 4ARM-20k-SGA (0.080 g, the
acceptable weight range is 0.0790 g to 0.0820 g), and 0.043 g of
freeze-dried phosphate buffer powder (0.043 g, the acceptable
weight range is 0.035 g to 0.052 g) were each carefully weighed out
and poured into the syringe. The syringe was then flushed
nitrogen/argon gas for about 10 seconds at a rate of 5 to 10 L/min
and the plunger was replaced to seal the contents. The syringe was
then flipped so that the cap was facing towards the ceiling. The
syringe cap was then loosened and the air space in the syringe was
minimized by expelling as much air as possible from the syringe.
Typical compressed powder volume is 0.2 mL. Then, the syringe cap
was tighten until the cap was finger tight.
[0233] The liquid component was prepared on a 500 mL batch size,
wherein 50 mL of commercial 2% chlohexidine solution, 450 mL of
distilled water, and 1.5 g of HPMC were poured in to sterile
container. The sterile container was then capped and shook
vigorously for 10 seconds. The solution was allowed to stand under
ambient conditions for 16 hours, thereby allowing for the foam to
dissipate and any remaining HPMC to dissolve.
[0234] The liquid/buffer syringe was prepared by removing the
plunger of the male Luer-lock syringe followed by capping the
syringe with the appropriate cap. 2.50 mL of the buffer/liquid
solution was transfered by pipette into the syringe. The syringe
was then flushed with nitrogen/argon gas for about 5 seconds at a
rate of 5 to 10 L/min. The plunger of the syringe was then replaced
to seal the contents. Then the syringe was flipped so that the cap
was facing towards the ceiling and the syringe cap was loosen and
air space was minimized by expelling as much air as possible from
the syringe. Then the syringe cap was tightened until the cap was
finger tight.
TABLE-US-00019 TABLE 17 Components used to fabricate the solid
components for the female syringe Components Technical Name
8ARM-20k-AA 8ARM PEG Acetate amine, HCl salt, MW 20k 8ARM-20k-NH2
8ARM PEG amine (hexaglycerol), HCl salt, MW 20k 4ARM-20k-SGA 4-arm
PEG succinimidyl glutaramide (pentaerythritol), MW 20k Commercial
2% chlorhexidine solution Freeze-dried phosphate buffer powder
TABLE-US-00020 TABLE 18 Materials used to fabricate kit including
vendor, part number and lot number. Vendor Description Vendor Part
# Catalog # 10 mL Luer-Lok Syringe BD CM-0003 309604 Non- Vented
Luer QOSINA CM-0004 65119 Dispenser Tip Cap, White 5 mL Female
Luer- QOSINA CM-0005 C3610 Lock Syringe, Purple PP Male Luer Lock
Cap, QOSINA CM-0006 11166 Non-Vented, PP
Example of Syringe Kit Preparation
[0235] Another syringe kit was developed where the solid
components, a mixture of white powders, are stored in one female
syringe. A standard male syringe is used to take up the drug
solution, such as one containing Kenalog. The two syringes are
connected and the contents mixed to produce a liquid polymer. The
liquid polymer is then delivered to the target site.
[0236] The components necessary to prepare the kit are disclosed in
Table 17 and Table 18. To prepare the powder components of the kit
to fill into the female syringe, the plunger of the 5 mL female
Luer-lock syringe was removed, and the syringe was capped with the
appropriate cap. 8ARM-20k-AA (0.0125 g, the acceptable weight range
is 0.012 g to 0.013 g), 8ARM-20k-NH2 (0.075 g, the acceptable
weight range is 0.007 g to 0.008 g), 4ARM-20k-SGA (0.040 g, the
acceptable weight range is 0.040 g to 0.042 g), and 0.018 g of
freeze-dried phosphate buffer powder (0.043 g, the acceptable
weight range is 0.017 g to 0.022 g) were each carefully weighed out
and poured into the syringe. The syringe was then flushed
nitrogen/argon gas for about 10 seconds at a rate of 5 to 10 L/min
and the plunger was replaced to seal the contents. The syringe was
then flipped so that the cap was facing towards the ceiling. The
syringe cap was then loosened and the air space in the syringe was
minimized by expelling as much air as possible from the syringe.
Then, the syringe cap was tightened until the cap was finger
tight.
Example 14
General Procedure for the Preparation of a Polyglycol-Based,
Biocompatible Hydrogel Polymer Matrix
[0237] A polyglycol-based, biocompatible pre-formulation is
prepared by mixing 0.028 g of 8ARM-AA-20K, 0.012 g of 8ARM-NH2-20K,
and 0.080 g of 4ARM-SGA-20K. 2.50 mL of culture medium is added to
the formulation. The formulation is mixed for about 10 seconds and
a 1 mL solution of the mixture is pipetted out using a mechanical
high precision pipette. The polyglycol-based, biocompatible
pre-formulation components polymerize to form a polyglycol-based,
biocompatible hydrogel polymer matrix. The polymerization time of 1
mL liquid is collected and then verified with the lack of flow for
the remaining liquids.
Example 15
General Procedure for the Preparation of a Polyglycol-Based,
Biocompatible Hydrogel Polymer Matrix and Stem Cells
[0238] A polyglycol-based, biocompatible pre-formulation is
prepared by mixing 0.0125 g of 8ARM-AA-20K, 0.0075 g of
8ARM-NH2-20K, and 0.040 g of 4ARM-SGA-20K. 1.0 mL of culture medium
is added to the formulation. The formulation is mixed for about 10
seconds and a 1 mL solution of the mixture is pipetted out using a
mechanical high precision pipette. The polyglycol-based,
biocompatible pre-formulation components polymerize to form a
polyglycol-based, biocompatible hydrogel polymer matrix. The
polymerization time of 1 mL liquid is collected and then verified
with the lack of flow for the remaining liquids. Various sized
slices of the polymerized polyglycol-based, biocompatible hydrogel
polymer matrix are placed in different wells of a 24 well plate.
0.5 mL of adult mesenchymal stem cells are seeded onto the polymer
matrices at various densities. The stem cells diffuse and become
incorporated into the polyglycol-based, biocompatible hydrogel
polymer matrix. Incorporation of the stem cells into the
polyglycol-based, biocompatible hydrogel polymer matrix is
demonstrated by removing a slice of the polymer matrix 10 days
after stem cell addition and using the slice to expand the cells in
culture. The incorporated stem cells remain viable, as demonstrated
by their ability to proliferate in culture.
Example 16
General Procedure for the Preparation of a Polyglycol-Based,
Biocompatible Hydrogel Polymer Matrix and Stem Cells
[0239] A polyglycol-based, biocompatible pre-formulation is
prepared by mixing 0.0125 g of 8ARM-AA-20K, 0.0075 g of
8ARM-NH2-20K, and 0.040 g of 4ARM-SGA-20K. 1.0 mL of culture medium
containing adult mesenchymal stem cells is added to the
formulation. The formulation is mixed for about 10 seconds and a 1
mL solution of the mixture is pipetted out using a mechanical high
precision pipette. The polyglycol-based, biocompatible
pre-formulation components polymerize to form a polyglycol-based,
biocompatible hydrogel polymer matrix. The polymerization time of 1
mL liquid is collected and then verified with the lack of flow for
the remaining liquids.
[0240] At any point during the combination of the polyglycol-based,
biocompatible pre-formulation compounds, additional components may
be added to the formulation. The formulation may be solid, liquid,
polymerized, gelled, or any combination thereof when the additional
component is added. The additional component may combine with or
diffuse through the formulation and become retained with the
formulation for a determined period of time. In one example, the
polyglycol-based, biocompatible hydrogel polymer matrix is formed,
followed by the addition of growth factors. The growth factors are
incorporated into the polyglycol-based, biocompatible hydrogel
polymer matrix. Additional components include, but are not limited
to, biomolecules, antibiotics, anti-cancers, anesthetics,
anti-virals, or immunosuppressive agents.
Example 17
Viability of Cells in a Polyglycol-Based, Biocompatible Hydrogel
Polymer Matrix
[0241] A single cell suspension of mesenchymal stem cells in D15
(DMEM, high glucose, 15% fetal bovine serum) is prepared and the
cells counted. 1 mL of cells at a 2.times.10.sup.4/mL density are
added to a 50 mL tube. The cells are maintained at room temperature
and prepared just before addition to a pre-formulation. A female
syringe containing a polyglycol-based, biocompatible
pre-formulation is prepared by mixing 0.0125 g of 8ARM-AA-20K,
0.0075 g of 8ARM-NH2-20K, and 0.040 g of 4ARM-SGA-20K in the female
syringe. An 18 G need is attached to a male syringe and the male
syringe is filled with 1 mL PBS. The next step is carried out
within 90-120 seconds. The needle is removed from the male syringe
and the male syringe is attached to the female syringe containing
the pre-formulation. The PBS is pushed from the male syringe into
the female syringe and the mixing process is started by repeatedly
pushing the PBS from one syringe to the other, with 20 strokes
being sufficient for mixing. After the final stroke, the entire
contents are pushed into the male syringe. An 18 G needle is
attached to the male syringe and the liquid pre-formulation is
ejected into the 50 mL tube containing the 1 mL of mesenchymal stem
cells. The cells are carefully mixed while the liquid
pre-formulation is being ejected into the tube. Care is made to
ensure that the cells are not mixed by aspiration with the needle
as this may induce cell stress.
[0242] Aliquots of the pre-formulation containing mesenchymal stem
cells are placed in chambers of a 4-chamber tissue culture glass
slide at 50, 100, 200 and 400 .mu.L. The pre-formulation is allowed
to gel for 2 minutes. 200 .mu.L of D15 is added to each chamber.
Three of these slides are prepared for three time points: 0, 2, and
24 hours. The cells are stained with membrane-permeant
3',6'-Di(O-acetyl)-2',7'-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescei-
n, tetraacetoxymethyl ester and membrane-impermeant ethidium
homodimer-1, 1 .mu.l/ml propidium iodide. The cells are imaged
using brightfield and fluorescence microscopy. Live cells fluoresce
green and dead cells fluoresce red. At the 2 hour time point, only
one dead cell was observed in multiple field views. One live cell
had a punctate cytoplasm. The remaining cells were viable and had
typical spheroid morphology in the hydrogel polymer matrix. At the
24 hour time point, more than 95% of the cells were viable.
Example 18
General Procedure to Determine the Properties of Cells in a
Polyglycol-Based, Biocompatible Hydrogel Polymer Matrix
[0243] The proliferation rate, viability and structural
characteristics of mesenchymal stem cells are evaluated after
incorporation with a biocompatible hydrogel polymer matrix.
[0244] To measure the rate of proliferation of mesenchymal stem
cells, a cell proliferation assay is performed. A biocompatible
pre-formulation comprising polyglycol-based compounds and a
suitable buffer, as described in Example 14, is prepared. The 100
.mu.l of the pre-formulation is coated on a 24 well plate to give a
coating of <5 mm thick. The stem cells are seeded onto the
coated plate at various cell densities (1.times.10.sup.3,
5.times.10.sup.3, 10.times.10.sup.3 and 20.times.10.sup.3 cells).
Cells are incubated in a growth medium at 37.degree. C., 5%
CO.sub.2. For each sample, a CellTiter 96.RTM. AQueous
Non-Radioactive (MTS) assay is performed at days 2, 7, and 10 after
seeding to confirm that the cells are proliferating. The growth
medium is removed from each well and replaced with 500 .mu.l of
fresh medium and incubate at 37.degree. C. for at least 1 hour in
5% CO.sub.2. 100 .mu.l of MTS reagent is added to each well and
incubated at 37.degree. C. for 3 hours, in 5% CO.sub.2. The
absorbance at 490 nm is measured using a microplate reader and
recorded. Wells with the formulation but without any cells are used
as blanks. Similarly, only media in the wells without any cells
serve as blanks. Each sample reading is obtained by subtracting the
blank. The graph of absorbance versus time is plotted. Absorbance
is directly proportional to the cell numbers, wherein a significant
increase in absorbance indicates cell viability and proliferation.
Fold change in proliferation is calculated.
[0245] To demonstrate the viability of adult mesenchymal stem
cells, a staining assay is performed at days 2, 7, and 10 on cells
which are seeded on a coated 24 well plate as described previously
in this example. The medium is removed and the cells are washed
twice with phosphate saline buffer. A 0.5 ml staining solution
comprising a mixture of celcein-am (10 .mu.g/ml) and propidium
iodide (100 .mu.g/ml) is added to each well and the plate is
incubated for 5-10 minutes at 37.degree. C. Cells are washed with
phosphate saline buffer and immediately imaged. Live cells
fluoresce green and dead cells fluoresce red.
[0246] To demonstrate that the adult mesenchymal stem cells
maintain their structure, a staining assay is performed on the
cells which are seeded a coated 24 well plate as described
previously in this example. The medium is removed and the cells are
washed twice with phosphate buffer. The cells are fixed with 4%
paraformaldehyde for 10 minutes at room temperature followed by two
washes with phosphate buffer. To the washed cells, cytoplasmic WGA
stain (wheat germ agglutinin; 488 green fluorescence) is added and
the cells and the cells are incubated for 10 minutes at room
temperature. The stain is removed and the cells are washed two
times with phosphate buffer. A nuclear TO-PRO-3 iodide stain (red
fluorescence) is added to the cells and the cells are incubated for
10 minutes at room temperature. The stain is removed and the cells
are washed two times with HBSS buffer. The anti-fade reagent
Pro-long gold is added to the cells and the cells are covered with
a coverslip. 3D confocal microscopy is performed to visualize the
structure and adherence of the cells. In general, the stem cells
maintain their physiochemical properties.
Example 19
Cell Elution from a Polyglycol-Based, Biocompatible Hydrogel
Polymer Matrix
[0247] A polyglycol-based, biocompatible hydrogel polymer matrix of
Example 15 is prepared. Additional polyglycol-based, biocompatible
hydrogel polymer matrices are prepared utilizing pre-formulation
compounds of Table 13 and cell. The polymer matrices are weighed
and placed in different Falcon tubes. Two ml of buffer/gm of the
polymer matrix are added in the falcon tubes. The falcon tubes are
placed in a water bath maintained at 37.degree. C. After 24 hours,
buffer is carefully removed and replaced with fresh buffer to
maintain a constant volume. The extraction process is repeated
until each polymer matrix is dissolved completely. The polymer
matrix is dissolved in two weeks.
[0248] The elution behavior of the cells with different
biocompatible pre-formulation components is tested. Cell elution
profiles vary with different biocompatible pre-formulation
components. Cells may diffuse while the polymer matrix is
maintained, released upon degradation of the polymer matrix or any
combination thereof. The composition of the biocompatible
pre-formulation components may be selected to control the release
of cells at a pre-determined time.
[0249] In some instances, the cell-containing polymer matrices
described herein further comprise additional components such as
buffers, growth factors, antibiotics, or anti-cancer agents. The
composition of the biocompatible pre-formulation components and
additional components may be varied to control the release of cells
and/or the additional components.
[0250] In some instances, the cells of any of the cell-containing
polymer matrices described in this example may be released from the
polymer matrix in a manner dependent on the pore-size of the
polymer matrix. In some instances, the cells remain viable after
release from the polymer matrix.
Example 20
A Polyglycol-Based, Biocompatible Pre-Formulation for Disease
Treatment
[0251] A polyglycol-based, biocompatible pre-formulation
comprising, 0.0125 g 8ARM-AA-20K, 0.0075 g 8ARM-NH2-20k, 0.040 g
4ARM-SGA-20K, mesenchymal stem cells, and a suitable culture medium
are combined in the presence of 1.0 mL water. The liquid
formulation is delivered via injection directly to a site of tissue
damage in the liver. The polyglycol-based, biocompatible
pre-formulation mixture polymerizes in vivo at the site of delivery
to form a polyglycol-based, biocompatible hydrogel polymer matrix
at the target site in 4 minutes. The polyglycol-based,
biocompatible hydrogel polymer matrix culture medium component is
configured to influence the physical, chemical and biological
environment surrounding the stem cells during and after
administration to a target site.
[0252] The polyglycol-based, biocompatible hydrogel polymer matrix
is retained at the target site, where the stem cells are released
over a period of two weeks. The released stem cells require
interaction and integration with the target tissue through
incorporation of appropriate physical and cellular signals.
Therefore, the polyglycol-based, biocompatible hydrogel polymer
matrix culture medium includes modifying factors, such as
biologically active proteins critical for successful tissue
generation. The mesenchymal stem cells begin to differentiate at
the target site between 7 and 14 days, resulting in improved liver
function.
Example 20
A Polyglycol-Based, Biocompatible Hydrogel Polymer Matrix for
Disease Treatment
[0253] A polyglycol-based, biocompatible hydrogel polymer matrix is
prepared by adding 1 mL of water to a pre-formulation comprising,
0.0125 g 8ARM-AA-20K, 0.0075 g 8ARM-NH2-20k, 0.040 g 4ARM-SGA-20K,
mesenchymal stem cells, and a suitable culture medium. After
gelling is complete, the hydrogel polymer matrix is delivered
directly to a site of tissue damage in the liver. The
polyglycol-based, biocompatible hydrogel polymer matrix culture
medium component is configured to influence the physical, chemical
and biological environment surrounding the stem cells during and
after administration to the target site in the liver.
[0254] The polyglycol-based, biocompatible hydrogel polymer matrix
is retained at the target site, where the stem cells are released
over a period of two weeks. The released stem cells require
interaction and integration with the target tissue through
incorporation of appropriate physical and cellular signals.
Therefore, the polyglycol-based, biocompatible hydrogel polymer
matrix culture medium includes modifying factors, such as
biologically active proteins critical for successful tissue
generation. The mesenchymal stem cells begin to differentiate at
the target site between 7 and 14 days, resulting in improved liver
function.
Example 21
A Polyglycol-Based, Biocompatible Polymer Matrix for Delivery of
Growth Factors
[0255] A polyglycol-based, biocompatible pre-formulation
comprising, 0.028 g 8ARM-AA-20K, 0.012 g 8ARM-NH2-20k, 0.08 g
4ARM-SGA-20K, growth factors, and a buffer are combined in the
presence of 2.5 mL water. The liquid formulation is delivered via
injection directly to a site of tissue damage. The
polyglycol-based, biocompatible pre-formulation mixture polymerizes
in vivo at the site of delivery to form a polyglycol-based,
biocompatible hydrogel polymer matrix at a target site. The
polyglycol-based, biocompatible hydrogel polymer matrix is
configured to release the growth factors at the target site. The
growth factors are configured to recruit cells from the body to the
polymer matrix site, wherein the recruited cells may form tissue
upon and throughout the polymer matrix.
[0256] An alternative to growth factor incorporation in a
polyglycol-based, biocompatible hydrogel polymer matrix is to
integrate DNA plasmids encoding a gene and mammalian promoter into
the polymer matrix. Delivery of the polyglycol-based, biocompatible
hydrogel polymer matrix with the DNA programs local cells to
produce their own growth factors.
Example 22
Pore Size Determination
[0257] The pore diameters are estimated from the molecular weight
per arm of the combined components. The pore diameter is calculated
based on the number of PEG units per arm and a carbon-carbon-carbon
bond length of 0.252 nm with a 110.degree. bond angle. This assumes
a fully extended chain that accounts for bonding angles and
complete reactivity of all functional end groups to form the pore
network. The pore diameter is further modified by a correlation
relating the pore size to the inverse of the biocompatible hydrogel
swelling ratio:
.xi..apprxeq.L*(V.sub.p/V.sub.s).sup.-1/3 (Equation 1)
where V.sub.p is the volume of polymer, V.sub.s is the volume of
the swollen gel, L is the calculated pore diameter, and .xi. is the
swollen pore diameter. Based on equilibrium swelling experiments,
the ratio of V.sub.p to V.sub.s is estimated to be around 0.5.
[0258] For the case of multi-component mixtures with a reactive
ester, the weighted average of each component with the ester is
used. For example, the pore sizes obtained from 4ARM-20k-AA with
4ARM-20k-SGA are averaged with the pore sizes obtained from
8ARM-20k-NH2 with 4ARM-20k-SGA for polymers comprised of
4ARM-20k-AA and 8ARM-20k-NH2 with 4ARM-20k-SGA.
* * * * *