U.S. patent application number 13/512913 was filed with the patent office on 2012-11-15 for active self-healing biomaterial system.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Kashappa-Goud Desai, Steven P. Schwendeman.
Application Number | 20120288537 13/512913 |
Document ID | / |
Family ID | 44304973 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120288537 |
Kind Code |
A1 |
Schwendeman; Steven P. ; et
al. |
November 15, 2012 |
ACTIVE SELF-HEALING BIOMATERIAL SYSTEM
Abstract
Methods and compositions are provided that load and encapsulate
an agent, such as a protein, in a porous self-healing polymer. A
delivery system includes a porous self-healing polymer, an ionic
affinity trap within the pores of the self-healing polymer, and an
agent associated with the ionic affinity trap. Methods of
encapsulating an agent in a polymer include providing a porous
self-healing polymer comprising an ionic affinity trap within the
pores. The polymer is incubated with an agent having an affinity
for the ionic affinity trap. At least a portion of the pores in the
polymer are then healed. Active encapsulation of macromolecules at
low concentrations may be achieved due to affinity of the agent for
the ionic affinity trap within the pores.
Inventors: |
Schwendeman; Steven P.;
(Superior Township, MI) ; Desai; Kashappa-Goud;
(Ann Arbor, MI) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
MICHIGAN
Ann Arbor
MI
|
Family ID: |
44304973 |
Appl. No.: |
13/512913 |
Filed: |
January 13, 2011 |
PCT Filed: |
January 13, 2011 |
PCT NO: |
PCT/US11/21166 |
371 Date: |
May 31, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61294666 |
Jan 13, 2010 |
|
|
|
Current U.S.
Class: |
424/400 ;
424/184.1; 424/94.61; 427/2.1; 514/1.1; 514/15.2; 514/44R;
514/772.3; 514/9.7; 521/182 |
Current CPC
Class: |
A61K 38/09 20130101;
A61K 9/1694 20130101; A61K 38/47 20130101; A61K 9/1647 20130101;
A61K 39/08 20130101; A61K 47/6921 20170801; A61P 37/00 20180101;
A61K 47/593 20170801; A61K 47/6937 20170801; A61P 35/00 20180101;
A61K 47/52 20170801; A61K 47/6923 20170801; A61K 38/38 20130101;
A61K 38/385 20130101; A61P 15/00 20180101 |
Class at
Publication: |
424/400 ;
514/772.3; 424/184.1; 514/1.1; 514/44.R; 514/15.2; 424/94.61;
514/9.7; 521/182; 427/2.1 |
International
Class: |
A61K 47/34 20060101
A61K047/34; A61K 39/00 20060101 A61K039/00; A61K 38/02 20060101
A61K038/02; A61K 48/00 20060101 A61K048/00; A61P 15/00 20060101
A61P015/00; B05D 5/00 20060101 B05D005/00; A61K 38/47 20060101
A61K038/47; A61K 38/08 20060101 A61K038/08; A61P 35/00 20060101
A61P035/00; C08G 63/08 20060101 C08G063/08; C08L 67/04 20060101
C08L067/04; A61K 9/00 20060101 A61K009/00; A61K 38/38 20060101
A61K038/38 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] This invention was made with U.S. government support under
grant number R21 EB008873 awarded by the National Institutes of
Health. The U.S. government has certain rights in the invention.
Claims
1. A delivery system comprising a solid polymer matrix comprising
an ionic affinity trap, the ionic affinity trap operable to sorb an
agent from an aqueous solution.
2. The delivery system of claim 1, further comprising an agent
sorbed to the ionic affinity trap.
3. The delivery system of claim 1, wherein the solid polymer matrix
comprises a self-healing polymer.
4. The delivery system of claim 3, wherein the self-healing polymer
comprises a pore that comprises the ionic affinity trap.
5. The delivery system of claim 4, wherein the self-healing polymer
comprises a plurality of pores comprising the ionic affinity
trap.
6. The delivery system of claim 5, wherein at least a portion of
the pores are interconnected.
7. The delivery system of claim 4, further comprising an agent
sorbed to the ionic affinity trap and wherein the pore partially or
fully encapsulates the agent and prevents the agent from exiting
the pore.
8. The delivery system of claim 1, wherein the solid polymer matrix
comprises a biodegradable polymer.
9. The delivery system of claim 1, wherein the solid polymer matrix
comprises a copolymer of lactic acid and glycolic acid.
10. The delivery system of claim 1, wherein the solid polymer
matrix comprises a microparticle or microsphere.
11. The delivery system of claim 1, wherein the ionic affinity trap
comprises a metal salt.
12. The delivery system of claim 1, wherein the ionic affinity trap
comprises aluminum hydroxide, aluminum phosphate, potassium
phosphate, magnesium carbonate, calcium phosphate, or combinations
thereof.
13. The delivery system of claim 1, wherein the ionic affinity trap
comprises an ionomer gel.
14. The delivery system of claim 1, wherein the ionic affinity trap
comprises ionized end groups of the polymer.
15. The delivery system of claim 14, wherein the ionized end groups
comprise carboxylate groups.
16. The delivery system of claim 2, wherein the agent comprises a
biomolecule, drug, or antigen.
17. The delivery system of claim 16, wherein the biomolecule
comprises a protein, peptide, proteoglycan, lipoprotein, or nucleic
acid.
18. The delivery system of claim 2, wherein the agent comprises an
immunocontraceptive.
19. The delivery system of claim 1, wherein the solid polymer
matrix further comprises a plasticizer.
20. A method of making a delivery system comprising sorbing an
agent to an ionic affinity trap, wherein a solid polymer matrix
comprises the ionic affinity trap and an aqueous solution comprises
the agent.
21. The method of claim 20, wherein the solid polymer matrix
comprises a self-healing polymer.
22. The method of claim 21, wherein the self-healing polymer
comprises a pore that comprises the ionic affinity trap.
23. The method of claim 22, wherein the self-healing polymer
comprises a plurality of pores comprising the ionic affinity
trap.
24. The method of claim 23, wherein at least a portion of the pores
are interconnected.
25. The method of claim 22, further comprising partially or fully
encapsulating the agent in the pore by increasing the temperature
of the self-healing polymer to about its T.sub.g or above.
26. The method of claim 20, wherein the solid polymer matrix
comprises a biodegradable polymer.
27. The method of claim 20, wherein the solid polymer matrix
comprises a copolymer of lactic acid and glycolic acid.
28. The method of claim 20, wherein the solid polymer matrix
comprises a microparticle or microsphere.
29. The method of claim 20, wherein the ionic affinity trap
comprises a metal salt.
30. The method of claim 20, wherein the ionic affinity trap
comprises aluminum hydroxide, aluminum phosphate, potassium
phosphate, magnesium carbonate, calcium phosphate, or combinations
thereof.
31. The method of claim 20, wherein the ionic affinity trap
comprises an ionomer gel.
32. The method of claim 20, wherein the ionic affinity trap
comprises ionized end groups of the self-healing polymer.
33. The method of claim 32, wherein the ionized end groups comprise
carboxylate groups.
34. The method of claim 20, wherein the agent comprises a
biomolecule, drug, or antigen.
35. The method of claim 34, wherein the biomolecule comprises a
protein, peptide, proteoglycan, lipoprotein, or nucleic acid.
36. The method of claim 20, wherein the agent comprises an
immunocontraceptive.
37. The method of claim 20, wherein the solid polymer matrix
further comprises a plasticizer.
38. The method of claim 20, wherein the aqueous solution comprises
less than about 1 mg/mL of the agent.
39. The method of claim 20, wherein at least 90% of the agent in
the sorbing step is sorbed to the ionic affinity trap.
Description
FIELD
[0002] The present technology relates to a delivery system with
high encapsulation efficiency for an agent such as a
biomolecule.
INTRODUCTION
[0003] Injectable biodegradable polymeric particles, such as
microspheres, provide a means to deliver and control the release of
molecules such as drugs, proteins, peptides, and vaccine antigens.
Once injected, the biodegradable polymeric particles can release
the molecule over the course of hours, days, or even weeks to
months, providing a distinct advantage over daily injections in
terms of patient acceptability and compliance. For example,
controlled release of a protein antigen can reduce the number of
doses in an immunization schedule and can optimize the desired
immune response via selective targeting of antigen to antigen
presenting cells. Several biodegradable polymers have been explored
for the microencapsulation and delivery of macromolecules.
Copolymers of lactic acid and glycolic acid (PLGA) are one type of
biodegradable polymer used in pharmaceutical products or medical
devices including several approved by the U.S. Food and Drug
Administration. For example, PLGAs are used in commercially
available controlled-release peptide delivery systems, including
the Lupron Depot.TM. (leuprolide acetate), Sandostatin LAR.TM.
(octreotide acetate), and Zoladex.TM. implant (goserelin
acetate).
[0004] Unfortunately, successful controlled release of
macromolecules such as proteins can be difficult. Stability of the
protein during encapsulation and stability during release in vivo
are two concerns. And slowly and completely releasing therapeutic
proteins in their native state from biodegradable polymeric
particles can pose a significant obstacle in the development of
controlled-release injectable depots. Methods for encapsulating
macromolecules in biodegradable polymers can involve harsh
processing conditions, including exposure to organic solvents,
excess heat, homogenization including mixing, sonication, and
high-speed agitation, and so forth, which alone or in combination
can denature and/or destabilize proteins and other macromolecules.
Additionally, drying and micronization of the macromolecule prior
to encapsulation may further destabilize the macromolecule.
[0005] What is more, hydrophilic macromolecules, including many
proteins, cannot readily diffuse through a hydrophobic polymer
phase, such as PLGA. The release of encapsulated protein drugs from
PLGA requires at some point the diffusion of the macromolecules
through water-filled pores and channels. For example, protein
release from PLGA microspheres can exhibit tri-phasic behavior.
First, protein on the surface or having access to the surface of
microspheres (i.e., in open pores) is released rapidly, providing
an initial burst release. Second, a time lag commonly exists as the
protein cannot diffuse through the polymer phase. Third, a
continuous release of protein occurs following polymer erosion so
that more pores and channels are formed allowing protein in
previously isolated pores to be released. There are few exceptions
to the low permeability rule (i.e., peptides and proteins cannot
diffuse through PLGA) not commonly known, which occur for example,
when lower MW PLGAs with acid end groups take up water. In this
case, the polymer may also sorb peptides of moderate MW deep into
the polymer phase itself.
[0006] Encapsulation methods employing self-healing polymers have
been developed to form biodegradable polymeric particles loaded
with various macromolecules; e.g., peptides, proteins, DNA, siRNA,
etc. U.S. Pat. Appl. Pub. 2008/0131478 to Schwendeman et al.
describes methods that obviate damaging stresses during
microencapsulation, which include forming a porous polymer, ideally
with a percolating pore network, incubating an aqueous solution of
the macromolecules below the glass transition temperature (T.sub.g)
of the polymer so that the macromolecule is taken up into the pores
of the polymer, and raising the temperature above the T.sub.g so
that the polymer pores close irreversibly encapsulating the
macromolecule. Other methods can be used to close the pores besides
temperature change. For example, exposure to solvent, such as
alcohol vapor, can be used to facilitate self-healing of the
polymer. Using this methodology, loadings of about 10% w/w or more
can be achieved.
[0007] Passive encapsulation methods rely on equilibration of the
protein between the solution outside the polymer and the aqueous
pores inside the polymer. Such methods may not provide high
encapsulation efficiency; i.e., mass macromolecule
encapsulated/mass of macromolecule charged to the system. As a
result, a significant proportion of the macromolecule to be loaded
remains in solution outside the polymer as the polymer pores close.
The macromolecule solution may have to be reused multiple times in
this case to avoid wasting the macromolecule. Furthermore, passive
encapsulation typically requires very high concentrations of the
macromolecule (e.g., >100 mg/mL) in order to achieve elevated
loading. Some macromolecules may have limited solubility,
prohibiting this method altogether.
[0008] Accordingly, a need exists for new methods of encapsulating
macromolecules in pore-containing polymers, such as PLGA, that can
increase the loading and encapsulation efficiency. It is desirable
that the method could operated without the need for organic solvent
or other harsh processing conditions during encapsulation, which
can denature proteins, for example, and destabilize macromolecules.
It is also desirable that there would be no need for micronization
of the macromolecule, such as protein or nucleic acid, before
encapsulation and is desirable that there would be no need for
drying, each of which can destabilize macromolecules. The
encapsulation method should be less expensive to carry out than
conventional methods, where cost may be a principal factor in the
slow development of controlled release injectable depots. For
example, passive loading of polymer particles may waste a
considerable amount of macromolecule, leaving a majority of the
macromolecule in the solution following pore closure.
SUMMARY
[0009] The present technology includes systems, methods, articles,
and compositions that relate to sorbing and/or encapsulating an
agent, such as a biomolecule, with a solid polymer, such as a
porous self-healing polymer, where the polymer may be in the form
of particles or microspheres. The delivery system may also take on
various forms, may be a portion of other forms, or may be coated
onto other forms, including various shapes or devices such as drug
eluting stents, sutures, screws, tissue engineering scaffolds, and
blood circulating nanoparticles, among others.
[0010] In some embodiments, a delivery system comprises a solid
polymer matrix comprising an ionic affinity trap, the ionic
affinity trap is operable to sorb an agent from an aqueous
solution. An agent can be sorbed to the ionic affinity trap. The
solid polymer matrix can be a self-healing polymer. The
self-healing polymer can comprise one or more pores that comprise
the ionic affinity trap, where at least a portion of the pores can
be interconnected. The delivery system can further include an agent
sorbed to the ionic affinity trap and wherein the pore partially or
fully encapsulates the agent and prevents the agent from exiting
the pore. The solid polymer matrix can comprises a biodegradable
polymer and can comprise a copolymer of lactic acid and glycolic
acid. The solid polymer matrix can take the form of a microparticle
or microsphere. The ionic affinity trap can comprise a metal salt,
such as aluminum hydroxide, aluminum phosphate, potassium
phosphate, magnesium carbonate, calcium phosphate, or combinations
thereof, and can comprise an ionomer gel. The ionic affinity trap
can comprise ionized end groups of the polymer, where the ionized
end groups can comprise carboxylate groups. The agent can comprise
a biomolecule, drug, or antigen, where the biomolecule can comprise
a protein, peptide, proteoglycan, lipoprotein, or nucleic acid. The
agent can also comprise an immunocontraceptive. The solid polymer
matrix can further comprise a plasticizer.
[0011] In some embodiments, a method of making a delivery system
comprises sorbing an agent to an ionic affinity trap, wherein a
solid polymer matrix comprises the ionic affinity trap and an
aqueous solution comprises the agent. The method can further
comprise partially or fully encapsulating an agent in a pore by
increasing the temperature of the self-healing polymer to about its
T.sub.g or above, where the solid polymer matrix includes a
self-healing polymer comprising the pore. The aqueous solution in
the sorbing step can comprise less than about 1 mg/mL of the agent.
At least 90% of the agent in the sorbing step can be sorbed to the
ionic affinity trap.
[0012] In some embodiments, a delivery system can include a
self-healing polymer comprising one or more pores, where the pore
or pores include an ionic affinity trap and an agent, such as a
biomolecule, associated with the ionic affinity trap. The delivery
system can be made by providing the self-healing polymer having one
or more pores, where the pore(s) includes an ionic affinity trap.
The self-healing polymer is contacted with an agent having affinity
for the ionic affinity trap so that the agent associates with the
ionic affinity trap. The self-healing polymer can then partially or
fully encapsulate the agent, preventing the agent from exiting the
pore(s). The delivery system can also include a self-healing
polymer where a portion of the self-healing polymer comprises
ionized end groups and an agent sorbed to the ionized end groups.
Such a delivery system can be made by contacting the self-healing
polymer with an agent having affinity for the ionized end groups
and optionally
[0013] Further areas of applicability will become apparent from the
description provided herein. The description and specific examples
in this summary are intended for purposes of illustration only and
are not intended to limit the scope of the present disclosure.
DRAWINGS
[0014] The drawings described herein are for illustrative purposes
only of selected embodiments and not all possible implementations,
and are not intended to limit the scope of the present
disclosure.
[0015] FIG. 1. Self-healing microencapsulation of large molecules
in PLGA microsphere injectable controlled-release depots. Scanning
electron micrographs of self-microencapsulating microspheres (SM-1,
Tables 1 and 2) before (A) and after (B) healing of polymer pores
in the presence of 230 mg/mL lysozyme at 42.5.degree. C. Laser
confocal fluorescent micrographs (C) of the cross-sectional
distribution of BSA-Coumarin (in white domains) after self-healing
encapsulation of the protein in microspheres (20-63 .mu.m in
diameter) (SM-2, Tables 1 and 2). Increasing protein loading by
increasing protein concentration exposed to SM-microspheres (D)
(SM-2, Tables 1 and 2) or SM-microsphere porosity (E) with an
increasing volume (25, 100, 200, and 350 .mu.L, open circles) of
inner water phase in the double emulsion or amount (0, 1.5, 4.3,
and 11% w/w MgCO.sub.3, closed circles) of porosigen (SM-3, Tables
1 and 2). Kinetics of self-encapsulation of large molecule in PLGA
microspheres (SM-3, Tables 1 and 2) after initiating self-healing
with increase of T>T.sub.g (F). SM microspheres were first
incubated at 4.degree. C. for 20 h and then at 42.5.degree. C.
(time=0 h) with 65 mg/mL dextran-FITC. Values represent
mean.+-.SEM, n=3 (F) or 5 (D and E) measurements.
[0016] FIG. 2. Assessment of lysozyme aggregation and leuprolide
controlled release after self-healing microencapsulation (SM)
without organic solvent. Insoluble aggregation recorded after
microencapsulation of encapsulation-labile lysozyme (A) according
to self-healing (Formulations A-D without organic solvent) and
standard solvent-evaporation (Formulations E and F with organic
solvent) processes. SM microspheres were prepared from 11
(Formulations A, and B) and 51 (Formulations C-F) kDa M.sub.w, PLGA
50/50, and in the presence (B, D, F) and absence (A, C, E) of 0.45
M sucrose in the aqueous lysozyme solution. A: Formulations A and
B, C and D, and E and F, respectively, correspond to SM-4, SM-3,
and TM-1 in Tables 1 and 2. In vitro and in vivo evaluation of
controlled-release leuprolide from SM-microspheres (SM-5, Tables 1
and 2). Slow release of leuprolide was observed in a physiological
buffer in vitro (B) and as indicated by serum testosterone
suppression in rats (C). Animals were injected subcutaneously once
with 1-month dose of soluble leuprolide (filled squares), once with
2-month dose of leuprolide acetate in SM-microspheres (open
diamonds), twice (day 0 and day 28) with commercial 1-month Lupron
Depot.TM. (open squares) or once with blank SM-microspheres
containing no drug (open triangles). Dose was based on 100
.mu.g/kg/day. Solid and dashed line respectively represents lower
testosterone detection limit (0.1 ng/mL) and castration level (0.5
ng/mL), respectively. Values represent mean.+-.SEM, n=5
measurements (A and B) or 6 animals (C).
[0017] FIG. 3. Active self-microencapsulation of ovalbumin (OVA)
and tetanus toxoid (TT) in Al(OH).sub.3 adjuvant-containing
SM-microspheres (ASM, Tables 1 and 2). Scanning electron
micrographs (A, B) depict the SM-microsphere morphology after
loading entire OVA mass from 0.5 mg/mL OVA solution and
self-healing at 37.degree. C. PLGA plasticized with 0% (A) and 5%
(B) diethyl phthalate (DEP). A and B formulations respectively
correspond to ASM-1 and ASM-3 in Tables 1-4. Controlled release of
OVA monomer (C) or antigenic TT (D) from active
self-microencapsulating microspheres relative to OVA or TT-loaded
aluminum adjuvant without polymer (closed squares, Al(OH).sub.3 no
PLGA; closed circles, 3.2% Al(OH).sub.3/3.5% trehalose/PLGA no
plasticizer (ASM-1, tables S1-S5); open circles, 3.2%
Al(OH).sub.3/3.5% trehalose/PLGA/5% DEP). Closed and open circles
respectively represent formulation ASM-1 and ASM-3 in Tables 1-5.
Values represent mean.+-.SEM, n=3 measurements.
[0018] FIG. 4. Goodness of self-encapsulation in PLGA microspheres
as indicated by elevated lysozyme loading (open bar chart) and
minimal initial burst (closed bar chart) of enzyme employing
diversity of pore-forming excipients/initial water phase volume to
create the PLGA 50/50 pore network. Formulations A, B, C, and D
were respectively prepared with 0, 1.5, 4.3, and 11% w/w MgCO.sub.3
and E, F, and G were respectively prepared with 25, 100, and 350
.mu.L initial water phase (SM-3, Tables 1 and 2). Values represent
mean.+-.SEM, n=3 measurements.
[0019] FIG. 5. Self-healing microencapsulation of leuprolide
acetate in PLGA 50:50 (M.sub.w=51 kDa) microspheres (SM-5, Tables 1
and 2). Scanning electron microscopy image of SM PLGA microspheres
before (A) and after (B) self-healing microencapsulation of
leuprolide acetate. Actual amount of leuprolide acetate
self-encapsulated by this formulation was 3.0.+-.0.2% (w/w
peptide/polymer matrix) (5 measurements).
[0020] FIG. 6. Protection of protein (BSA and lysozyme) against
acid-induced instability during release from self-microencapsulated
PLGA 50:50 (M.sub.w=51 kDa) microspheres (SM-3, Tables 1 and 2).
(A) Effect of MgCO.sub.3 (porosigen/stabilizer) loading (3 ( ) and
4.5 (.DELTA.) % w/w) and addition of sucrose (0 (.box-solid.) and
0.45 (.largecircle.) M) in protein loading solution on cumulative
release of BSA from self-microencapsulated microspheres. Actual BSA
loading in , .DELTA., .box-solid., and .largecircle. SM
formulations was 4.25.+-.0.05, 5.65.+-.0.06, 7.26.+-.0.09, and
5.54.+-.0.04%, respectively. Initial water phase (200 .mu.L) was
PBS (pH 7.4) with (.box-solid. and .largecircle.) or without
trehalose (500 mg in 1 g PBS). (B) Effect of MgCO.sub.3
(porosigen/stabilizer) loading (0 ( ), 1.5 (.DELTA.), 4.5
(.diamond-solid.), and 11 (.quadrature.) % w/w) on cumulative
release of lysozyme from self-microencapsulated microspheres.
Actual lysozyme loading in , .DELTA., .diamond-solid., and
.quadrature.SM formulations was 4.2.+-.0.2, 6.4.+-.0.1, 9.8.+-.0.3,
and 8.7.+-.0.4%, respectively. In vitro release studies were
conducted in PBST at 37.degree. C. and symbols represent
mean.+-.SEM (three measurements).
[0021] FIG. 7. Effect of encapsulation of Al(OH).sub.3 and blending
of hydrophobic plasticizer (diethyl phthalate (DEP)) on the
self-healing phenomenon of PLGA 50:50 (M.sub.w=51 kDa)
microspheres. Surface morphology of blank (3.5% w/w trehalose
loaded), ASM-1, ASM-2, and ASM-3 PLGA microspheres before and after
self-healing microencapsulation process. ASM-1, ASM-2, and ASM-3
PLGA microsphere formulations consists of 3.2% w/w Al(OH).sub.3 and
3.5% w/w trehalose as porosigen and Al(OH).sub.3 lyophilization
stabilizer. ASM-2 and ASM-3 contain 2.5 and 5% w/w DEP,
respectively. Blank and ASM-1 PLGA microspheres were incubated at
25 and 43.degree. C. for 48 h. ASM-2 and ASM-3 PLGA microspheres
were incubated for 48, 24, and 30 h at 10, 25, and 37.degree. C.,
respectively. Preparation process of blank ASM PLGA microspheres is
given in Table 1.
[0022] FIG. 8. Evaluation of quality of active protein
self-microencapsulation in Al(OH).sub.3--PLGA 50:50 (M.sub.w=51
kDa) (ASM PLGA) microspheres in 190 mM sodium citrate solution.
Cumulative ovalbumin released as a function of time from
unencapsulated Al(OH).sub.3 gel (control) (.box-solid.) and with
(.largecircle.) (ASM-3, Tables 3 and 4) and without ( ) (ASM-1,
Tables 3 and 4) 5% w/w DEP in Al(OH).sub.3--PLGA microsphere
formulations. In vitro release studies were conducted at 37.degree.
C. and symbols represent mean.+-.SEM (three measurements).
[0023] FIG. 9. Photomicrographs of 3.2 wt % alhydrogel/3.8 wt %
trehalose/PLGA microparticles before (A) and after (B) self-healing
at 25.degree. C. for 24 h and 43.degree. C. for 48 h.
[0024] FIG. 10. Photomicrographs of 3.2 wt % alhydrogel/3.8 wt %
trehalose/2.5 wt % DEP/PLGA microparticles before (A) and after (B)
self-healing at 10.degree. C. for 48 h, 25.degree. C. for 24 h, and
37.degree. C. for 30 h.
[0025] FIG. 11. Photomicrographs of 3.2 wt % Alhydrogel/3.8 wt %
Trehalose/5 wt % DEP/PLGA microparticles before (A) and after (B)
self-healing.
[0026] FIG. 12. Evaluation of in vitro release of leuprolide
acetate (LA) from LA-PLGA particles. Cumulative amount of LA
released as a function of incubation time in PBST at 37.degree. C.
The actual loading of LA in LA-PLGA particles was about 17 wt %.
Symbols represent mean.+-.SE, n=3.
[0027] FIG. 13. Prolonged serum testosterone suppression by
leuprolide acetate (LA)-PLGA particles in male Sprague-Dawley rats.
Effect of dosing interval (2.times., 3.times., and 4.times.) of
LA-PLGA particles on serum testosterone suppression. Animals were
injected subcutaneously with 1.times. (day 0) soluble leuprolide (
), LA-PLGA particles with different dosing interval (2.times.: day
0, 14, 28, and 42 (.tangle-solidup.), 3.times.: day 21 and 42
(.diamond.), and 4.times.: day 0 and 28 ()), and blank PLGA
particles (.largecircle.) in a liquid vehicle (1% w/v
carboxymethylcellulose and 2% w/v mannitol) along with 2.times.
(day 0 and 28) commercial 1-month Lupron Depot (.quadrature.). The
dose of leuprolide acetate was 100 .mu.g/kg/day. Solid and dashed
line respectively represents lower testosterone detection limit and
castration level. Symbols represent mean.+-.SEM, n=6. Lowest
detection limit of testosterone was 0.1 ng/mL.
DETAILED DESCRIPTION
[0028] The following description of technology is merely exemplary
in nature of the subject matter, manufacture and use of one or more
inventions, and is not intended to limit the scope, application, or
uses of any specific invention claimed in this application or in
such other applications as may be filed claiming priority to this
application, or patents issuing therefrom. A non-limiting
discussion of terms and phrases intended to aid understanding of
the present technology is provided at the end of this Detailed
Description.
[0029] Preparation of controlled-release polymeric biomaterials
generally requires an organic solvent during drug
microencapsulation, limiting both biomaterial design and
applications for large molecular drugs. The present technology
provides a new microencapsulation paradigm based on spontaneous
self-healing of polymers. For example, biomolecules such as
peptides, proteins, or polysaccharides dissolved in aqueous
solution can be self-microencapsulated in poly(lactic-co-glycolic
acid) (PLGA) by placing the biomacromolecule solution in contact
with solid PLGA, preformed with an interconnected pore-network, at
below the polymer glass transition temperature (T.sub.g), and then
healing the pores at a temperature at or above the T.sub.g. Healed
polymers can then slowly release the biomacromolecules under
physiological conditions for over 1 month. Benefits of the present
approach include improved compatibility with biotechnology-derived
drugs, reduced manufacturing cost and residual organic solvent, the
ability to create new biomaterial architectures, and facile use
among non-formulation scientists and clinicians.
[0030] Modern synthetic polymeric biomaterials are widely used to
slowly release medicines over days to years after administration to
the body. These polymers are configured in numerous biomedical and
pharmaceutical three-dimensional forms (e.g., spheres, rods,
coatings, porous matrices) including micro- to millimeter scale
injectable depots, drug-eluting stents, scaffolds for engineering
tissues, and blood-circulating nanometer scale particles and can be
made biodegradable or nondegradable. Until now, drugs, particularly
peptides and proteins, are most commonly microencapsulated by first
combining drug with a polymer dissolved in organic solvent. Before
or after this combination the drug is either micronized (e.g., by
homogenization, sonication, or grinding) or molecularly dissolved
in the solvent, to yield drug domains, which later become dispersed
in the final polymer matrix. Both steps can compromise stability of
encapsulated proteins and other biomolecules. The organic solvent
is removed to clinically acceptable levels and the polymer is dried
before use. Described here is a new self-microencapsulation
paradigm based on the polymer's own spontaneous "self-healing"
capacity in aqueous media. Features of this new approach include a
simple mixing process (e.g., as mixing naked DNA to lipofectin gene
delivery vector), the absence of exposure of drug to organic
solvent during encapsulation (e.g., as supercritical fluid polymer
processing), and mild processing conditions (e.g., as
spray-congealing for commercial manufacture of PLGA-encapsulated
growth hormone).
[0031] To illustrate this new paradigm, injectable
self-microencapsulating (SM) microsphere (SM-1, Table 1) depots of
biocompatible copolymers of lactic and glycolic acids (PLGA) were
prepared by a standard emulsion-based method with leachable
trehalose to create interconnected porous network in the polymer,
but without including an agent such as a biomolecule or drug. After
leaching the sugar, pores on the scale of 250 to 2500 nm were
easily viewed by electron microscopy (FIG. 1A). The dry
microspheres were incubated at 4.degree. C. (<<hydrated
T.sub.g.about.30.degree. C.) in concentrated aqueous lysozyme
solution at 230 mg/mL for 48 h to allow the protein to enter the
open polymer pores. Self-healing of the pores was initiated without
organic solvent by raising the temperature to about or greater than
the T.sub.g to 42.5.degree. C. for 44 h (SM-1, Table 2), resulting
in lysozyme-encapsulated microspheres with 3.8.+-.0.1% protein
loading (w/w protein/polymer matrix, 5 measurements) and a
nonporous polymer surface (FIG. 1B).
[0032] Large molecules penetrate deep within the polymer matrix
prepared by this method, as viewed by laser scanning confocal
micrographs of healed SM microspheres prepared with fluorescent
coumarin-labelled BSA (FIG. 1C). Moreover, molecules as large as 2
million Da were found to enter the polymer pore network and become
self-encapsulated, as indicated by the similar loading in the
polymer of 2 MDa and 4 kDa fluorescent-labeled dextrans. Protein
loading determined after extensive washing of SM microspheres was
found easily adjustable, for example, as seen by the sensitivity of
BSA (FIG. 1D) and lysozyme (FIG. 1E) loading, respectively, to
initial protein loading solution concentration (SM-2, Tables 1 and
2) and dry SM polymer porosity (SM-3, Tables 1 and 2). To test the
goodness of encapsulation, SM microspheres prepared by several
different conditions were loaded with protein and incubated under
physiological conditions (in PBS+0.02% Tween 80, pH 7.4 at
37.degree. C.) for 48 h to observe the "initial burst release" of
protein (FIG. 4), which is often too high with poorly encapsulated
material. SM microspheres with elevated protein loading of
1.2.+-.0.1 to 9.8.+-.0.3% (5 measurements) and optimal porosigen
loading (e.g., 1.5-4.5% (w/w magnesium carbonate/polymer matrix), 5
measurements) typically exhibited an optimal initial burst release
of protein (<20% release). Importantly, the measured loading and
initial burst values were within the desirable range as established
by clinically used PLGA depots and required loading times were on
the order of 12 hours (FIG. 1F).
[0033] As expected by the mild encapsulation conditions
(37-43.degree. C. temperature exposure) by the new approach--no
harsh mixing or organic solvent exposure--protein stability was
also improved with SM microspheres relative to microspheres
prepared by traditional emulsion-based microencapsulation
techniques (e.g., water-in-oil-in-water--solvent evaporation,
w/o/w, as used for the Lupron Depot). For example, using the model
enzyme, lysozyme, well-established to undergo aggregation during
solvent evaporation, the potential stability improvement of the
enzyme in SM microspheres was evaluated relative to solvent
evaporation control groups. In formulations with two different MW
polymers with and without addition of protein-stabilizing sucrose,
the stability of lysozyme was improved with SM microspheres in each
case (FIG. 2A), and when loading by self-healing in the presence of
sucrose, negligible aggregation or activity losses of the enzyme
were detected.
[0034] Desirable polypeptides were also successfully
self-microencapsulated by PLGAs and released slowly and
continuously over a period of >1 month. For example, the most
commonly delivered peptide from PLGA depots, leuprolide acetate,
used to suppress testosterone in prostate cancer patients to
inhibit growth of the hormone-dependent cancer, was loaded in SM
PLGA microspheres employing an ionic affinity trap, ZnCO.sub.3, to
create pores for self-encapsulation and to facilitate continuous
release of peptide. The resulting SM microspheres (FIG. 5)
encapsulated 3.0.+-.0.2% (w/w peptide/polymer matrix) leuprolide
acetate (5 measurements) and released the peptide in vitro slowly
and continuously for 2 months (FIG. 2B). After administration of a
single injection of the same formulation in rats, slow release of
leuprolide acetate was observed, as indicated by the steady
suppression of testosterone (FIG. 2C) (owing to down-regulation of
LHRH receptors) before escaping castration levels after 6-weeks.
Similar behavior was observed after two injections of the 1-month
Lupron Depot.TM. formulation, whereas both negative control groups,
leuprolide acetate-free SM-microspheres and a 1-month dose of
solution leuprolide, were ineffective to suppress testosterone.
Model proteins, bovine serum albumin and lysozyme, were also slowly
released, albeit in a first-order fashion (FIG. 6), without any
signs of classic acid-induced aggregation of BSA and with full
monomeric and enzymatic activity recovery of lysozyme in the
polymer after 1-month release incubation. Note that in this
example, the leuprolide does not freely diffuse across the polymer
phase (which does not have acidic end groups), and thus the peptide
likely distributes to the polymer pores during encapsulation.
[0035] Ultimate success of microencapsulation of expensive biotech
drugs requires minimal drug losses during encapsulation. In a
single batch process with SM microspheres, the percentage of drug
from the loading solution retained within the final particles,
i.e., the encapsulation efficiency (EE), is low (.about.1.5-13%) by
the passive process. However, as with minimal or no peptide or
protein damage occurs upon self-microencapsulation, the loading
solution could be reasonably recycled multiple times with
concentration adjustment. A similar issue was resolved in the
marketed Doxil.TM. stealth liposomes by the active loading of
doxorubicin via precipitating the drug with ammonium sulfate as it
diffused into the empty liposome.
[0036] We investigated active loading strategies using two vaccine
antigens (ovalbumin (OVA) and tetanus toxoid (TT)). For example,
OVA or TT protein antigens were loaded into SM PLGA containing, as
an ionic affinity trap, lyophilization-stabilized Al(OH).sub.3
adjuvant (ASM, Table 1), which absorbed the antigen into the
polymer matrix from surrounding 0.5 or 0.8 or 1 mg/mL antigen
solution, with 98.+-.1% EE (6 measurements) and 1.0.+-.0.05% OVA
loading (6 measurements) (ASM-3, tables S3 and S4) or 87.+-.0.4% EE
(3 measurements) and 1.6.+-.0.03% TT loading (Table 5). Extent of
self-healing of polymer pores was also easily enhanced at
37.degree. C. in the active-loading microspheres by addition of a
plasticizer, diethyl phthalate (DEP), to the polymer to further
mobilize the hydrated polymer chains (FIG. 7). After
self-microencapsulation, decreased surfaces pores are visible with
plasticizer added (FIGS. 3A and B). Vaccine antigens were
effectively self-encapsulated with the active loading strategy in
both preparations, as indicated by slow-release of the antigens
relative to unencapsulated Al(OH).sub.3 in 190 mM sodium citrate
(FIG. 8), phosphate buffered saline (PBS) (FIG. 3C), and PBS+0.02%
Tween.RTM. 80+0.2% BSA (FIG. 3D). Hence, it is now possible to
self-microencapsulate in biodegradable polymers the entire mass of
bioactive macromolecules from a low concentration aqueous solution
by simple mixing and heating of the solution with an SM-polymer
matrix. Also noteworthy were a) the absent requirement of a drying
step after microencapsulation of OVA and TT, as drying can cause
irreversible damage to proteins, and b) the complete release of
antigenically active TT without any apparent common formaldehyde or
acid-induced antigen instability.
[0037] Spontaneous self-healing in homogenous polymer systems has
been described in nano-scale cracks of solid rocket propellants,
following bullet holes in plastic plates, film formation from latex
particles, and across lap joints of polymer films. The process
mechanism, which has found to be ubiquitous to polymers in the
vicinity of their T.sub.g or above has been analyzed in detail to
involve multiple elements such as: a) polymer chain interdiffusion,
b) minimization of energetically unfavorable interfacial area, and
c) transfer of energy stored in a defect. We first observed
spontaneous pore closing in PLGA microspheres that encapsulated a
water-soluble polypeptide during the initial burst release of
peptide less than 24 hours after exposure of the polymer to aqueous
media at physiological temperature; the resulting closure of the
peptide release route initiated a lag phase in release
characteristic of this polymer when above a critical MW. Initial
studies thereafter to demonstrate the self-encapsulation paradigm
in aqueous media attempted to obtain high loading with low initial
burst (as seen in FIG. 1F) suffered from poor interconnected
networks or pore networks which would not close (likely due to very
high porosity of the polymer). We found that polymer matrices with
moderate porosity and defects .about.2 .mu.m of less (FIG. 1B)
could be healed in aqueous solution when pores were created by
sugar or salt similarly as those observed during the initial
peptide release. Consistent with previous mechanistic analysis,
self-encapsulation requires a minimum temperature for polymer-chain
mobility to occur over reasonable time scales (FIG. 1F).
[0038] The benefits and uses of the self-microencapsulating
paradigm are far reaching. For example, a clinician can mix sterile
SM microspheres with an injectable solution of vaccine (e.g.,
tetanus toxoid) before injecting into women of child-bearing age,
providing improved immunity for their unborn children against
neonatal infection. New biomaterial architectures (e.g.,
drug-eluting stent coatings) that release process-sensitive large
molecules as previously unchartered formulation conditions (e.g.,
high temperature, reactive molecules, organic solvent) can be used
to create the SM polymer delivery system without concern of
damaging the encapsulated macromolecule. For manufacturing, a
mixture of several different SM microsphere formulations, each
having distinct design characteristics (release kinetics, size,
surface biofunctional groups) can be combined for a drug of
interest in a single sterile mixing step. With the absence of
aseptic processing of organic solvents, this paradigm can have
significant cost savings, which was a critical factor in halting
production of the Nutropin Depot, the first and only FDA approved
injectable protein depot. The simplicity of self-microencapsulation
can also significantly facilitate various controlled release
approaches, providing more rapid advancement of controlled release
products and technology.
[0039] The following materials and methods were employed to
demonstrate the present technology.
[0040] Materials: Poly(D,L-lactic-co-glycolic acid) (PLGA) 50:50
with an i.v.=0.20 dl/g in hexafluoro isopropanol at 30.degree. C.
(Lot #A07-044, end-group=1-dodecyl ester, weight average molecular
weight (M.sub.w)=11 kDa) of Lactel Absorbable Polymers was
purchased from DURECT Corporation (Cupertino, Calif., USA). PLGA
50:50 with an i.v.=0.19 dl/g in chloroform at 30.degree. C. (Lot
#1158-515, end-group=methyl ester, M.sub.w=19 kDa) and 0.57 dl/g in
hexafluoroisopropanol at 30.degree. C. (Lot #W3066-603,
end-group=lauryl ester, M.sub.w=51 kDa) were purchased from
Lakeshore Biomaterials (Birmingham, Ala., USA).
.alpha.,.alpha.-Trehalose dihydrate was purchased from Pfanstiehl
(Waukegon, Ill., USA), zinc carbonate (ZnCO.sub.3) was from ICN
Biomedicals Inc., and 7-methoxycoumarin-3-carbonyl azide was from
Invitrogen Corporation (Carlsbad, Calif., USA). Tetrahydrofuron
(THF) was purchased from Fisher-Scientific (Pittsburgh, Pa., USA).
Tetramethyl-rhodamine (TMR)-dextran was purchased from Invitrogen
Corporation (Carlsbad, Calif., USA). Tetanus toxoid (TT) (3120
Lf/mL) and equine tetanus antitoxin were received as gift samples
respectively from Serum Institute of India Ltd. (Pune, India) and
U.S. Food and Drug Administration (Silver Spring, USA). Human
tetanus immune globulin (HyperTET.TM. S/D, 250 units) was purchased
from Talecris Biotherapeutics, Inc. (Research Triangle Park, N.C.,
USA). Poly(vinyl alcohol) (PVA) (9-10 kDa, 80% mol hydrolyzed),
Alhydrogel.TM. aluminum hydroxide adjuvant gel (Al(OH).sub.3),
bovine serum albumin (BSA) fraction V, ovalbumin (OVA), lysozyme
from chicken egg white, sodium hydroxide (NaOH), magnesium
carbonate (MgCO.sub.3), BSA labeled with fluorescein isothiocyanate
(BSA-FITC), sodium citrate, p-nitrophenyl phosphate liquid
substrate, and phthaldialdehyde reagent (containing 1 mg
o-phthaldialdehyde per mL solution of 2-mercaptoethanol as the
sulfhydryl moiety) were purchased from Sigma-Aldrich (St. Louis,
Mo., USA). Leuprolide acetate (Batch #091203) was purchased from
Shanghai Shjnj Modern Pharmaceutical Technology Co., Ltd (Shanghai,
China). Male Sprague-Dawley rats were procured from Charles River
Laboratories. Isoflurane was purchased from Baxter Healthcare
Corporation (Deerfield, Ill., USA). B-D Microtainer.TM. blood
collection and serum separation tubes were purchased from Becton,
Dickinson and Company (Franklin Lakes, N.J., USA). Goat anti-human
IgG-alkaline phosphatase was purchased from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, Pa., USA). All other common salts,
reagents, and solvents were purchased from Sigma-Aldrich. Size
exclusion-high performance liquid chromatography (SE-HPLC) columns
(TSK gel G3000SWx1 and TSK gel G2000SWx1 columns, Tosoh Biosciences
LLC, Montgomeryville, Pa., USA), Shodex Protein KW-G size exclusion
chromatography guard column (Showa Denko, N.Y., USA), Nova-Pak C18
column (4 .mu.m, 3.9.times.150 mm) and Bonda-Pak C18 guard column
(4 .mu.m) (Waters Corporation, Milford, Mass., USA) were used.
[0041] Conjugating BSA to a pH-insensitive fluorescent coumarin:
About 1.2 g BSA was dissolved in 40 mL of 0.2 M sodium bicarbonate
(pH 4.5). To this, 2 mL of 10 mg/mL 7-methoxycoumarin-3-carbonyl
azide in dimethyl sulfoxide (DMSO) was added while stirring. The
solution was stirred continuously at room temperature in darkness
for 90 min. To quench the reaction, 4 mL of 1.5 M hydroxylamine
hydrochloride was added and then the solution was extensively
dialyzed using a 25,000 Da M.sub.w cut-off membrane against
degassed distilled water at 4.degree. C.
[0042] Preparation of self-microencapsulating (SM) PLGA
microspheres: Various formulations of self-microencapsulating PLGA
50:50 (M.sub.w=11, 51, and 19 kDa) microspheres (Table 1) were
prepared by a double emulsion (water-in-oil-in-water
(W/O/W))-solvent evaporation method without drug. Briefly, polymer
solutions of various PLGAs were first prepared by dissolving the
required amount of polymer in 1 mL methylene chloride
(CH.sub.2Cl.sub.2) in a 5- or 10-mL syringe or 10-mL glass tube.
Suitable initial water phase (WP) was added to the corresponding
polymer solution and immediately homogenized as per the first
homogenization conditions specified in Table 1 using a Tempest
IQ.sup.2 homogenizer (The VirTis Company, Gardiner, N.Y., USA)
equipped with a 10 mm shaft in an ice water bath to create the
respective first emulsion. Two mL of aqueous 5% PVA was added to
the first emulsion of all the formulations, vortexed or homogenized
as per the second homogenization/vortexing conditions (Table 1),
and the resulting emulsion was injected into 100 mL of chilled (ASM
microspheres) or non-chilled (TM-1 and SM-1 to SM-5 microspheres)
0.5% PVA solution under continuous stirring. Microspheres were
stirred 3 h at room temperature, and collected with sieves to
separate by size (20-63 and 63-90 .mu.m) and washed thoroughly with
distilled water to help remove residual PVA. Collected SM PLGA
microspheres were then freeze-dried using a freeze drier (Labconco
Corporation, Kansas City, Mo., USA) and stored at -20.degree. C.
until further use.
TABLE-US-00001 TABLE 1 Formulation conditions for preparing
traditional (lysozyme encapsulated) and various types of SM PLGA
50:50 microspheres. PLGA 50:50 Conc.(mg polymer in 1 WP Formulation
Initial water phase M.sub.w mL CH.sub.2Cl.sub.2) + volume First
Second homog./ code (WP) composition (kDa) excipient (.mu.L) homog.
vortexing SM-1 500 mg trehalose in 51 320 175 20,000 rpm Homog. at
6000 1 g PBS for 1.5 min rpm for 45 s SM-2 300 mg/mL BSA in 19 700
150 10,000 rpm Vortexing for 15 s PBS for 1 min SM-3 0 or 500 mg 51
320 + 0, 4.8, 25, 100, 17,000 rpm Homog. at 6000 trehalose in 1 g
PBS 14.4, and 39.5 200, and for 1 min rpm for 25 s mg MgCO.sub.3
350 SM-4 500 mg trehalose in 11 1100 175 20,000 rpm Homog. at 6000
1 g PBS for 1.5 min rpm for 45 s TM-1 200 mg/mL 51 320 110 20,000
rpm Homog. at 6000 lysozyme with or for 1.5 min rpm for 45 s
without 0.45M sucrose in water SM-5 1 g PBS or 500 mg 51 320 + 0,
3.5, 200 10,000 rpm Vortexing for 15 s trehalose in 1 g PBS and 10
mg for 1 min ZnCO.sub.3 ASM 300 .mu.L of 25 mM 51 250 + 0, 7, 200
17,000 rpm Vortexing for 50 s succinate buffer (pH and 14.2 mg for
1 min 4.0) containing 13.1- DEP 13.8 mg Al(OH).sub.3 gel and
14.1-15 mg trehalose SM: self-microencapsulating PLGA microsphere
formulation; TM: traditional PLGA microsphere formulation; ASM:
active self-microencapsulating PLGA microsphere formulation;
CH.sub.2Cl.sub.2: methylene chloride; M.sub.w: molecular weight;
PBS: phosphate buffered saline (pH 7.4); MgCO.sub.3: magnesium
carbonate; ZnCO.sub.3: zinc carbonate; DEP: diethyl phthalate;
Conc.: concentration
TABLE-US-00002 TABLE 2 Conditions for investigating passive and
active self- microencapsulation of enzyme/protein/peptide by
various types of PLGA 50:50 SM microspheres. SM Concentration of
Incubation Enzyme/protein/ microspheres loading solution:
temperature/duration peptide self- added volume (.degree. C./h) FC
encapsulated (mg) (mg/mL: mL) Loading Self-healing SM-1 Lysozyme
200 or 225 200 or 230:1 or 1.4 4/48 42.5/44 SM-2 BSA-coumarin 125
43 or 79 or 115 or 4/24 or 48 37 or 42/18 or 157 or 175 or 204:0.8
24 or 1 SM-3 BSA or Lysozyme 50 or 100 1 (TMR-Dextran or 4/24 or 48
(TMR- 42.5/48 or 72 or TMR-Dextran (FITC-Dextran/ FITC-Dextran) or
65 Dextran or FITC- (TMR-Dextran or FITC-Dextran TMR-Dextran)
(FITC-Dextran) or Dextran) or 4/16 or FITC- 80 or 150 (BSA/ 200 or
300 (BSA or or 72 (BSA or Dextran) or Lysozyme) Lysozyme with or
Lysozyme) 43/46 or 48 without 0.45M (BSA or sucrose):1 Lysozyme)
SM-4 Lysozyme 200 250 with or without 4/24 37/12 0.45M sucrose:1
SM-5 Leuprolide acetate 1000 127:4 4/42 43/48 ASM OVA or TT 20 0.5
or 1.0:0.4 (OVA) 10/48 (OVA) or 37/30 (OVA) or or 0.8:0.5 (TT) 24
(TT) + 25/24 38/40 (TT) (OVA and TT) FC: formulation code; SM:
self-microencapsulating PLGA microsphere formulation; ASM: active
self-microencapsulating PLGA microsphere formulation; OVA:
ovalbumin; TT: tetanus toxoid.
[0043] Passive and active self-healing microencapsulation of large
molecules (lysozyme, BSA, leuprolide, OVA and TT) by PLGA
Microspheres: The passive self-microencapsulation paradigm was
studied using all the microspheres formulations given in Table 1,
except Al(OH).sub.3-encapsulated PLGA microspheres, which were used
for testing active self-microencapsulation of OVA and TT. Briefly,
microspheres of 20-63 .mu.m size were incubated with chilled
(4.degree. C.) enzyme/protein/peptide solution on a rocking
platform (VWR Scientific, West Chester, Pa., USA) at 4.degree. C.
(loading conditions) for a specified period of time, as listed in
Table 2. After completion of loading duration, microspheres were
then transferred on a rigged rotator (Glas-Col, Terre Haute, Ind.,
USA) to prevent interparticle healing in an incubator maintained at
specified temperature for specified duration (self-healing
conditions), as listed in Table 2. Microspheres were then removed,
washed 10 times with distilled and deionized water (ddH.sub.2O),
centrifuged at 3000-3800 rpm for 5-10 min, and freeze-dried. To
test active self-microencapsulation, about 21 mg
Al(OH).sub.3-encapsulated PLGA microspheres of 20-63 .mu.m size
were incubated with low protein (OVA or TT) concentration solution
(0.5-1.0 mg/mL, volume=0.4 or 0.5 mL) on a rigged rotator for a
specified duration at 10 and 25.degree. C. for active protein
loading and then at 37 or 38.degree. C. for self-healing (Table 2).
Microspheres were then removed, washed one time with ddH.sub.2O,
centrifuged with conditions given above and supernatant was
removed. Microspheres were used without drying.
[0044] Preparation of lysozyme encapsulated PLGA microspheres by a
traditional encapsulation (TE) method: As summarized in Table 1,
one hundred ten .mu.L of 200 mg/mL aqueous lysozyme solution with
or without 0.45 M sucrose was added to 320 mg PLGA (50:50,
M.sub.w=51 kDa) in 1 mL CH.sub.2Cl.sub.2 and immediately
homogenized in a 5-mL-syringe at 20,000 rpm for 1.5 min, creating
the first emulsion. Two mL of 5% PVA was immediately added to the
tube and the mixture was then homogenized for 45 s at 6,000 rpm and
the resultant emulsion was injected into 100 mL of 0.5% PVA under
continuous stirring. Microspheres were stirred for 3 h at room
temperature, and collected with sieves to separate by size (20-63
and 63-90 .mu.m) and washed thoroughly with ddH.sub.2O to help
remove residual PVA.
[0045] Coomassie (modified Bradford) protein assay: A modified
Bradford assay was used to determine protein concentrations.
Briefly, appropriate volume of standard or sample was mixed with
Coomassie Plus.TM. reagent (Thermo Fisher Scientific, Rockford,
Ill., USA) in a 96-well plate (Nalge Nunc International, Rochester,
N.Y., USA). Then, the absorbance was read at 595 nm within 30 min
using a Dynex II MRX microplate reader (Dynex Technology Inc.,
Chantilly, Va., USA).
[0046] Size exclusion-high performance liquid chromatography
(SE-HPLC) analysis of protein: Samples were injected into the TSK
Gel G3000SW (lysozyme, BSA and BSA-Coumarin) or G2000 SW (OVA)
column (7.8 mm i.d..times.30 cm long) column (Tosoh Biosciences
LLC, Montgomeryville, Pa., USA) attached with a Shodex.TM. Protein
KW-G guard column (Showa Denko, N.Y., USA), eluted by 0.05 M
potassium phosphate containing 0.2 M NaCl (pH 7.0) for (lysozyme
and OVA) or PBS (pH 7.4) for (BSA and BSA-Coumarin) at a flow rate
of 0.9 for (lysozyme, BSA and BSA-Coumarin) or 0.7 (OVA) mL/min. UV
detection at 215 and 280 nm and fluorescence detection with
excitation and emission wavelengths of 278 nm and 350 nm for BSA,
OVA and lysozyme, and 384 nm and 480 nm for BSA-Coumarin were
used.
[0047] High performance liquid chromatography (HPLC) analysis of
leuprolide acetate: Analysis of leuprolide acetate was accomplished
by HPLC, with a gradient of acetonitrile (Solvent A) and 0.05 M
sodium phosphate buffer, pH 7.0 (Solvent B) on a Nova-Pak C18
column (4 .mu.m, 3.9.times.150 mm, Waters Corporation, Milford,
Mass., USA). The gradient method was 0 min (20% A), 6 min (30% A),
9.5 min (37% A), 11.5 min (37% A), 16.5 min (50% A), and 19 min
(20% A), followed by a 2 min recovery. UV detection was measured at
215 nm and 280 nm, and fluorescence detection was performed at
excitation and emission wavelengths of 278 and 350 nm,
respectively.
[0048] Determination of active loading by protein disappearance
from the loading solution: To follow active loading (i.e., mass
fraction of encapsulated species in PLGA) as a function of time and
temperature of incubation, after various stages of the incubation,
the protein/active SM (Al(OH).sub.3-encapsulated) PLGA microsphere
mixtures were passed through a low protein binding PVDF membrane
filter (Millipore, Bedford, Massachusetts, USA) and remaining
protein (OVA or TT) in solution was analyzed by the modified
Bradford assay.
[0049] Determination of loading by polymer removal and recovery of
residual protein/peptide: For determination of soluble lysozyme,
BSA, and BSA-Coumarin, PLGA microspheres were dissolved in acetone
and dispersed for 1 h, centrifuged at 13,000 rpm for 10 min and the
supernatant was removed. Centrifugation/supernatant removal was
repeated 3-fold, and the residual solvent was removed via
concentrator (Vacufuge.TM. concentrator 5301 (Eppendorf
International, Hamburg, Germany)). The remaining protein was then
dissolved in 10 mM potassium phosphate buffer, pH 7.0 (lysozyme) or
PBS, pH 7.4 (BSA and BSA-Coumarin) and analyzed by SE-HPLC.
[0050] Leuprolide acetate content in self-encapsulated PLGA
microspheres was determined by two-phase extraction. Briefly, about
5 mg leuprolide acetate self-encapsulated PLGA microspheres (n=5)
were weighed into 5-mL glass vials. To these vials, 1 mL of
methylene chloride and 2 mL of 50 mM sodium acetate (pH 4.0) were
added, followed by vortexing for 1 min. One and a half mL of buffer
layer was removed, replaced with 1.5 mL of same buffer (2
extractions) or 50 mM sodium acetate+1M NaCl (3 extractions) and
extracted. The content of leuprolide acetate in each extracted
fraction was then analyzed and quantified by HPLC. Five extractions
were found to be sufficient to remove leuprolide completely from
PLGA microspheres as the HPLC peak of leuprolide acetate
disappeared by the 6.sup.th extraction.
[0051] To determine the OVA content in active self-encapsulated
PLGA microspheres, microspheres were dissolved in ethyl acetate and
centrifuged at 6,000 rpm for 5 min. The supernatant polymer
solution was removed and the aluminum hydroxide-OVA sediment was
washed twice with ethyl acetate. The sediment was then dried at
30.degree. C. using the Vacufuge.TM. to remove ethyl acetate. One
milliliter of 190 mM sodium citrate solution was added to the dried
aluminum hydroxide-OVA pellet, mixed thoroughly, and incubated at
37.degree. C. for 3 days with constant agitation. In control
studies, this duration was determined to be sufficient for complete
elution of OVA from the aluminum hydroxide gel. Then, the samples
were centrifuged at 6,000 rpm for 5 min and soluble OVA amount in
supernatant was analyzed by the SE-HPLC. To the remaining residue,
a reducing and denaturing solution (10 mM dl-dithiothreitol+6 M
urea+1 mM EDTA) was added to dissolve any aggregate and
centrifuged. The content of insoluble aggregate of OVA in
supernatant was analyzed by the modified Bradford assay (S1).
[0052] Enzyme-linked immunosorbent assay (ELISA): Antigenically
active TT was determined by the ELISA. Except for final incubation
step with p-nitrophenyl phosphate liquid substrate, all initial
ELISA steps were performed at room temperature. Briefly, 100 .mu.L
of 2-3 international units (IU)/mL of equine tetanus antitoxin in
PBS (pH 7.2) was added to 96-well microtitration plates (Nalge Nunc
International, Rochester, N.Y., USA) and incubated overnight. The
plates were washed 3-5 times between all steps with PBS containing
0.05% Tween.TM. 20 (pH 7.2). Phosphate blocking buffer (PBB,
PBS/0.5% BSA/0.05% Brij.TM. 35, pH 7.4) was used as a diluent for
all TT samples and antibodies (except equine tetanus antitoxin
above). Standard TT with known concentration and test samples were
diluted at 2-fold steps in coated plates using PBB as a diluent.
The plates were held for 2 h and washed. Then, 100 .mu.L of human
anti-TT IgG (HyperTET.TM. S/D, 1:5000 dilution) was added and
allowed to react for 2 h followed by 100 .mu.L of goat anti-human
IgG-alkaline phosphatase diluted 1:20000 in PBB for another 2 h.
The plates were washed and 100 .mu.l of p-nitrophenyl phosphate
liquid substrate was added. After 30 min incubation at 37.degree.
C., the absorbance was read at 405 nm on a Dynex II MRX microplate
reader (Dynex Technology Inc., Chantilly, Va., USA) equipped with
Revelation 4.21 Software. Log/Logit curve fitting model was used to
plot the standard curve and calculate unknown concentration of TT
in test samples. Evaluation of in vitro release of protein and
peptide from SM and TE PLGA microspheres: In vitro release of large
molecules from passive and active self-encapsulated PLGA
microspheres was determined by quantifying either the amount
released into the release medium (BSA, lysozyme, TT, and OVA) or
the amount of encapsulated species directly remaining in the
polymer (leuprolide acetate). Briefly, .about.4-10 (passive
BSA/lysozyme/leuprolide acetate SM PLGA microspheres) or 20 (active
OVA or TT self-encapsulated Al(OH).sub.3--PLGA microspheres) mg of
microspheres were incubated in either 0.5-1.5 mL of phosphate
buffered saline (PBS)+0.02% Tween 80 (PBST) (pH 7.4) or both PBS
and 190 mM sodium citrate (active OVA self-encapsulated
Al(OH).sub.3--PLGA microspheres) or PBST+0.2% BSA (active TT
self-encapsulated Al(OH).sub.3--PLGA microspheres) at 37.degree. C.
under constant agitation (100 rpm/min). Release medium was removed
and replaced with fresh buffer at pre-selected time points. Soluble
BSA/OVA/lysozyme in release media was quantified by SE-HPLC and
modified Bradford protein assay. Release of antigenic TT was
determined by ELISA. In order to determine the in vitro release of
leuprolide acetate, peptide remaining in the polymer after
incubation of specified period was determined by the method
described in the loading analysis. Cumulative amount of peptide
released was then calculated by subtracting the remaining amount of
peptide in the polymer from initial peptide content (S2).
[0053] Determination of soluble and insoluble lysozyme loading by
amino acid analysis (AAA): Determination of total and soluble
lysozyme content in microspheres and soluble lysozyme content in
other solution-based samples was performed by AAA. Briefly,
microspheres (.about.4 mg), soluble protein solutions, and standard
samples were weighed into clear glass ampoules in a total volume of
1.5 ml 6 N HCl. Ampoules were then sealed under light vacuum and
incubated at 110.degree. C. for 25 h. The hydrolyzate from each
vial was completely emptied into microcentrifuge tubes and each
vial was rinsed with 250 .mu.L water and emptied into respective
microcentrifuge tube. The resulting solution was then evaporated
under vacuum at room temperature. About 1 mL of 1.0 M sodium
bicarbonate buffer (pH 9.5) was weighed into each tube to
neutralize the remaining acid. For individual amino acid analysis,
a weighed amount of 350 .mu.L of hydrolyzed protein solution and
350 .mu.L of o-phthaldialdehyde reagent solution were combined in a
microcentrifuge tube, vortexed for 15 s, and immediately injected
onto a C18 column fitted with a guard column (total time from
mixing to injection<1 min), using a previously reported HPLC
method for AAA with o-phthaldialdehyde reagent (S3). Samples were
eluted (gradient elution) with mobile phases of methanol:water
(65:35) (A) and methanol:THF:50 mM phosphoric acid (20:20:960)
(titrated to pH 7.5 with 10 N NaOH) (B) at a flow rate of 1.4
mL/min. Gradient elution conditions were 40% A for 0.5 min, 17 min
concave gradient to 50% A, 15 min linear gradient to 100% A, 5 min
isocratic elution with 100% A, 7.5 min linear gradient to 40% A,
and isocratic 40% A for 5 min. Amino acids were detected by
measuring fluorescence respectively at an excitation and emission
wavelength of 350 and 455 nm. Protein and standards were quantified
using the average of the 3 individual amino acids standards found
to be stable under hydrolysis conditions: alanine, phenylalanine,
and lysine.
[0054] Scanning electron microscopy (SEM): Surface morphology of
microspheres was examined by taking SEM images using a Hitachi
53200N scanning electron microscope (Hitachi, Tokyo, Japan).
Briefly, microspheres were fixed previously on a brass stub using
double-sided adhesive tape and then were made electrically
conductive by coating, in a vacuum, with a thin layer of gold
(approximately 3 to 5 nm) for 60 s at 40 W. The surface view images
of microspheres were taken at an excitation voltage of 5-10 kV.
[0055] Confocal microscopy: Distribution of self-microencapsulated
protein in the PLGA microspheres was observed by taking confocal
images of BSA-coumarin loaded PLGA microspheres. Briefly, 1 mg of
BSA-coumarin loaded PLGA microspheres were suspended in 80 .mu.L of
water. A droplet of this suspension was placed on a clean glass
slide, a glass cover slip placed on top of the droplet, and excess
of water was removed. Samples were imaged at an excitation/emission
wavelength of 384/480 nm using a confocal microscope (Olympus
America Inc., Center Valley, Pa., USA).
[0056] Determination of polymer matrix porosity of SM
microencapsulating microspheres: Measurement of polymer matrix
porosity of blank SM microencapsulating PLGA microspheres was done
by Porous Materials, Inc. (Ithaca, N.Y., USA) using an AMP-60K-A-1
mercury porosimeter, generating pore volume versus pressure data.
The pore volume was reported as volume per gram microspheres
(cc/g). Total microsphere volume was calculated as the sum of the
pore volume and the polymer volume, where the polymer density (1.25
g/cc for 51 kDa PLGA 50:50, provided by manufacturer) and weight of
the porosimetry sample were used to calculate the pore volume.
Percent porosity was calculated as the pore volume per total
microsphere volume. Pressure associated with microspheres' packing
and surface wetting, before mercury intrusion into the pores had
taken place, was not calculated into the final pore volume as has
been reported previously (S4). The method of determination of
porosity utilized large amount of microspheres sample (.about.250
mg) and there was no significant difference (p>0.05) among the
different measurements (three measurements) of the same
formulation. Hence, only one test was run for the measurement of
polymer matrix porosity of various SM microspheres
formulations.
[0057] Kinetics of self-healing microencapsulation: About 50 mg of
SM PLGA 50:50 (M.sub.w=51 kDa) microspheres were placed into
separate tubes of 65 mg/mL dextran-FITC (10,000 M.sub.w).
Microspheres were incubated at 4.degree. C. for 20 h, and then
transferred to 42.5.degree. C. for 72 h, with approximately 10% of
microspheres removed at preset time points while replacing the
volume post sampling with fresh dextran-FITC solutions. Sample
microspheres were washed 10-fold with distilled water, with
centrifugation at 3200 rpm for 10 min to collect the microspheres
after each wash. The dextran-FITC was extracted using acetone to
dissolve the PLGA and concentrating the insoluble dextran-FITC
using centrifugation (10,000 rpm at 10 min), and repeating 3-fold.
Dextran-FITC was dissolved in PBS, pH 7.4, and loading was
determined via HPLC with fluorescence (without column separation)
using 20 or 40 .mu.L injection volume and a 1 mL/min PBS, pH 7.4
mobile phase. The fluorescence of the dextran-FITC was measured
respectively at an excitation and emission wavelength of 490 and
520 nm.
[0058] Evaluation of testosterone suppression in rats following
injection of leuprolide acetate self-encapsulated PLGA
microspheres: The ability of PLGA self-healing microencapsulation
to provide long-term in vivo release was evaluated by assessing
long-term testosterone suppression after a single injection of
leuprolide acetate self-encapsulated PLGA microspheres in male
Sprague-Dawley rats (S5-S8). The treatment of experimental animals
was in accordance with University committee on use and care of
animals (University of Michigan UCUCA), and all NIH guidelines for
the care and use of laboratory animals. Male Sprague-Dawley rats of
6 weeks old were housed in cages and given free access to standard
laboratory food and water, and allowed one week to acclimate prior
to study initiation. Animals were anesthetized with 2-4% isoflurane
administered by a calibrated vaporizer (Midmark, Orchard Park,
N.Y., USA). The leuprolide acetate self-encapsulated PLGA
microspheres (1.times.2-month dose), leuprolide acetate solution
(1.times.1-month dose), and blank SM PLGA microspheres without drug
(1.times. dose) in a liquid vehicle (1% w/v carboxymethylcellulose
and 2% w/v mannitol), and commercial 1-month Lupron Depot (Abbott
Laboratories, North Chicago, Ill., USA) (2.times. dosing at days 0
and 28) were subcutaneously injected at the back (lower neck
portion) of rats (6 animals/study group). Total dose of leuprolide
acetate was based on 100 .mu.g/kg/day. Animal body weight
considered for dosing leuprolide acetate was 425 g which is
projected body weight of male Sprague Dawley rat at midpoint (day
28) of the study (as per the weight (g)/age (weeks) curve given by
Charles River Laboratories). Blood samples were collected via
jugular vein stick before (day -7 and 0 for baseline testosterone
level) and after (1, 7, 14, 21, 28, 35, 42, 49, and 56 days)
injection of preparations. The collected blood samples were
immediately transferred to B-D Microtainer.TM. blood collection and
serum separation tubes previously incubated in ice, centrifuged at
8,000 rpm for 10 min, and then the serum was removed and stored in
microcentrifuge tubes at -20.degree. C. until further use. Serum
testosterone levels were assayed by radioimmunoassay using a
TESTOSTERONE Double Antibody-125I RIA Kit (MP Biomedicals LLC.,
Solon, Ohio, USA) at the University of Pennsylvania Diabetes Center
(Philadelphia, Pa., USA). Lowest detection limit of testosterone
was 0.1 ng/mL. In case of samples which exhibited testosterone
level below the detection limit, a 0.1 ng/mL value was used for
statistical evaluation and plotting the curve.
[0059] Sterilization of active SM (Al(OH).sub.3--PLGA) PLGA
microspheres with gamma irradiation: Active SM PLGA microspheres
were irradiated by using .sup.60Co as irradiation source (Michigan
Memorial Phoenix Project, University of Michigan) at 2.5 MRad dose
and 0.37 MRad/h dose rate. Briefly, about 250 mg active SM PLGA
microspheres were freeze-dried, placed in 5-mL ampoules and then
ampoules were sealed under vacuum. All the samples were irradiated
at room temperature.
[0060] Statistical Analysis: The results are expressed as
mean.+-.standard error of mean (n=3 or 5 or 6). An unpaired
Student's t-test was used to assess statistical significance
between numerous SM PLGA microsphere formulations with respect to
polymer porosity, protein and peptide loading, stability, and in
vitro release, and in vivo testosterone level. Results were
considered statistically significant if p<0.05.
[0061] Goodness of self-encapsulation--Effect of pore-forming
excipients/initial water phase volume on lysozyme loading and
initial burst release: The goodness of self-encapsulation in PLGA
microspheres as indicated by elevated lysozyme loading (open bar
chart) and minimal initial burst (closed bar chart) of enzyme
employing diversity of pore-forming excipients/initial water phase
volume to create the PLGA 50/50 pore network is shown in FIG.
4.
[0062] Limitations on molecular size--Self-microencapsulation of
dextran blue: As suggested by the open pore distribution in FIG. 1
(before and after loading lysozyme in SM microspheres) very large
molecules can penetrate the polymer pore network and become
encapsulated. Therefore, we tested the virtual upper limit of
molecular size of biomacromolecules by encapsulating TMR-dextran
(M.sub.w=2 MDa) relative to low molecular weight 4 kDa FITC-dextran
in SM-PLGA 50:50 (M.sub.w=51 kDa) microspheres (SM-3, Tables 1 and
2). At a constant solution concentration of 1 mg/mL, which is just
below the solubility of the TMR-dextran, both dyes were
successfully encapsulated at very similar levels as indicated by
the following data determined experimentally (five measurements):
SM PLGA microspheres self-encapsulated 0.30.+-.0.01 and
0.22.+-.0.01% w/w TMR-dextran and FITC-dextran 4 kDa,
respectively.
[0063] Self-healing microencapsulation of leuprolide acetate in
PLGA 50:50 (M.sub.w=51 kDa) microspheres (SM-5, Tables 1 and 2) is
shown in FIG. 5.
[0064] Protection of protein during microencapsulation--Improved
stability of lysozyme through self-microencapsulation: As lysozyme
is a common model protein for testing protein damage during
microencapsulation, we self-microencapsulated this enzyme and
monitored its loading in terms of monomeric, total, and
enzymatically active protein content. The stability of lysozyme
encapsulated via self-healing microencapsulation (SM-3 and SM-4,
Tables 1 and 2) was compared with the enzyme encapsulated via a
traditional w/o/w process (TM-1, Tables 1 and 2). Combined soluble
and insoluble lysozyme encapsulated was measured via AAA, and the
soluble fractions were determined by SE-HPLC.
[0065] Addition of sucrose, as a differentially soluble material,
either to inner water phase in the double emulsion (TE method) or
loading solution (self-microencapsulation method) resulted in
reduced lysozyme loading (% w/w lysozyme/polymer matrix). For
example, the lysozyme loading into medium M.sub.w PLGA (M.sub.w=51
kDa) microspheres prepared by TE with and without sucrose in WP was
0.65.+-.0.01 and 4.86.+-.0.04%, respectively (three measurements).
Whereas lysozyme loading into medium M.sub.w PLGA (M.sub.w=51 kDa)
microspheres by the self-encapsulation technique with and without
sucrose in loading solution was 3.39.+-.0.03 and 5.17.+-.0.08%,
respectively (three measurements).
[0066] The percentage (% w/w) of loaded lysozyme that exists as
intact soluble monomer was slightly higher for the
self-microencapsulated microsphere (SM-3, Tables 1 and 2)
formulations than the TM-1 formulations. In the absence of sucrose,
TM-1 and SM PLGA (M.sub.w=51 kDa) microspheres had 85.+-.2 and
90.+-.2% intact monomer (three measurements), respectively. In the
presence of sucrose in the loading solution, SM PLGA (M.sub.w=51
kDa) microspheres had 98.+-.2% of the loaded protein as intact
monomer (three measurements). Importantly, the fraction of total
lysozyme loaded as insoluble aggregates was significantly less
(p<0.05) for self-microencapsulation technique when compared to
TE method. For example, lysozyme loaded via TE method (TM-1, Tables
1 and 2) underwent 40.+-.4% and 12.+-.2% insoluble aggregation
(three measurements) with or without sucrose in the inner water
phase, respectively (FIGS. 2F and E). By contrast, the
self-microencapsulation technique respectively showed 8.+-.3 and
1.+-.2% insoluble aggregation (three measurements) without and with
sucrose in loading solution (FIGS. 2C and D) for the SM PLGA
(M.sub.w=51 kDa) microsphere formulation (SM-3, Tables 1 and
2).
[0067] Protection of protein (BSA and lysozyme) against
acid-induced protein instability during release from
self-encapsulated PLGA microspheres: The success of controlled
protein delivery vehicles based on PLGA significantly depends upon
the ability of the polymer to retain and release the stable form of
proteins under physiological conditions over extended release
times. Co-encapsulation of MgCO.sub.3 along with proteins in PLGA
50:50 by the TE method has been used to stabilize proteins against
the acidic polymer microclimate and released the proteins in a
stable form compared to control formulations without an ionic
affinity trap. As BSA is a common model protein for assessing
protein damage due to acid produced by the PLGA polyester during
long-term controlled release, we self-microencapsulated the protein
into SM-PLGA 50:50 microspheres containing MgCO.sub.3 as an
acid-neutralizer and porosigen with and without trehalose as an
additional porosigen (SM-3, Tables 1 and 2) and monitored its
loading, release from microspheres and acid-induced aggregated
protein content in the polymer. Blank SM PLGA 50:50 (M.sub.w=51
kDa) microspheres containing 3 and 4.5% w/w (MgCO.sub.3/polymer
matrix) MgCO.sub.3 self-microencapsulated 4.25.+-.0.05 and
5.65.+-.0.06% w/w (protein/polymer matrix), respectively (three
measurements). Both the self-microencapsulated formulations
exhibited a high initial burst (49-54% BSA release after 1-day),
which was then substantially reduced by co-encapsulation of
trehalose along with MgCO.sub.3 during the preparation of SM
microspheres (FIG. 6A). Importantly, analysis of the remaining
protein in the microspheres after 28 days of in vitro release
provided a mass balance (total recovery) between 109.+-.4 to
121.+-.2% (three measurements) and out of which <2% BSA was
aggregated.
[0068] Another model protein, lysozyme, was similarly
self-encapsulated in blank PLGA 50:50 (M.sub.w=51 kDa) microspheres
(SM-3, Tables 1 and 2) containing 0, 1.5, 4.3, 11.0% w/w
(MgCO.sub.3/polymer matrix). The amount of lysozyme
self-encapsulated in 0, 1.5, 4.3, 11.0% w/w MgCO.sub.3 loaded blank
PLGA 50:50 microspheres was 4.5.+-.0.2, 6.4.+-.0.1, 9.8.+-.0.3, and
8.7.+-.0.4% w/w (lysozyme/polymer matrix), respectively (three
measurements). The release rate of lysozyme from above mentioned
formulations was directly related to the amount of ionic affinity
trap (MgCO.sub.3) loaded into the blank PLGA 50:50 microspheres
(FIG. 6B). For example, after 28 days, the cumulative amount of
lysozyme released from self-microencapsulated microspheres
formulations was 30.+-.1, 41.+-.1, 51.+-.4, and 57.+-.4%
respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO.sub.3 loaded blank
PLGA 50:50 microspheres. The amount of protein remaining in the
microspheres after 28 days of release, including soluble monomer,
soluble aggregates, and insoluble aggregates, was similarly
quantified and >90% mass balance (total recovery) was achieved
for all four formulations. For example, total amount of lysozyme
recovered from self-microencapsulated microspheres formulations was
92.+-.2, 91.+-.1, 99.+-.4, and 110.+-.4% (three measurements)
respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO.sub.3 loaded PLGA
50:50 microspheres. Importantly, the total amount of soluble
protein, both released over 28 days and recovered as residual
soluble monomer, was significantly higher (p<0.05) for 4.3 and
11.0% w/w MgCO.sub.3-based self-encapsulated microspheres
formulations. For example, total (released+residual) amount of
soluble protein recovered for 0, 1.5, 4.3, and 11.0% w/w MgCO.sub.3
loaded PLGA 50:50 microspheres was 83.+-.2, 85.0.+-.1, 95.+-.4, and
107.+-.4%, respectively. Furthermore, the specific activity of the
residual lysozyme remaining in the self-encapsulated microspheres
after 28 days of release was analyzed. The specific activity was
calculated based upon the total amount of soluble protein analyzed,
both monomer and aggregated. The specific activity, given as the
percentage of the specific activity of the native, standard
lysozyme was 102.+-.6, 116.+-.19, 100.+-.5, and 97.+-.5%
respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO.sub.3 loaded PLGA
50:50 microspheres. Thus, the soluble lysozyme retained in the
self-microencapsulated microspheres after 28 days of release was
still completely active within experimental error for all
formulations.
[0069] Kinetics of PLGA self-encapsulation: In order to determine
the time required for self-healing of PLGA to encapsulate large
molecules, SM PLGA microspheres (SM-3, Table 1) were loaded with
FITC-dextran (SM-3, Table 2) and the loaded biomacromolecular dye
was analyzed at various times before and after initiating
self-encapsulation by increasing temperature from 4.degree. C.
(T<T.sub.g) to 42.5.degree. C. (T>T.sub.g). At each time
point, microspheres were washed extensively with ddH.sub.2O to
remove any unencapsulated dye. As shown in FIG. 1F, only background
levels of dextran were loaded when at the low temperature for 20 h.
However, after increasing temperature to 42.degree. C. (at time 0)
loading of two separate, but equivalent, formulations both
increased steadily over 12 h reaching a maximal and steady loading
value for 72 h. Hence, without any manipulation of the polymer
(e.g., by plasticization) self-healing time for this formulation
was on the order of 12 h. In addition, loading was completely
reproducible for the two identical formulations.
[0070] PLGA self-healing microencapsulation provides long-term
delivery of bioactive large molecules in vivo--Long-term
testosterone suppression in rats after single injection of
leuprolide acetate self-encapsulated PLGA microspheres: Leuprolide
acetate (M.sub.w=1209.6 Da) is commonly used in the treatment of
hormone-dependent cancers (e.g., prostate cancer) and gynecologic
disorders (e.g., endometriosis and precocious puberty).
Commercially available injectable PLGA microsphere-based
formulation of leuprolide (Lupron Depot.TM.) is prepared by the
traditional (water-in-oil-water) encapsulation method. The ability
of PLGA self-healing microencapsulation to provide long-term in
vivo release was evaluated by assessing long-term testosterone
suppression in male Sprague-Dawley rats (six rats/study group) in
comparison with commercial Lupron Depot.TM. and negative controls
(FIG. 2C). The initial serum testosterone level (1-2 ng/mL)
increased to .about.5 ng/mL after one day of subcutaneous injection
of Lupron Depot.TM., leuprolide self-encapsulated PLGA microspheres
and leuprolide solution. This initial elevation is typical to
treatment with luteinizing hormone-releasing hormone (LHRH)
agonists (e.g., leuprolide acetate) resulting from initial
stimulation of pituitary LHRH receptors and increased release of
luteinizing hormone (LH), thereby stimulating testicular
steroidogenesis and release of gonadotropins. The testosterone
levels fell below the castration level (0.5 ng/mL) within a week
and remained under that level for 6-7 weeks following a single
injection of leuprolide acetate self-encapsulated PLGA microspheres
and 8 weeks after two injections (day 0 and 28) of Lupron
Depot.TM.. Importantly, there was no significant serum testosterone
levels difference (p<0.05) between self-encapsulated PLGA
microspheres and commercial Lupron Depot.TM. over a period of 6
weeks, indicating equivalent potential of new PLGA self-healing
microencapsulation paradigm to provide long-term in vivo delivery
of peptide drugs. In contrast, leuprolide solution control because
of the short serum half-life of the peptide, failed to provide
testosterone suppression below the castration level during the
whole study duration. However, there was a significant serum
testosterone levels difference (p>0.05) between negative
controls (blank SM PLGA microspheres and leuprolide solution) and
long-term leuprolide delivery vehicles (self-encapsulated PLGA
microspheres and commercial Lupron Depot.TM.) over a period of 6-8
weeks.
[0071] Active self-microencapsulation of protein with high
encapsulation efficiency by Al(OH).sub.3--PLGA microspheres--Effect
of hydrophobic plasticizer: The encapsulation efficiency of SM PLGA
microspheres by the passive self-encapsulation method is 1.5-13%.
In addition, as partitioning of the proteins between polymer pores
and external solutions should be close to unity when loading by the
passive process, high external protein concentrations (e.g.,
>.about.100 mg/mL) were required previously to achieve
loading>1%. Therefore, to increase the encapsulation of
efficiency of SM PLGA microspheres and to reduce external protein
concentration, we added a known protein sorbing ionic affinity
trap, Al(OH).sub.3, in SM PLGA microspheres (ASM PLGA microspheres,
Table 1) and tested their potential to self-microencapsulate
different vaccine antigens (ovalbumin (OVA) and tetanus toxoid
(TT)), from low aqueous protein concentrations (0.5 or 0.8 or 1.0
mg/mL) (Tables 2-5).
[0072] As shown in Table 3, all the three ASM (ASM-1, ASM-2, and
ASM-3) PLGA microspheres sorbed the OVA from surrounding protein
solution within the incubation time compared blank porous PLGA
microspheres, suggesting active self-microencapsulation of protein
by ASM PLGA microspheres. Negative loading values were observed
with porous blank (no Al(OH).sub.3) microspheres which can be
attributed to the uptake of small amounts of water by the polymer
leaving behind more concentrated protein solution.
TABLE-US-00003 TABLE 3 OVA mass loss kinetics as a function of
incubation time from OVA/active SM Al(OH).sub.3-PLGA 50:50 (M.sub.w
= 51 kDa) microspheres mixture. Formulation code Temp. Remaining
OVA mass and t IM.sup.a in solution.sup.a (.mu.g)* OVA loaded
(.mu.g)* .degree. C. h (.mu.g) Blank ASM-1 ASM-2 ASM-3 Blank ASM-1
ASM-2 ASM-3 10 3 404 415 .+-. 5 325 .+-. 3 317 .+-. 6 319 .+-. 3
-11 .+-. 5 79 .+-. 3 87 .+-. 6 85 .+-. 3 6 404 415 .+-. 3 321 .+-.
4 310 .+-. 4 310 .+-. 1 -11 .+-. 3 83 .+-. 4 94 .+-. 4 94 .+-. 1 24
404 416 .+-. 2 315 .+-. 3 300 .+-. 10 301 .+-. 6 -12 .+-. 2 89 .+-.
3 104 .+-. 10 103 .+-. 6 48 404 425 .+-. 3 285 .+-. 3 285 .+-. 7
284 .+-. 4 -21 .+-. 3 119 .+-. 3 119 .+-. 7 120 .+-. 4 25 + 37 24 +
30 404 419 .+-. 6 203 .+-. 4 212 .+-. 5 197 .+-. 3 -15 .+-. 6 201
.+-. 4 192 .+-. 5 208 .+-. 3 Temp and t: incubation temperature
(.degree. C. ) and duration (h); IM: initial OVA mass in solution
.sup.avolume = 0.4 mL; *Mean .+-. SE, n = 6 ASM-1: 3.2% w/w
Al(OH).sub.3/3.5% w/w trehalose/0% w/w diethyl phthalate (DEP)/PLGA
microspheres ASM-2: 3.2% w/w Al(OH).sub.3/3.5% w/w trehalose/2.5%
w/w DEP/PLGA microspheres ASM-3: 3.2% w/w Al(OH).sub.3/3.5% w/w
trehalose/5% w/w DEP/PLGA microspheres
TABLE-US-00004 TABLE 4 OVA loading and self-microencapsulation
efficiency of active SM Al(OH).sub.3-PLGA 50:50 (M.sub.w = 51 kDa)
microspheres at different initial loading concentration. Initial
OVA Remaining OVA recovered Encapsulation OVA mass.sup.a OVA
mass.sup.a OVA loaded from polymer efficiency.sup.b Loading.sup.b
FC (.mu.g) (.mu.g)* (.mu.g)* (.mu.g)* (%)* (% w/w)* ASM-1 202.1 7.1
.+-. 2.4 195.0 .+-. 2.4 194.1 .+-. 2.2 96.1 .+-. 1.1 1.00 .+-. 0.01
404.1 203.2 .+-. 4.3 200.9 .+-. 4.3 197.9 .+-. 3.1 49.0 .+-. 0.8
0.98 .+-. 0.02 ASM-2 202.1 4.4 .+-. 0.4 197.7 .+-. 0.4 196.2 .+-.
0.3 97.1 .+-. 0.2 0.97 .+-. 0.01 404.1 211.8 .+-. 5.4 192.3 .+-.
5.4 190.5 .+-. 5.6 47.1 .+-. 1.4 0.90 .+-. 0.03 ASM-3 205.7 2.5
.+-. 1.6 203.2 .+-. 1.6 202.1 .+-. 1.3 98.3 .+-. 1.1 1.00 .+-. 0.05
404.1 196.6 .+-. 2.7 207.5 .+-. 2.7 203.5 .+-. 3.4 50.4 .+-. 1.7
1.00 .+-. 0.03 FC: formulation code; .sup.avolume = 0.4 mL; *Mean
.+-. SE, n = 6; .sup.bbased on the OVA content in the polymer
ASM-1: 3.2% w/w Al(OH).sub.3/3.5% w/w trehalose/0% w/w diethyl
phthalate (DEP)/PLGA microspheres ASM-2: 3.2% w/w Al(OH).sub.3/3.5%
w/w trehalose/2.5% w/w DEP/PLGA microspheres ASM-3: 3.2% w/w
Al(OH).sub.3/3.5% w/w trehalose/5% w/w % DEP/PLGA microspheres
[0073] With 400 .mu.g initial incubation mass of OVA from 1 mg/mL
OVA, the active ASM PLGA microspheres self-microencapsulated about
200 .mu.g (self-microencapsulation capacity=1% w/w (OVA/polymer
matrix)) thereby exhibiting self-microencapsulation efficiency of
about 50% (Table 4).
[0074] The similar loading of the three preparations (ASM-1, ASM-2,
and ASM-3) also indicated that the protein loading was governed by
the effective capacity of the Al(OH).sub.3 for sorbing OVA when the
external OVA content exceeded the Al(OH).sub.3 sorbing capacity
available in the polymer. Therefore, the encapsulation efficiency
was further increased by decreasing external OVA content to roughly
that encapsulated (-200 .mu.g) previously, that is, by incubating
microspheres with 200 .mu.g OVA from 0.5 mg/mL OVA. As expected,
ASM-1, ASM-2, and ASM-3 microspheres self-microencapsulated almost
the entire OVA mass, thereby exhibiting extraordinary
self-microencapsulation efficiency (96-98%) (Table 4). The
potential of active SM PLGA microspheres to actively
self-microencapsulate very sensitive vaccine antigen (TT) after
sterilization of blank microspheres with gamma radiations was
investigated and compared with the results obtained prior to
irradiation (Table 5). There was no significant difference in
active loading of TT before and after sterilization of active SM
PLGA microspheres, indicating the effectiveness of this novel
strategy to self-encapsulate vaccine antigens after terminal
sterilization of microspheres. For example, with 400 .mu.g initial
incubation mass of TT from 0.8 mg/mL loading solution, all the
active SM PLGA microsphere formulations self-encapsulated TT
equivalent to about 1.6% w/w polymer loading and 87% encapsulation
efficiency (3 measurements).
TABLE-US-00005 TABLE 5 Effect of gamma irradiation of active self-
microencapsulating PLGA microspheres on active loading and
encapsulation efficiency of tetanus toxoid (TT). Initial TT
Remaining Encapsulation mass.sup.a TT mass.sup.a TT loaded TT
Loading.sup.b efficiency.sup.b (.mu.g) (.mu.g)* (.mu.g)* (% w/w)*
(%)* Unencapsulated Al(OH).sub.3.sup.c 400 0.0 400.0 .+-. 0.0 -- --
Before irradiation ASM-1 400 53.7 .+-. 1.9 346.3 .+-. 1.9 1.62 .+-.
0.02 86.6 .+-. 0.5 ASM-2 400 52.2 .+-. 1.4 347.8 .+-. 1.4 1.66 .+-.
0.03 86.9 .+-. 0.4 After irradiation.sup.d ASM-1 400 52.8 .+-. 1.2
347.2 .+-. 1.2 1.64 .+-. 0.03 86.8 .+-. 0.3 ASM-2 400 53.2 .+-. 0.6
346.8 .+-. 0.6 1.61 .+-. 0.03 86.7 .+-. 0.2 .sup.avolume = 0.5 mL;
*Mean .+-. SE, n = 3; .sup.bbased on the TT mass loss from
solution; .sup.cmass of Al(OH).sub.3 = 0.6 mg ASM-1: 3.2% w/w
Al(OH).sub.3/3.5% w/w trehalose/0% w/w diethyl phthalate (DEP)/PLGA
microspheres ASM-2: 3.2% w/w Al(OH).sub.3/3.5% w/w trehalose/5% w/w
DEP/PLGA microspheres; .sup.dirradiation dose and dose rate were
2.5 MRad at 0.37 MRad/h.
[0075] The effect of encapsulation of Al(OH).sub.3 and blending of
hydrophobic plasticizer (diethyl phthalate (DEP)) on the
self-healing phenomenon of PLGA 50:50 (M.sub.w=51 kDa) microspheres
is shown in FIG. 7.
[0076] It is noted that the Al(OH).sub.3 gel appeared to suppress
self-healing as pores still appeared after incubation of the active
SM PLGA microspheres at 43.degree. C. (FIG. 7) as compared to full
healed microspheres prepared without Al(OH).sub.3 gel (FIG. 7).
Hence, we sought to increase PLGA mobility by incorporating a
hydrophobic plasticizer in the polymer. For example, diethyl
phthalate (DEP) was blended with the polymer while preparing blank
Al(OH).sub.3--PLGA microspheres. As expected, with increasing
amount of hydrophobic plasticizer in PLGA, pore-closing
(self-healing) at 37.degree. C. was clearly visible in FIG. 7,
indicating that any suppression of self-healing by Al(OH).sub.3
could be overcome by the hydrophobic plasticizer.
[0077] It can be emphasized that SM PLGA microspheres with
respectively 3.2, 3.5, and 5% w/w of Al(OH).sub.3, trehalose and
DEP were found to be an optimal formulation for active self-healing
microencapsulation of protein at physiological temperature
(37.degree. C.) with high encapsulation efficiency. Moreover, the
successful employment of DEP to reduce the required temperature for
self-healing opens-up the door to self-microencapsulate
temperature-sensitive molecules in higher M.sub.w PLGA at or below
physiological temperature.
[0078] Evaluation of quality of active protein
self-microencapsulation in Al(OH).sub.3--PLGA microspheres:
Evaluation of quality of active protein self-encapsulation in
Al(OH).sub.3--PLGA (M.sub.w=51 kDa) (ASM PLGA) microspheres was
tested first in 190 mM sodium citrate solution, a buffer commonly
used to elute protein antigens completely from Al(OH).sub.3
adjuvant within 3 days. The release of OVA from active
self-encapsulated Al(OH).sub.3--PLGA microspheres was significantly
different (p<0.05) compared to unencapsulated Al(OH).sub.3 (FIG.
8). For example, ASM-3 PLGA microspheres (3.2% w/w
Al(OH).sub.3/3.5% w/w trehalose/5% w/w DEP/PLGA microspheres)
largely retained OVA (i.e., 60-73%) after 1-day of exposure to the
citrate buffer, whereas unencapsulated Al(OH).sub.3 released all
the protein (97.+-.0.8% release). In addition, ASM-3 PLGA
microspheres released OVA slowly in a controlled manner over a
period of 10 days (48.+-.4.4% release (three measurements) after 10
days), indicating an effective active self-encapsulation of protein
in Al(OH).sub.3--PLGA microspheres. After 10 days of release
duration, the remaining OVA (soluble and insoluble) in ASM-3 PLGA
microspheres was recovered as described in the OVA recovery method.
After 10 days of release, 47.+-.6.4% soluble monomer and
5.8.+-.0.8% insoluble aggregate (covalent and non-covalent) was
recovered from ASM-3 PLGA microspheres with a total recovery of
100.8.+-.0.8% (three measurements).
[0079] Long-term controlled release of proteins from ASM PLGA
(Al(OH).sub.3--PLGA) microspheres: The potential of PLGA active
self-encapsulation to provide long-term release of stable proteins
was evaluated by assessing OVA monomer and antigenic TT release
from unencapsulated Al(OH).sub.3 and active self-encapsulated
without (ASM-1) and with (ASM-3) 5% w/w DEP-Al(OH).sub.3--PLGA
microspheres (Tables 3-5) in PBS (pH 7.4) (FIG. 3C) or PBS+0.02%
Tween.TM. 80+0.2% BSA (FIG. 3D) at 37.degree. C. The release of OVA
monomer or antigenic TT from ASM-3 PLGA microspheres (3.2% w/w
Al(OH).sub.3/3.5% w/w trehalose/5% w/w DEP/PLGA microspheres) was
also significantly different (p<0.05) than unencapsulated
Al(OH).sub.3 (FIGS. 3C and D). For example, OVA-Al(OH).sub.3
control gel exhibited 76, 90, and 99% OVA monomer release and
TT-Al(OH).sub.3 control gel exhibited 87, 95, and 98% antigenic TT
release respectively after 1, 3, and 7 days. In contrast, ASM-3
PLGA microspheres exhibited very less initial burst (17% OVA
monomer or 32% antigenic TT release after 1 day) and provided slow
and continuous release of OVA monomer or antigenic TT over a period
of 28 days (49 and 68% OVA monomer or 83 and 99% antigenic TT
release respectively after 14 and 28 days). After 28 days of
release, 19.+-.3% soluble OVA monomer and 10.+-.2% insoluble OVA
aggregate (covalent and non-covalent) was recovered from ASM-3 PLGA
microspheres with a total recovery of 98.+-.3% (three
measurements).
[0080] As the microencapsulation of vaccine antigens in
Al(OH).sub.3--PLGA microspheres can be easily performed by simple
of mixing of vaccine antigens/Al(OH).sub.3--PLGA microspheres and
heating the mixture to physiological temperature (no harsh
manufacturing conditions), this new approach opens-up a new mode
for sustained vaccine delivery similar to a injectable peptide
formulation (e.g., Lupron depot), thereby improving the stability
and efficacy of vaccine antigens. Note that injectable PLGA
microspheres represent an approach to control the release of
vaccine antigens to reduce the number of doses in the immunization
schedule and optimize the desired immune response via selective
targeting of antigen to antigen presenting cells.
[0081] Accordingly, the present technology provides delivery
systems having high encapsulation efficiencies for one or more
various agents. The delivery system can include an ionic affinity
trap, such as a metal salt, in combination with a solid polymer
matrix to enable a large portion or substantially the entire amount
of an agent outside the polymer to enter pores in the polymer, for
example, where the agent is subsequently encapsulated following
self-healing of the polymer.
[0082] The delivery system includes a solid polymer matrix. The
solid polymer matrix can include a polymer, such as a self-healing
polymer, and can include one or more pores, an ionic affinity trap,
an agent, and anything else associated with the delivery system.
For example, the solid polymer matrix can include a self-healing
polymer that includes one or more pores including an ionic affinity
trap that can be used to sorb an agent, where the pores are then
partially or completely closed to encapsulate the agent and prevent
it from leaving the pore(s). The polymer can include a porous
self-healing polymer that is able to alter its shape following a
treatment. For example, open pores within the self-healing polymer
can partially or completely close following a temperature change to
sequester contents of the pores. Encapsulation efficiencies greater
than 99% can be achieved. A further advantage is that the solution
concentration of the agent to be encapsulated can be less than the
concentration used in passive methods; e.g., about 1 mg/mL of agent
in the present methods versus roughly 100 mg/mL or greater amounts
of agent in passive methods. This opens the door to encapsulate an
agent having a low aqueous solubility and/or an agent that is only
available in limited quantities. Likewise, the amount of residual
agent left in the solution following encapsulation can be
significantly decreased, thereby making the present methods more
proficient and economical. In some embodiments, the delivery system
comprises a self-healing polymer, where a portion of the
self-healing polymer comprises ionized end groups and an agent
sorbed to the ionized end groups.
[0083] Various embodiments of the present technology can include
the following aspects. The self-healing polymer can be
biodegradable and can degrade or erode over time. The ionic
affinity trap may also act as an adjuvant in some cases. The
delivery system can further include the use of a plasticizer to
plasticize the polymeric material and manipulate the temperature at
which self-healing begins to occur. Control of these properties can
be important for encapsulation of certain agents that may be
damaged at elevated temperatures (e.g., 43.degree. C. or higher)
commonly used with moderate molecular weight self-healing
polymers.
[0084] In some embodiments, the delivery system can include a
porous self-healing polymer, optionally a differentially soluble
material such as a saccharide or disaccharide, and one or more
ionic affinity traps and plasticizers. The differentially soluble
material can be employed to obtain a porous self-healing polymer
network and/or to stabilize the polymeric material. The ionic
affinity trap and plasticizer can improve the encapsulation
efficiency and the self-healing property of the polymer,
respectively. A self-healing polymer having a porous network, such
as PLGA microspheres, can be prepared using established methods,
such as those described in U.S. Pat. Appl. Pub. 2008/0131478 to
Schwendeman et al.; Sophocleous et al., J. of Controlled Release
137 (2009) 179-184; Kang et al., Molecular Pharmaceutics, vol. 4,
no. 1, 104-118 (2007); Cui et al., Vaccine 25 (2007) 500-509; and
Jiang et al., Advanced Drug Delivery Reviews 57 (2005) 391-410.
[0085] In accordance with the present methods, a solution
comprising an agent is placed in contact with a self-healing
polymer having pores or one that can form pores when in contact
with the solution. At the same time or following a soaking period,
the self-healing polymer experiences a condition that causes
spontaneous polymer chain rearrangement, which in turn causes the
accessible pores (pores having access to the polymer surface) to
close. The agent becomes entrapped, encapsulated, or absorbed
within the self-healing polymer when these pores close.
[0086] In some embodiments, the self-healing polymer can be:
poly(dicyclopentadiene); poly(dimethyl siloxane); poly(diethoxy
siloxane); furan-maleimide-based polymers; dicyclopentadiene-based
polymers; anthracene-maleimide based polymers;
1,1,1-tris-(cinnamoyloxymethyl)ethane (TCE)-based polymers;
poly(ethylene-co-butylene); methyl methacrylate (MMA) embedded
polypropylene fibers; epoxy with a urea-formaldehyde microcapsule;
ionomers including hydrocarbon polymers bearing pendant carboxylic
acid groups that are either partially or completely neutralized
with metal or quaternary ammonium ions (e.g., Surlyn 8920, Surlyn
8940, Nucrel 960, and Nucrel 925); epoxy resins-diglycidyl ether of
bisphenol A, diglycidyl ether of bisphenol F; and combinations
thereof. Also included are self-healing polymeric materials having
reactive furfuryl functionality, including poly(furfuryl
methacrylate) and poly(furfuryl methacrylate)-co-poly(methyl
methacrylate), as described by Kavitha et al., Applied Materials
& Interfaces, vol. 1, no. 7, 1427-1436 (2009). Also included
are self-healing polymeric materials based on furan-functionalized,
alternating thermosetting poly ketones and bis-maleimide, as
described by Zhang et al., Macromolecules 2009, 42, 1906-1912. The
polymeric material can also be a biodegradable material. And the
polymeric material can be in several forms, including particles
such as particles, including microspheres, and various shapes of
tissue engineering scaffolds.
[0087] In some embodiments, the self-healing polymer can be
copolymers of lactic acid and glycolic acid (PLGA) and related
copolymers, including any polymer containing a polyester with
lactic and/or glycolic acid repeat units. The polymers may be made
using any method, and may be linear, star, branched, cross-linked,
or any configuration so long as the polymer has lactic and/or
glycolic repeat units, which may be liberated by hydrolysis. In
accordance with the methods described herein, the pore-containing
polymers may be preformed prior to the encapsulation step; i.e.,
the microparticles, microspheres, tissue engineering scaffold,
nanoparticles, drug-eluting stent, suture, or screw may be formed
according to known methods prior to contact with the agent to be
encapsulated.
[0088] PLGA is a polyester composed of one or more of three
different hydroxy acid monomers, d-lactic, l-lactic, and/or
glycolic acids. In general, the polymer, can be made to be highly
crystalline (e.g., poly(l-lactic acid)), or completely amorphous
(e.g., poly(d,l-lactic-co-glycolic acid)), can be processed into
most any shape and size (e.g., down to <200 nm), and can
encapsulate molecules of virtually any size. PLGA microspheres and
other injectable implants have an established safety record and are
used in several different marketed products from various companies
worldwide. For example, these controlled-release products are
capable of controlling the release of peptides and proteins slowly
and continuously from about 1 to 6 months, or even longer.
[0089] In addition to the depot effect, smaller PLGA microparticles
(e.g., less than 10 .mu.m) have demonstrated adjuvant activity via
their uptake by macrophages and dendritic cells (DCs), and their
localization in lymph nodes, and to induce cytotoxic T lymphocyte
(CTL) responses. For example, despite the biocompatibility of PLGA,
the mild inflammatory response produced by PLGA microspheres is
hypothesized as being involved in their adjuvant characteristics.
Most significant are reports of long-lasting antibody responses,
many neutralizing above protective levels, in numerous animal
models following a single dose of PLGA microparticle encapsulated
antigens including displays of immunological memory after 1 year of
immunization and protection against challenge.
[0090] PLGA can be in the form of particles, such as microspheres,
which can be prepared using a double emulsion-solvent evaporation
microencapsulation method. For example, PLGA along with a
saccharide or disaccharide, such as trehalose, and CH.sub.2Cl.sub.2
can be homogenized at 10,000 rpm using a Tempest IQ2 homogenizer
(The VirTis Company, Gardiner, N.Y.) equipped with a 10 mm shaft in
an ice/water bath for 1 min to prepare the first emulsion. Two
milliliters of 5% (w/v) PVA solution can then be immediately added
to the first emulsion and the mixture vortexed (Genie 2, Fisher
Scientific Industries, Inc., Bohemia, N.Y.) for 15 sec. to produce
the w/o/w double emulsion. The resulting emulsion can be poured
into 100 mL of 0.5% (w/v) PVA solution under rapid stirring and
hardened at room temperature for about 3 hours. Hardened
microspheres can be collected by centrifugation, washed three times
with purified water, and freeze-dried. For freeze-drying, samples
can be flash-frozen in liquid nitrogen and placed on a Freezone 6
freeze-drying system (Labcono, Kansas City, Mo.) at
133.times.10.sup.-3 mbar or less vacuum at a condenser temperature
of -46.degree. C. for 48 hours. Three percent MgCO.sub.3 powder
(w/w of polymer) can suspended in the polymer solution before
encapsulation when ionic affinity trap-containing PLGA microspheres
are desired.
[0091] Morphology and size distribution of the microspheres can by
ascertained by scanning electron microscopy. For example,
microspheres can be first coated with gold for 200 sec. by a vacuum
coater (Desk II, Denton Vacuum, Inc., Hill, N.J.). Microsphere
morphology can then be observed using a scanning electron
microscope (S3200N Variable Pressure SEM, Hitachi) with a voltage
of 15 keV. For size distribution analysis, the size of more than
200 particles can be measured from SEM micrographs and the
weight-averaged mean radius of the microspheres can be calculated.
To observe the microsphere cross section, polymer specimens can be
pre-cut by a razor blade on a glass slide before coating with
gold.
[0092] In some embodiments, the delivery system can be prepared as
follows. PLGA microparticles loaded with an ionic affinity trap
(e.g., Alhydrogel) can be prepared by a water/oil/water (w/o/w)
emulsion method. Briefly, alhydrogel can be concentrated to the
desired concentration by removing water. Trehalose solutions
(double the required concentration) can be prepared by dissolving
the required amount in 50 mM succinate buffer. Alhydrogel and
trehalose can then be mixed at 1:1 (v/v) ratio. Polymer solution (1
mL) can be prepared by dissolving the required amount of PLGA (250
or 350 mg/mL) in methylene chloride. About 200 or 300 .mu.L of
initial water phase can be added to PLGA solution followed by
homogenization at 17,000 rpm for 60 seconds. Then, 2 mL of 5% PVA
(9-10 k, 80% hydrolyzed) can be immediately added to the mixture
and vortexed for 50 seconds. The resulting emulsion can then be
poured into 100 mL of 0.5% PVA (9-10 k, 80% hydrolyzed) under
continuous stirring. The resulting microspheres can be stirred for
about 3 hours at room temperature, collected and washed thoroughly
by passing through sieves of different mesh size. Microspheres can
then be flash-frozen with liquid nitrogen and immediately
freeze-dried.
[0093] Before encapsulation of an agent, the self-healing material
comprising the self-healing polymer can be acceptably terminally
sterilized (e.g., by gamma irradiation) with little or no loss in
polymer molecular weight. A sterile solution of agent and sterile
microspheres of polymeric material can then be combined to effect
encapsulation. In some cases, a sterile agent solution can be added
to sterile and dry microspheres.
[0094] During microsphere formation, the polymer can be subjected
to numerous stresses (e.g., excess heat, mixing, etc.) that
normally cannot be used after loading the agent because certain
agents (e.g., peptide/protein/DNA) may degrade under such
conditions. In addition, the element of control over the ultimate
polymeric material morphology and the kind of microsphere prepared
can be vastly increased if encapsulation is performed after
microsphere (scaffold) preparation.
[0095] In some embodiments, the self-healing polymer can be in
various configurations, and is not limited to particles or
microspheres, for example. The self-healing polymer can be formed
in various shapes and articles of various sizes. For example, the
self-healing polymer can be used as a polymer coating on
drug-eluting stents, prepared as nanopartices, and formed into
tissue engineering scaffolds, including shapes that replace
portions of tissue or shapes that conform to various tissues. For
example, the materials and methods provided herein are also
applicable to self-healing materials used as tissue engineering
scaffolds, as well as any type of biomaterial or any other polymer
encapsulation system (e.g., agricultural) that requires the need to
encapsulate molecules that do not strongly partition into the
polymer phase, but instead are encapsulated within the pores of a
self-healing polymer. This is particularly useful for aqueous-based
materials such as biological materials present in their native
state in aqueous solution.
[0096] To load the microspheres, the microspheres can be incubated
in an aqueous solution comprising an agent, where the agent can be
at a fairly low concentration (e.g., about 0.5 to 1 mg/mL). The
aqueous mixture of polymeric material and agent can then be
incubated between about 10.degree. C. to about 43.degree. C. over a
period of time, for example, ranging from hours to days. During
this incubation, the porous network of the polymeric material heals
thereby encapsulating the agent with a high encapsulation
efficiency (e.g., >99%). Incubation temperature for loading the
agent into the delivery system varies as the composition of the
polymeric material varies; e.g., with or without an plasticizer.
The incubating temperature can be tuned from about 10.degree. C. to
about 43.degree. C. to achieve a very high encapsulation efficiency
of the agent and to improve the self-healing property of the
polymer. With the use of plasticizer, for example, self-healing of
a moderate molecular weight polymer can occur readily at about
37.degree. C. instead of about 43.degree. C.
[0097] Thus, the self-healing polymer can be combined with a
solution comprising an agent at a relatively low concentration,
where nearly the entire amount of agent is taken up into the
polymer pores, and the agent is then encapsulated within the
polymer following self-healing of the polymer, where each step
includes applying an appropriate temperature adjustment.
[0098] In some embodiments, the encapsulation efficiency (weight of
agent encapsulated/weight agent in solution exposed to polymer) is
greater than 50%. In some embodiments, the encapsulation efficiency
is greater than 60%. In some embodiments, the encapsulation
efficiency is greater than 70%. In some embodiments, the
encapsulation efficiency is greater than 80%. In some embodiments,
the encapsulation efficiency is greater than 90%. In some
embodiments, the encapsulation efficiency is greater than 95%. And
in some embodiments, the encapsulation efficiency is greater than
99%.
[0099] A differentially soluble material, such as a saccharide or
similar material, can be used in forming the pores in the
self-healing polymeric material. For example, after forming
microparticles from a mixture of saccharide and polymer, the
saccharide portion can be dissolved without dissolving the polymer
to leave empty pores in the polymer. Useful saccharides include
disaccharides such as trehalose, sucrose, and lactose. Other
saccharides that can be used include polysaccharides, such as
dextran, and glycosaminoglycans, such as heparin. The saccharide
can be used to stabilize an ionomer gel used as the ionic affinity
trap, for example, as described by A L Clausi, S A Merkley, J F
Carpenter, T W Randolph, J Pharm Sci 97, 2049 (2008). Other
pore-forming saccharides that can be used include mannose and
mannitol.
[0100] In some embodiments, the differentially soluble material can
include a water-soluble osmotic material in order to create the
pores in a porous self-healing polymer. For example, Mg and Al
salts can be used to create a percolating pore network; i.e., pores
that interconnect with the surface of the polymer. Other basic
salts are described by Zhu et al. in Pharmaceutical Research, Vol.
17, No. 3, 2000. For example, useful components for making pores
and/or stabilizing proteins include those described in S. E.
Bondos, A. Bicknell, Analytical Biochemistry 316 (2003) 223-231.
Examples of such materials that may promote protein solubility
include: kosmotropes including MgSO.sub.4 at 0-0.4 M,
NH.sub.4SO.sub.4 at 0-0.3 M, Na.sub.2SO.sub.4 at 0-0.2 M,
Cs.sub.2SO.sub.4 at 0-0.2 M; weak kosmotropes including NaCl 0-1 M,
KCl 0-1M; Chaotropes including CaCl.sub.2 0-0.2 M, MgCl2 0-0.2 M,
LiCl 0-0.8 M, RbCl 0-0.8 M, NaSCN 0-0.2 M, NaI 0-0.4 M, NaClO4
0-0.4 M, NaBr 0-0.4 M, Urea 0-1.5 M; Amino acids including glycine
0.5-2%, L-arginine 0-5 M; sugars and polyhydric alcohols including
Sucrose 0-1M, Glucose 0-2 M, Lactose 0.1-0.5 M, Ethylene glycol
0-60% v/v, Xylitol 0-30% w/v, Mannitol 0-15% w/v, Inositol 0-10%
w/v, Sorbitol 0-40% w/v, Glycerol 5-40% v/v; Detergents including
Tween 80 0-0.2% w/v, Tween 20 0-120 lM, and Nonidet P-40 0-1%; and
combinations thereof.
[0101] Ionic affinity traps used to sorb an agent in the present
delivery systems include bases such as metal salts and metal
hydroxides. The ionic affinity trap may also be in the form of a
gel and can include various ionomers. For example, aluminum
hydroxide and calcium phosphate gels are known as ionomers. Of the
various metal salts and hydroxides, aluminum hydroxide and aluminum
phosphate are two such ionic affinity traps that are particularly
useful. Some ionic affinity traps may further act as an adjuvant to
stimulate the immune system and increase the response to a vaccine.
For example, where the agent is a macromolecule such as a protein
antigen, the ionic affinity trap may also perform as an adjuvant
when the delivery system is used for vaccination.
[0102] In some embodiments, the ionic affinity trap can include
aluminum hydroxide and/or aluminum phosphate. Commercial forms of
aluminum hydroxide (Alhydrogel.TM., 2%) and aluminum phosphate
(Adju-Phos.TM.) are available from Accurate Chemical and Scientific
Corporation (Westbury, N.Y.). These aluminum materials can also act
as adjuvants when the present delivery system is used with or as a
vaccine. Aluminum hydroxide and aluminum phosphate can be prepared
by exposing aqueous solutions of aluminum ions to alkaline
conditions under very controlled circumstances, which in the case
of aluminum phosphate takes place in the presence of phosphate
ions. Various soluble aluminum salts can be used for the production
of the ionic affinity trap, but the experimental
conditions--temperature, concentration and even rate of addition of
reagents--can strongly influence the results. Other metal salts
that can be used as an ionic affinity trap include those disclosed
in Zhu et al. in Pharmaceutical Research, Vol. 17, No. 3, 2000.
[0103] Colloidal or sub-colloidal suspensions of aluminum hydroxide
can be characterized by particle size distribution, electrical
charge, and the hydrated colloid nature of the precipitate formed.
Alterations of the preparation recipe can give rise to various
forms of aluminum hydroxide which differ with respect to their
physico-chemical characteristics, stability and protein
adsorption.
[0104] Several models for the structure of aluminum hydroxide
exist. One model takes form in a ring-structure of six members,
each member consisting of an Al.sup.3+ surrounded by six
coordinated water molecules in an octohedral shape. The coordinated
water molecules are oriented with the oxygen toward the aluminum
ion. The high charge of the Al.sup.3+ is believed to weaken the
bond between oxygen and hydrogen thus facilitating the removal of
protons, especially under alkaline conditions. Deprotonization,
thus facilitated by the alkalinity, is believed to lead to the
initial formation of dimers by dihydroxyl bridges between
octohedras and later to the formation of the six-membered
ring-structure and even larger structures. In this process, the
ratio of aluminium to hydroxide approaches 1:3. On the basis of
this model the chemical formula Al(OH).sub.3 is misleadingly
simple. The model thus described is a generalized model that does
not consider crystalline forms or inclusion of other ions. When
inclusion of other ions, originating from the salts used in the
preparation, is taken into consideration aluminium hydroxide
precipitated from aluminium chloride can be described as
Al(OH).sub.2.55(Cl).sub.0.45, existing as a polymer of ten fused
six-membered rings and if precipitated from sulphate as
Al(OH).sub.2.30(SO4).sub.0.35 and based on three fused such
rings.
[0105] When X-ray crystallography and IR spectroscopy are applied
to aluminium gel preparations, a boehmite-like (aluminium
oxyhydroxide) pattern is seen in preparations known as aluminium
hydroxide, whereas commercialized aluminium phosphate gel adjuvant
is identified as amorphous aluminium hydroxyphosphate. It is
possible to calculate an average primary crystallite size of 4.5
nm.times.2.2 nm.times.10 nm for boehmite preparations.
[0106] The ionic affinity trap can bind agents such as proteins,
including protein antigens, where the ionic affinity trap can be an
aluminum salt adjuvant. Without the use of such adjuvants, proteins
may be only weakly immunogenic. Aluminum salts are currently the
only adjuvants generally approved for use in vaccines for humans.
Despite their approved use, the mechanism of action is still poorly
understood. Among a variety of nonmutually exclusive proposed
mechanisms, roles as depots for antigen induction of inflammatory
responses and delivery of antigen into antigen presenting cells are
proposed.
[0107] The two most common aluminum salts employed as adjuvants are
the phosphate and hydroxide forms. The salts themselves have been
well characterized with aluminum hydroxide (Alhydrogel.TM.) can
usually be found in a crystalline state, whereas aluminum phosphate
can exist in an amorphous form. The points of zero charge
(analogous to the isoelectric point of a macromolecule) are 4.0-5.5
and about 11 for the phosphate and hydroxide salts, respectively.
In general, proteins seem to better adsorb to the oppositely
charged salts through simple electrostatic effects, although apolar
and ion displacement interactions may play a role as well.
[0108] What happens to the structure and stability of proteins when
they are adsorbed onto the surface of these two commonly employed
aluminum salts is not well characterized. When proteins are
adsorbed to solid surfaces, highly polar (including charged)
interfaces tend to minimally perturb protein structure and
stability, although exceptions are known. In contrast, more apolar
surfaces often significantly alter the structure and stability of
many proteins. The effect of the aluminum salt on protein structure
and stability is important from several perspectives. For example,
to the extent that epitopes are conformational in nature, their
retention (or alteration) may be critical for vaccine
immunogenicity. The stability of protein antigens is equally
important when they are stored for long periods prior to their use.
The latter strongly impacts the utility of vaccines for use in the
developing world, where shipping under cold conditions can be
problematic, and in the use of vaccines against potential
bioterrorism agents, where storage for long periods in centralized
locations may be critical for their effectiveness.
[0109] In some embodiments, the ionic affinity trap can be calcium
phosphate. Calcium phosphate can function as an adjuvant and can be
used to potentiate the immune response of vaccines and to prepare
adsorbed allergen extracts. It can be well tolerated and readily
resorbed by the body and it is believed to potentiate the immune
response by the depot mechanism whereby the antigen is adsorbed
during the preparation of the vaccine and slowly released following
administration. Calcium phosphate is also believed to act by
presenting the adsorbed antigen to antigen presenting cells as a
particulate antigen.
[0110] Calcium phosphate has a molecular composition close to
Ca.sub.3(PO.sub.4).sub.2, where the calcium/phosphorus molar ratio
(Ca/P) can vary from 1.35 to 1.83 depending on the rate of mixing
during the precipitation reaction. The properties of the
precipitate are strongly dependent on the precipitation conditions.
For example, calcium phosphate precipitated by rapid mixing can
adsorb about 100% of diphtheria toxoid while calcium phosphate
precipitated by slow mixing can adsorb about 58% of the same dose
of diphtheria toxoid.
[0111] Preparations of calcium phosphate are available from Reheis
Inc. (Berkeley Heights, N.J.) and Brenntag Biosector
(Frederikssund, Denmark).
[0112] Although its name suggests that it is
Ca.sub.3(PO.sub.4).sub.2, X-ray diffraction, FTIR spectroscopy,
thermal analysis, and the Ca/P molar ratio identify commercial
calcium phosphate as non-stoichiometric hydroxyapatite,
Ca.sub.10-x(HPO4).sub.x (PO4).sub.6-x(OH).sub.2-x, where x varies
from 0 to 2. The surface charge is also pH-dependent (point of zero
charge=5.5). Consequently, commercial calcium phosphate exhibits a
negative surface charge at physiological pH and electrostatically
adsorbs positively charged materials, such as positively charged
antigens. The presence of hydroxyls can further allow calcium
phosphate to adsorb phosphorylated antigens by ligand exchange with
surface hydroxyls.
[0113] Another useful ionic affinity trap is aluminum phosphate.
And in some embodiments, the ionic affinity trap can be alum, which
includes aluminum and potassium.
[0114] Other examples of ionic affinity traps include extracellular
matrix-like materials, including dextran sulfate, chitosan, and
hyaluronic acid.
[0115] In some embodiments, there are a number of different ways to
actively load the polymer. (1) Layer-by-layer assembly based on
charge. For example, start with a negatively charged agent, such as
heparin, inside the polymer pores and then bind heparin-binding
growth factors; e.g., fibroblast growth factors and vascular
endothelial growth factors. Alternating incubations of growth
factor and heparin create a network of growth factor stabilized in
between heparin layers; i.e., heparin-growth factor-heparin-growth
factor and so on. (2) Creating a gradient of a substance from
inside the polymer to outside that causes the protein to
precipitate in the polymer; e.g., with ammonium sulfate inside the
polymer. (3) Placing a nucleating agent for crystallization inside
the polymer at a concentration above saturation; e.g., using
features as described in U.S. Pat. No. 5,869,604. This includes
using exogenous nucleating agents such as minerals, transition
metal ions such as copper and lead, highly absorbent structures
such as zeolites, preformed crystal seeds of amino acids, and
preformed crystal seeds of polypeptides other than the agent being
loaded into the pores. (4) Creating a Donnan equilibrium (e.g.,
charged species that can not escape the pores), forming a pH
gradient, precipitating the protein at its isoelectric pH. (5)
Addition of a counterion to the pores of the polymer that causes
the protein to come out of solution as a stabilized insoluble salt;
e.g., Zn.sup.2+ insulin or Zn.sup.2+ growth hormone using Zn
acetate in the polymer. (6) Placing a porous polymer with evacuated
pores in contact with a solution comprising the agent and releasing
pressure so that the solution fills the pores.
[0116] A plasticizer can be used to plasticize the polymeric
material and manipulate the temperature at which self-healing
begins to occur. Useful plasticizers include diethyl phthalate,
tributyl acetylcitrate, and similar compounds. Other useful
plasticizers include those that (1) cause a pore network to form
(e.g., by osmotic pressure), (2) stabilize the encapsulated
molecule, and (3) cause the encapsulated molecule to preferentially
distribute inside the polymer pores (e.g., either in solution, in
solid state, or sorbed to a structure of some kind) relative to the
outside solution.
[0117] The agent to be encapsulated may be any material, compound,
or biomolecule of interest that can associate with the ionic
affinity trap. The methods provided herein are particularly useful
for agents that would be subject to degradation when exposed to
conditions used in preparing pore-containing polymers. Examples of
agents include biomolecules such as proteins, peptides,
proteoglycans, lipoproteins, and nucleic acids, such as RNA and
DNA. Some non-limiting examples of proteins that may be used with
the present methods include bovine serum albumen, hen egg-white
lysosome, ribonuclease A, growth hormone, tetanus toxoid,
erythropoietin, insulin-like growth factor-I, vascular endothelial
growth factor, bone morphogenetic protein, and basic fibroblast
growth factor.
[0118] In some embodiments, the agent can be a small molecule or a
large colloidal particle (e.g., virus), or any bioactive substance,
such as a biomacromolecule. The only caveat is that the agent to be
encapsulated should have an affinity for the ionic affinity trap.
In this way, the agent can be present at a low concentration (e.g.,
1 mg/mL or lower) so that the ionic affinity trap acts to bind and
effectively load the porous polymeric material with the agent.
Loading of the porous polymeric material is therefore not dependent
on passive diffusion, which typically requires a high concentration
of agent to be loaded in order to obtain the desired loading
level.
[0119] The present technology may also be used for small molecules,
e.g., drugs used in drug-eluting stents or in nanoparticle
delivery. Hydrophilic molecules can be problematic to encapsulate
in drug-eluting stents by conventional methods. The present
technology may also be used to encapsulate nanoparticulate
materials (e.g., viruses) without drying the polymer. For example,
some materials may be denatured or degraded in whole or part by a
drying process and hence the present methods can avoid drying is
such instances.
[0120] To further illustrate the present technology, another
example of PLGA microspheres loaded with ovalbumin can be prepared
as follows to demonstrate an embodiment of a delivery system
constructed in accordance with the present technology. The effects
of formulation and incubation parameters on agent loading were
ascertained indicated in Tables 6-13. Ovalbumin is used as a model
to demonstrate loading of a protein antigen, for example. The
following parameter effects were determined.
TABLE-US-00006 TABLE 6 Effect of alhydrogel loading (theoretical).
Incubation Temperature Ovalbumin Adsorbed (.mu.g)* and Time
Alhydrogel Loading (Theoretical) (wt %) T (.degree. C.) t (h) Blank
0.7 1.3 3.2 25 3 -0.7 .+-. 3.8 42.4 .+-. 2.4 70.9 .+-. 7.9 87.6
.+-. 2.8 6 -4.0 .+-. 2.7 62.5 .+-. 4.9 73.0 .+-. 1.0 91.2 .+-. 1.7
24 -7.1 .+-. 9.3 65.3 .+-. 9.9 77.8 .+-. 6.3 95.4 .+-. 1.9 *Mean
.+-. S.E Concentration of PLGA: 350 mg/mL; Initial water phase: 200
.mu.L Trehalose loading: 7.8 wt %; Microparticles size: 20-63
.mu.m
TABLE-US-00007 TABLE 7 Effect of trehalose loading (theoretical).
Incubation Temperature Ovalbumin Adsorbed (.mu.g)* and Time
Trehalose Loading (Theoretical) (wt %) T (.degree. C.) t (h) 0 1.9
3.8 10.4 25 3 73.0 .+-. 7.1 75.1 .+-. 1.4 85.0 .+-. 2.8 84.2 .+-.
3.1 6 73.4 .+-. 5.3 82.7 .+-. 8.2 99.2 .+-. 3.3 85.5 .+-. 3.3 24
80.0 .+-. 2.8 104.2 .+-. 1.8 111.0 .+-. 5.5 88.0 .+-. 7.9 *Mean
.+-. S.E Concentration of PLGA: 250 mg/mL; Initial water phase: 200
.mu.L Alhydrogel loading: 2.9 to 3.2 wt %; Microparticles size:
20-63 .mu.m
TABLE-US-00008 TABLE 8 Effect of volume of initial water phase.
Ovalbumin Incubation Adsorbed (.mu.g)* Temperature Volume of
Initial and Time Water Phase (.mu.L) T (.degree. C.) t (h) 200 300
25 3 70.9 .+-. 7.9 50.7 .+-. 0.4 6 73.0 .+-. 1.0 68.1 .+-. 4.7 24
77.8 .+-. 6.3 73.2 .+-. 4.6 *Mean .+-. S.E Concentration of PLGA:
350 mg/mL; Alhydrogel loading: 1.35 and 1.93 wt %; Trehalose
loading: 7.8 wt %; Microparticles size: 20-63 .mu.m
TABLE-US-00009 TABLE 9 Effect of size of microparticles. Ovalbumin
Incubation Adsorbed (.mu.g)* Temperature Size of and Time
Microparticles (.mu.m) T (.degree. C.) t (h) 20-63 63-90 25 3 75.4
.+-. 12.9 29.1 .+-. 9.0 6 79.5 .+-. 10.7 47.0 .+-. 9.7 24 92.3 .+-.
5.5 59.2 .+-. 4.3 *Mean .+-. S.E Concentration of PLGA: 350 mg/mL;
Alhydrogel loading: 1.35 wt % Trehalose loading: 14.4 wt %; Initial
water phase: 200 .mu.L
TABLE-US-00010 TABLE 10 Effect of concentration of PLGA. Ovalbumin
Incubation Adsorbed (.mu.g)* Temperature Concentration and Time of
PLGA (mg/mL) T (.degree. C.) t (h) 250 350 25 3 84.2 .+-. 3.1 78.0
.+-. 5.8 6 85.5 .+-. 3.3 84.1 .+-. 2.3 24 88.0 .+-. 7.9 87.8 .+-.
4.1 *Mean .+-. S.E Alhydrogel loading: 2.9 wt %; Trehalose loading:
14.4 wt % Initial water phase: 200 .mu.L; Microparticles size:
20-63 .mu.m
TABLE-US-00011 TABLE 11 Effect of incubation temperature. Ovalbumin
Incubation Adsorbed (.mu.g)* Temperature Incubation and Time
Temperature (.degree. C.) T (.degree. C.) t (h) 10 25 25 3 78.9
.+-. 2.6 85.0 .+-. 2.8 6 83.3 .+-. 3.4 99.2 .+-. 3.3 24 89.8 .+-.
2.5 111.0 .+-. 5.5 *Mean .+-. S.E Concentration of PLGA: 250 mg/mL;
Alhydrogel loading: 3.2 wt %; Trehalose loading: 3.8 wt %; Initial
water phase: 200 .mu.L; Microparticles size: 20-63 .mu.m
TABLE-US-00012 TABLE 12 Effect of adjuvant type. Incubation
Ovalbumin Temperature Adsorbed (.mu.g)* and Time Adjuvant Type
Aluminum Calcium T (.degree. C.) t (h) Hydroxide Phosphate 25 3
78.9 .+-. 2.6 55.7 .+-. 5.4 6 83.3 .+-. 3.4 58.7 .+-. 5.0 24 89.8
.+-. 2.5 82.0 .+-. 9.7 *Mean .+-. S.E Concentration of PLGA: 250
mg/mL; Alhydrogel loading: 3.2 wt %; Calcium Phosphate loading: 3.4
wt %; Trehalose loading: 3.8 wt %; Initial water phase: 200 .mu.L;
Microparticles size: 20-63 .mu.m
TABLE-US-00013 TABLE 13 Recovery of ovalbumin after release study
in 194 mM Sodium citrate. Ovalbumin Insoluble Ovalbumin Recovered
aggregate Released from (covalent after 10 polymer and Total days
after 10 days noncovalent) Recovery Formulation (%) (%) (%) (%) 3.2
wt % 78.3 .+-. 4.6 21.0 .+-. 4.2 1.3 .+-. 0.2 100.6 .+-. 0.2
Alhydrogel/ PLGA 3.1 wt % 47.9 .+-. 4.4 47.1 .+-. 6.4 5.8 .+-. 0.8
100.8 .+-. 0.8 Alhydrogel/5 wt % DEP/PLA
[0121] Self-healing of microparticles loaded with ovalbumin is
further illustrated in FIGS. 9-11. FIG. 9 depicts 3.2 wt %
Alhydrogel/3.8 wt % Trehalose/PLGA microparticles before (A) and
after (B) self-healing by incubating at about 25.degree. C. for
about 24 hours and about 43.degree. C. for about 48 hours. FIG. 10
depicts 3.2 wt % Alhydrogel/3.8 wt % Trehalose/2.5 wt % DEP/PLGA
microparticles before (A) and after (B) self-healing by incubating
at about 10.degree. C. for about 48 hours, at about 25.degree. C.
for about 24 hours, and at about 37.degree. C. for about 30 hours.
FIG. 11 depicts 3.2 wt % Alhydrogel/3.8 wt % Trehalose/5 wt %
DEP/PLGA microparticles before (A) and after (B) self-healing.
[0122] In another example of the present technology, a delivery
system is used to microencapsulate Leuprolide, a potent agonistic
analogue of luteinizing hormone-releasing hormone, inhibits the
secretion of pituitary gonadotropin when administered chronically
in therapeutic doses. Microsphere depot formulations of leuprolide
can be developed for long-term testosterone suppression.
[0123] In some embodiments, the present technology can employ
"sorption loading." Basically, this involves taking ground PLGA of
relatively low MW (from the manufacturer), which has ionized end
group (in this case carboxylate, but could be made anything), and
then incubating low peptide solution concentrations. The ionic
interaction causes the peptide to "sorb" to the polymer. The full
nature of this sorption is not fully understood, other than it
requires the ionized end group and a high enough temperature for
polymer chain mobility. It could be mostly at the surface, i.e.,
adsorption, or mostly in the bulk, i.e., absorption. The sorption
group in FIG. 13 is generated with injections every two, three, or
four weeks at the same dosage as the Lupron Depot. The polymer is
of low MW and takes up more water than the polymers generated for
most self-healing microencapsulation methods. In this example, we
expect the peptide sorbs by penetration directly into the polymer
phase and combines with carboxylic end groups (the ionic affinity
trap), as the acid end-group PLGA takes up sufficient water for
peptide permeation.
[0124] Evaluation of long-term testosterone suppression ability of
leuprolide acetate (LA)-PLGA particles in male Sprague-Dawley rats
was performed as follows.
[0125] The efficacy of LA-PLGA particles to provide long-term in
vivo LA release was evaluated by assessing long-term testosterone
suppression ability of LA-PLGA particles in male Sprague-Dawley
rats. The treatment of experimental animals was in accordance with
University committee on use and care of animals (University of
Michigan UCUCA), and all NIH guidelines for the care and use of
laboratory animals. Male Sprague-Dawley rats of 6 weeks old were
housed in cages and given free access to standard laboratory food
and water, and allowed one week to acclimate prior to study
initiation. Animals were anesthetized with 2-4% isoflurane
administered by a calibrated vaporizer (Midmark, Orchard Park,
N.Y., USA). The leuprolide acetate (1.times.) and LA-PLGA particles
(2.times. (day 0, 14, 28, and 42), 3.times. (day 0, 21, and 42),
and 4.times. (day 0 and 28) in a liquid vehicle (1% w/v
carboxymethylcellulose and 2% w/v mannitol), and commercial Lupron
Depot (2.times. (day 0 and 28)) were subcutaneously injected at the
back (lower neck portion) of rats (6 animals/study group). The dose
of leuprolide acetate was 100 .mu.g/kg/day. Animal body weight
considered for dosing leuprolide acetate was 425 g which is
projected body weight of male Sprague Dawley rat at midpoint (day
28) of the study (as per the weight (g)/age (weeks) curve given by
Charles River Laboratories). Blood samples were collected via
jugular vein stick before (day -7 and 0 for baseline testosterone
level) and after (1, 7, 14, 21, 28, 35, 42, 49, and 56 days)
injection of preparations. The collected blood samples were
immediately transferred to B-D Microtainer.TM. blood collection and
serum separation tubes previously incubated in ice, centrifuged at
8,000 rpm for 10 min, and then the serum was removed and stored in
microcentrifuge tubes at -20.degree. C. until further use. Serum
testosterone levels were assayed by Radioimmunoassay using a
TESTOSTERONE Double Antibody-125I RIA Kit (MP Biomedicals LLC.,
Solon, OH, USA) at the University of Pennsylvania Diabetes Center
(Philadelphia, Pa., USA). Lowest detection limit of testosterone
was 0.1 ng/mL.
[0126] The following methods were employed. Aspects of leuprolide
acetate (LA) sorption by PLGA particles include the following.
Previously grinded and sieved (20-63 .mu.m) PLGA was used for the
absorption of LA to PLGA. Solution (3 mM) of LA in HEPES (0.1 M, pH
7.4) was added to PLGA particles (1 mL/10 mg particles) and
incubated on a rotary shaker at 37.degree. C. After 6 h of
incubation, LA equivalent to 1 mM was added to boost the
concentration gradient and hence LA absorption to PLGA. After 24 h
incubation, LA/PLGA particles mixture was centrifuged at 8000 rpm
for 10 min and supernatant was removed. The residual particles were
washed three times with deionized water (1 mL water/10 mg
particles) and then freeze-dried. LA absorbed PLGA particles were
passed through sieves to obtain 20-63 .mu.m and stored at
-20.degree. C. until further use.
[0127] Aspects of determining leuprolide acetate loading in
leuprolide acetate-PLGA particles include the following. LA content
in LA-PLGA particles was determined by two-phase extraction.
Briefly, about 10 mg LA absorbed PLGA 50:50 (Resomer.RTM. RG 502H)
particles (n=3) were weighed into 5 mL glass vials. To these vials,
1 mL of methylene chloride and 2 mL of 50 mM sodium acetate (pH
4.0) were added, followed by vortexing for 1 min. One and half
milliliter of buffer layer was removed, replaced with 1.5 mL of
same buffer (5 extractions) or 50 mM sodium acetate+1 M NaCl (6
extractions) and similarly extracted for 11 times. The content of
LA in each extracted fraction was then analyzed and quantified by
HPLC. Eleven extractions found to be sufficient to remove LA
completely from LA-PLGA complexes as the peak of LA disappeared in
12.sup.th extraction.
[0128] Evaluation of in vitro release of leuprolide acetate from
LA-PLGA particles was as follows. In vitro release of LA was
performed under perfect sink condition. Briefly, about 10 mg
LA-PLGA particles were weighed (n=3) into Eppendorf tubes and 1 mL
of PBST+0.02% sodium azide was added. Eppendorf tubes were then
incubated at 37.degree. C. on a rotary shaker at 240 rpm. At
specified time points (1-10 days and every two days thereafter),
tubes were centrifuged at 8,000 rpm for 5 min and 1 mL supernatant
was removed and then replaced with pre-warmed (37.degree. C.)
release medium. Analysis of LA in release samples was performed by
HPLC.
[0129] The following results were obtained.
TABLE-US-00014 TABLE 14 Evaluation of leuprolide acetate (LA)
content in LA-PLGA particles. LA loading (wt %) Amino acid LA
loading Extraction analysis efficiency LA absorbed Fraction of
method method (%) (.mu.mol/g PLGA) acids occupied.sup.a 17.2 .+-.
2.2 17.0 .+-. 2.7 33.2 .+-. 0.1 140.8 .+-. 10.9 0.68 .+-. 0.05
.sup.aFraction of acids occupied = LA absorbed/total acids of
polymer (.mu.mol/g PLGA).
[0130] In vitro release of leuprolide acetate from LA-PLGA
particles is shown in FIG. 12.
[0131] In vivo testosterone suppression in male Sprague-Dawley rats
by leuprolide acetate-PLGA particles is shown in FIG. 13.
[0132] Benefits of the current technology include: a) the ability
to prepare agent encapsulated products in a straightforward manner,
b) reduction in the cost of manufacturing as loss of expensive
agents during encapsulation is reduced and a smaller quantity of
agent (about 0.5 to 1 mg/mL) can be used to achieve very high
encapsulation efficiency (99% or more), c) can be used with
multiple agents, d) can be used at point-of-care, and e) allows
terminal sterilization of the delivery system prior to agent
loading (i.e., no aseptic manufacturing with organic solvents is
required).
[0133] Example embodiments are provided so that this disclosure
will be thorough, and will fully convey the scope to those who are
skilled in the art. Numerous specific details are set forth such as
examples of specific components, devices, and methods, to provide a
thorough understanding of embodiments of the present disclosure. It
will be apparent to those skilled in the art that specific details
need not be employed, that example embodiments may be embodied in
many different forms, and that neither should be construed to limit
the scope of the disclosure. In some example embodiments,
well-known processes, well-known device structures, and well-known
technologies are not described in detail. Equivalent changes,
modifications and variations of some embodiments, materials,
compositions and methods can be made within the scope of the
present technology, with substantially similar results.
[0134] The following non-limiting discussion of terminology is
provided with respect to the present technology.
[0135] The headings (such as "Introduction" and "Summary") and
sub-headings used herein are intended only for general organization
of topics within the present disclosure, and are not intended to
limit the disclosure of the technology or any aspect thereof. In
particular, subject matter disclosed in the "Introduction" may
include novel technology and may not constitute a recitation of
prior art. Subject matter disclosed in the "Summary" is not an
exhaustive or complete disclosure of the entire scope of the
technology or any embodiments thereof. Classification or discussion
of a material within a section of this specification as having a
particular utility is made for convenience, and no inference should
be drawn that the material must necessarily or solely function in
accordance with its classification herein when it is used in any
given composition.
[0136] The description and specific examples, while indicating
embodiments of the technology, are intended for purposes of
illustration only and are not intended to limit the scope of the
technology. Moreover, recitation of multiple embodiments having
stated features is not intended to exclude other embodiments having
additional features, or other embodiments incorporating different
combinations of the stated features. Specific examples are provided
for illustrative purposes of how to make and use the compositions
and methods of this technology and, unless explicitly stated
otherwise, are not intended to be a representation that given
embodiments of this technology have, or have not, been made or
tested.
[0137] As used herein, the words "desire" or "desirable" refer to
embodiments of the technology that afford certain benefits, under
certain circumstances. However, other embodiments may also be
desirable, under the same or other circumstances. Furthermore, the
recitation of one or more desired embodiments does not imply that
other embodiments are not useful, and is not intended to exclude
other embodiments from the scope of the technology.
[0138] As used herein, the word "include," and its variants, is
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that may also be
useful in the materials, compositions, devices, and methods of this
technology. Similarly, the terms "can" and "may" and their variants
are intended to be non-limiting, such that recitation that an
embodiment can or may comprise certain elements or features does
not exclude other embodiments of the present technology that do not
contain those elements or features.
[0139] Although the open-ended term "comprising," as a synonym of
non-restrictive terms such as including, containing, or having, is
used herein to describe and claim embodiments of the present
technology, embodiments may alternatively be described using more
limiting terms such as "consisting of" or "consisting essentially
of." Thus, for any given embodiment reciting materials, components
or process steps, the present technology also specifically includes
embodiments consisting of, or consisting essentially of, such
materials, components or processes excluding additional materials,
components or processes (for consisting of) and excluding
additional materials, components or processes affecting the
significant properties of the embodiment (for consisting
essentially of), even though such additional materials, components
or processes are not explicitly recited in this application. For
example, recitation of a composition or process reciting elements
A, B and C specifically envisions embodiments consisting of, and
consisting essentially of, A, B and C, excluding an element D that
may be recited in the art, even though element D is not explicitly
described as being excluded herein.
[0140] As referred to herein, all compositional percentages are by
weight of the total composition, unless otherwise specified.
Disclosures of ranges are, unless specified otherwise, inclusive of
endpoints and include all distinct values and further divided
ranges within the entire range. Thus, for example, a range of "from
A to B" or "from about A to about B" is inclusive of A and of B.
Disclosure of values and ranges of values for specific parameters
(such as temperatures, molecular weights, weight percentages, etc.)
are not exclusive of other values and ranges of values useful
herein. It is envisioned that two or more specific exemplified
values for a given parameter may define endpoints for a range of
values that may be claimed for the parameter. For example, if
Parameter X is exemplified herein to have value A and also
exemplified to have value Z, it is envisioned that Parameter X may
have a range of values from about A to about Z. Similarly, it is
envisioned that disclosure of two or more ranges of values for a
parameter (whether such ranges are nested, overlapping or distinct)
subsume all possible combination of ranges for the value that might
be claimed using endpoints of the disclosed ranges. For example, if
Parameter X is exemplified herein to have values in the range of
1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may
have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10,
2-8, 2-3, 3-10, and 3-9.
[0141] "A" and "an" as used herein indicate "at least one" of the
item is present; a plurality of such items may be present, when
possible. "About" when applied to values indicates that the
calculation or the measurement allows some slight imprecision in
the value (with some approach to exactness in the value;
approximately or reasonably close to the value; nearly). If, for
some reason, the imprecision provided by "about" is not otherwise
understood in the art with this ordinary meaning, then "about" as
used herein indicates at least variations that may arise from
ordinary methods of measuring or using such parameters.
[0142] When an element or layer is referred to as being "on,"
"engaged to," "connected to" or "coupled to" another element or
layer, it may be directly on, engaged, connected or coupled to the
other element or layer, or intervening elements or layers may be
present. In contrast, when an element is referred to as being
"directly on," "directly engaged to," "directly connected to" or
"directly coupled to" another element or layer, there may be no
intervening elements or layers present. Other words used to
describe the relationship between elements should be interpreted in
a like fashion (e.g., "between" versus "directly between,"
"adjacent" versus "directly adjacent," etc.). As used herein, the
term "and/or" includes any and all combinations of one or more of
the associated listed items.
* * * * *