U.S. patent application number 11/741018 was filed with the patent office on 2007-08-23 for thermosensitive and biodegradable microgel and a method for the preparation thereof.
Invention is credited to Jiandong DING, Biaobing Wang, Ying Zhang, Wen Zhu.
Application Number | 20070196425 11/741018 |
Document ID | / |
Family ID | 28050420 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196425 |
Kind Code |
A1 |
DING; Jiandong ; et
al. |
August 23, 2007 |
THERMOSENSITIVE AND BIODEGRADABLE MICROGEL AND A METHOD FOR THE
PREPARATION THEREOF
Abstract
The present invention provides a thermosensitive and
biodegradable microgel and a method of synthesizing such microgels.
The thermosensitive and biodegradable microgel is synthesized from
a macromer comprising a thermosensitive block polymer
co-polymerized with a biodegradable moiety encapped with a
cross-linkable or polymerizable moiety at either end. The microgels
of the present invention are synthesized by inverse suspension
polymerization of the macromers. The microgels are biodegradable
into components that are non-toxic and easily removed from the
body. The microgel of the present invention is temperature
sensitive and is "intelligent" as well as biodegradable. The
microgels are preferably used for the controlled release of a drug
or in tissue engineering. Most preferably, the microgels are
suitable for the control release of biologically active substances
such as proteins.
Inventors: |
DING; Jiandong; (Shanghai,
CN) ; Zhu; Wen; (Shanghai, CN) ; Wang;
Biaobing; (Shanghai, CN) ; Zhang; Ying;
(Shanghai, CN) |
Correspondence
Address: |
Morgan & Finnegan L.L.P;Maria C.H. Lin
Three World Financial Center
New York
NY
10281-2101
US
|
Family ID: |
28050420 |
Appl. No.: |
11/741018 |
Filed: |
April 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10762029 |
Jan 20, 2004 |
7226617 |
|
|
11741018 |
Apr 27, 2007 |
|
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Current U.S.
Class: |
424/423 ;
424/486 |
Current CPC
Class: |
A61K 9/0024 20130101;
A61K 9/06 20130101; A61K 9/19 20130101; A61K 47/34 20130101 |
Class at
Publication: |
424/423 ;
424/486 |
International
Class: |
A61K 9/14 20060101
A61K009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 8, 2003 |
CN |
03115319.4 |
Claims
1. A thermosensitive and biodegradable microgel_wherein a) the
microgel has a chemically cross-linked network comprising at least
one negative temperature-sensitive macromolecule and one
biodegradable group represented by structural formula. wherein
represents chemical cross-linked bonds; F(DC).sub.2 represents a
polymeric chain between the cross-linked bonds; F is a
temperature-sensitive block copolymer of poly(ethylene oxide) (PEO)
and poly(propylene oxide) (PPO); D is a biodegradable moiety; and C
is a cross-linked moiety resulting from C*, a cross-linkable
moiety; and b) the microgel is in the form of microparticles
prepared by the following steps: i) co-polymerizing a biocompatible
polymer or oligomer F with an internal ester D by ring opening
polymerization of D in the presence of F using a catalyst; wherein
F is a temperature-sensitive polymer or oligomer; D is a
biodegradable moiety; ii) end capping the copolymer with a
crosslinkable moiety C* to provide a macromer with crosslinkable
ends; iii) cross linking the crosslinkable end(s) of the macromer
to form the microparticles of the microgel by inverse suspension
polymerization, wherein the continuous phase is a water-insoluble
organic solvent and the dispersion phase consists of a hydrophilic
medium in which the macromer is present; c. the microgel being
useful for encapsulating a drug post polymerization; and d. the
drug encapsulated microgel being injectable into the body.
2. The thermosensitive and biodegradable microgel of claim 1,
wherein the temperature-sensitive polymer or oligomer F is a
tri-block copolymer of poly(ethylene oxide) (PEO) and
poly(propylene oxide) (PPO), having the formula ##STR4## wherein X
is from 1 to 300, Y from 1 to 300.
3. The thermosensitive and biodegradable microgel of claim 1,
wherein D is a biodegradable oligoester or polyester, or is a
biodegradable random or block co-oligoester or co-polyester
selected from the group consisting of (R.sub.1).sub.m,
(R.sub.2).sub.n, and (R.sub.3).sub.l, wherein R.sub.1, R.sub.2, and
R.sub.3 respectively are represented by: ##STR5## wherein
m=0.about.100, n=0.about.100, l=0.about.100, and not all of m, n
and l can be zero.
4. The thermosensitive and biodegradable microgel of claim 1,
wherein the cross-linked moiety C is ##STR6## wherein R can be --H,
--CH.sub.3 or alkyl.
5. The thermosensitive and biodegradable microgel of claim 1,
wherein the temperature-sensitive polymer or oligomer F is used as
the principal constituent in an amount based on the microgel in the
range of 51 wt %.about.99.9 wt %.
6. The thermosensitive and biodegradable microgel of claim 1,
wherein the chemically cross-linked network is obtained by
crosslinking a mixture of different block copolymers, F and D and
comprising a crosslinked moiety C.
7. The thermosensitive and biodegradable microgel of claim 1,
wherein 0 wt %.about.49 wt % of the chemically cross-linked network
comprises a dangling unpolymerized end.
8. The thermosensitive and biodegradable microgel of claim 1,
wherein the size of the microgel particles is in the range of 5 nm
to 5 mm.
9. The thermosensitive and biodegradable microgel of claim 1,
wherein the microgel is in a bulk state or admixed with a
solvent.
10. A method of loading a substance into the network of the
thermosensitive and biodegradable microgel of claim 1 by absorbing
the substance into the network of the microgel by allowing the
microgel to swell in a solution containing the substance at a low
temperature below the phase transition temperature of the
microgel.
11. The method of claim 10, wherein the substance absorbed in the
gel was entrapped by increasing the temperature, or by drying the
hydrogel, or by increasing the temperature and then drying the
hydrogel.
12. The method of claim 10, wherein the absorbed or entrapped
substance from the microgels was released into a solution at a
temperature above the phase transition temperature of the
microgel.
13. The method of claim 10, wherein the phase transition
temperature is below the temperature of a human or a warm-blood
animal.
14. The method of claim 10, wherein the swollen microgel is loaded
with a substance and entrapped therein at a temperature above the
phase transition temperature of the microgel for a time sufficient
to cause further gelation of the network to prevent leakage of the
entrapped substance.
15. The method of claim 10, wherein the loaded substance is a pure
substance of a mixture which does not chemically react with the
microgel.
16. The method of claim 10, wherein the loaded substance is
biologically active.
17. The method of claim 16, wherein the solvent for the
biologically active substance is water, an aqueous solution or
PBS.
18. The method of claim 16, wherein the biologically active
substance is a biomacromolecule or a derivative thereof.
19. The method of claim 18, wherein the biologically active
substance is a protein.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a polymeric biodegradable
material that is suitable for use in a pharmaceutical formulation.
More particularly, it is directed to a thermosensitive and
biodegradable microgel, a method of preparation of the
biodegradable microgel and a unique application of the
material.
BACKGROUND OF THE INVENTION
[0002] Controlled Release of Recombinant Proteins
[0003] With the rapid advances made in biotechnology and genetic
engineering, a growing number of proteins and peptides have been
produced by recombinant DNA technology for use as pharmaceutical
therapeutic agents. Such proteins include erythropoietin (EPO),
granulocyte-colony-stimulating factor (G-CSF and GM-CSF),
interferons (alpha, beta, gamma, and consensus), insulin and
interleukin-1 etc. In addition to these proteins, several hundred
other proteins are currently undergoing clinical trials as drugs.
Because proteins have generally short in vivo half-lives and
negligible oral bioavailability, they are typically administered by
frequent injection, a procedure that is hard to accept for most
patients. It has been conjectured that the employment of controlled
release technology for the administration of such therapeutic
agents can alleviate such a problem to a degree. Hence, it is
highly desirable to develop sustained-release systems.
[0004] Tissue engineering is another area of biomedical research
that is much pursued. The goal of tissue engineering is to employ
the techniques of modern biotechnology to regenerate or replace
lost or damaged tissues and organs. In addition to cells and their
scaffolds, a class of proteins called growth factors are frequently
required for tissue engineering. These include nerve growth factor
(NGF), platelet-derived growth factor (PDGF), fibroblast growth
factor (FGF), epidermal growth factor (EGF), tumor necrosis factor
(TNF) etc. Growth factors, most of which are globular, are
effective in the process for supplying the oxygen and nutrients
which are necessary for the survival of the transplanted cells in
organ transplants (Lanza et al., 2000).
[0005] Cell growth factors are usually injected locally and active
targeting of the agent is not absolutely necessary. However, it has
been found that direct single-time injection of a growth factor
solution into a regeneration site is less effective, as the
injected growth factor rapidly diffuses away from the site.
Repeated injection is, of course, inconvenient. Novel drug delivery
systems are thus desired. The projection is that controlled
delivery systems for recombinant proteins will be a major
technology in tissue engineering during the next century.
(Yasuhiko, (2000) Pharmaceutical Science & Technology Today, 3:
80-89)
[0006] Microparticles as Controlled-Release Carriers
[0007] Microparticles has held promises in many areas of medicine.
In recent years, microparticles have garnered growing attention and
have been the subject of investigation as an ideal drug carrier. Up
to the present, microparticles have found application in more than
thirty different drugs, including antipyretic analgesic,
antibiotic, fibrin and anticancer drugs, etc. Biomolecules such as
proteins, enzymes, hormones and peptides, are sensitive and easily
degraded. The most promising controlled-release approach would be
to encapsulate such materials within microparticles. A principal
advantage of formulating these sensitive biomolecules in
microparticles is that they may be administrated by injection, and
does not require formal surgical procedure for their
administration.
[0008] Materials that are useful for making into microparticles can
be grouped into three categories: natural polymers such as glutin,
alginate and chitosan; semi-synthetic polymers such as
carboxymethyl cellulose, cellulose acetate phthalate, methyl
cellulose and ethyl cellulose; and synthetic polymers such as
polyamide, poly(acrylic acid), poly(vinyl alcohol), polycarbonate,
poly(amino acid), poly(lactic acid), poly(lactide-co-glycolide) and
poly(d,l-lactide)-poly(ethylene glycol) copolymer. Natural polymers
are abundant and usually biodegradable. However, the principal
disadvantage is in the difficulty of their modification and
purification. There are significant batch to batch and source to
source variations, due to the need to isolate these materials from
living organisms.
[0009] The requirements for materials used for encapsulation are
suitable drug release rates, stability, non-toxicity, absence of
interference with the pharmacological action, strength, suitable
hydrophilicity, plasticity, permeability and solubility. In
addition, it would be desirable to have microparticles made from
biodegradable polymers to eliminate the need for their removal
after the agent has been released. Synthetic polymers are available
in a wide range of compositions with readily adjustable properties.
Therefore, much attention has been paid to the use of biodegradable
materials. Synthetic polyesters have, especially, been widely
investigated. See. Blanco et al., (1998) Eur. J. Pharm. Biopharm.
45: 285-294, Zhu et al., (1999) Eur. Polym. J. 35: 1821-1828.
[0010] Up to the present, most researchers have concentrated on the
use of hydrophobic biodegradable polymers. Singh et al., (2001) J
Control. Release 70: 21-28, reported the in vitro and in vivo
release behavior of polylactide-co-glycolide microparticles with
entrapped insulin growth factor (rhlGF-I). Jain et al., (2000) Eur.
J. Pharm. Biopharm, 50: 257-262, investigated the release behavior
of bovine heart cytochrome C and heart skeletal muscle myoglobin
from injectable PLGA microparticles.
[0011] There are various problems associated with the applications
of such polymers. The problems are: the difficulty of the
homogeneous dispersal of the hydrophilic drug within the polymer
matrices, the inability of certain macromolecules to diffuse out
through the polymer matrix, the unpredictability of drug release
behavior, the deterioration of the drug, e.g., denaturation caused
by the presence of organic solvents, and irritation to the organism
due to side effects caused by the presence of organic solvents.
[0012] Degradable polymers containing water-soluble polymers have
also been investigated. Copolymerization of lactide, glycolide and
caprolactone with the polyether such as polyethylene glycol (PEG)
was expected to partially overcome the above drawbacks, while
taking advantage of the virtues of both biodegradable and
hydrophilic polymers. Sawhney et al., (1990) J Biomed. Mater. Res,
24(10): 1397-1411, Casey et al., (U.S. Pat. No. 4,716,203)
describes the synthesis of a block copolymer of PGA (poly(glycolic
acid)) and PEG. Cho et al (2001) J. Control. Release 76: 275-284.
applied the W/O/W double emulsion method to prepare PLLA-PEG
copolymer microparticles, where bovine serum albumin (BSA) was used
as the model drug. Although the copolymers have improved
hydrophilicity, most of the biodegradable synthetic polymers
reported so far can only be processed in organic solvents which are
harmful to protein activity. For these reasons, it is desirable to
have a hydrogel as a preferable candidate as a protein drug
carrier.
[0013] Use of Hydrogel as Protein Carriers
[0014] Hydrogels have been intensely investigated as protein drug
vehicles because of their excellent biocompatibility and
hydrophilicity. Compared with other synthetic biomaterials such as
PLGA, hydrogels more closely resemble natural living tissues
because of their high water contents and soft and rubbery
consistency. The nature of hydrogels minimizes irritation to
surrounding tissues. Furthermore, hydrogels are useful in
protecting the drug from hostile environments, e.g., the presence
of enzymes or the low pH in the stomach. Some biodegradable
hydrogels have been reported, Sawhney et al., (1993)
Macromolecules, 26:581-587, and Hubbell et al., U.S. Pat. Nos.
5,986,043, 6,060,582, and 6,306,922. However, the hydrogels were
synthesized via ultraviolet polymerization or photopolymerization,
which are not suitable for entrapping ultraviolet-sensitive
proteins.
[0015] There are many different physical forms of hydrogels, such
as microgel, bulk gel etc. Since microparticles are injectable,
microgel would be a good carrier for proteins. Drug delivery
systems in the form of microparticles may enable the release of the
therapeutic agent in a specified area or over a specified time
period. Kim S W et al., (1997) Nature 388: 860-862, studied
injectable hydrogel. During the process of loading drug, the use of
any organic solvent which can denature the protein was avoided.
However, in this case, bulk gels instead of gel particles were
used, and the encapsulation temperature was higher than human body
temperature.
[0016] Cross-linked glutin and collagen have also been employed as
a hydrogel to encapsulate peptides of opposite charges. Alginates
have been shown to be able to encapsulate biological materials.
Lim, U.S. Pat. No. 4.352,883. But the rates of degradation of both
kinds of gels were not easily controlled over a wide range of
conditions.
[0017] In addition to swelling, some hydrogels also show changes in
response to stimuli. The present inventors explored these
characteristics for developing novel ways for drug loading and drug
release.
[0018] There have been many studies on materials for drug release.
These materials are either merely biodegradable as reported by
Cohen et al., (1991) Pharm. Res., 8: 713-720, Langer et al., (1998)
Nature 392: 5-10, Bawa et al., (1985) J. Control. Release, 1:
259-267, Li et al., (2002) J. Polym. Sci. part A: Polym. Chem.
40(24): 4550-4555, Fu J. et al., (1997, 1998) Chemical Journal of
Chinese Universities, 18(10): 1706-1710, 19(5):813-816; or merely
responsive to environmental stimuli, see e.g., Wu et al., (1995,
1996) Macromolecules 28(15): 5388-5390, 29(5):1574-1578, Wu, U.S.
Pat. No. 6,030,634; Zhou et al., (2003) J. Polym. Sci. part A:
Polym. Chem. 41(1):152-159. There have been a few reports about a
material having a combination of both properties, Shah, U.S. Pat.
No. 6,541,033, and Shah et al., U.S. Pat. No. 0,099,709. From these
few reports, the use of biodegradable and thermosensitive microgels
in a drug delivery system is still quite limited.
[0019] RealGel.TM. is a block copolymer with
temperature-sensitivity and degradability, Zentner et al., (2001)
J. Control. Release, 72: 203-215. It is not a chemical gel but a
physical gel. The term of "chemical gel" represents a gel in which
gellation is due to chemical crosslinking, while the term of
"physical gel" denotes a gel in which gellation is induced by
physical parameter such as temperature. In addition, this gel is a
liquid at high temperature, and forms a semi-solid gel when it can
be used to encapsulate a drug at a lower temperature. However, the
positive temperature sensitivity may lead to denaturation of the
protein when it is being encapsulated at a high temperature before
gelation. A negative temperature sensitive biodegradable chemical
hydrogel has been reported by Hubbell et al., (2000) U.S. Pat. No.
6,129,761, but it was a bulk gel, wherein an ultraviolet initiation
method was used. There are difficulties in applying this type of
bulk gel in an inverse suspension polymerization process.
[0020] In accordance with the present invention, a thermosensitive,
biodegradable microgel for the sustained delivery of drugs is
provided. The drug is released at a controlled rate from the
microgel, which eventually biodegrade into non-toxic products. The
rate of degradation can also be adjusted by adjusting the
composition of the biodegradable groups.
[0021] Drug Loading
[0022] In order to encapsulate protein molecules within
microparticles, most researchers utilize a solvent evaporation
method because this method is useful for achieving a high level of
encapsulation. There are basically two different approaches for
encapsulation: a water/oil/water double emulsion or a single
emulsion in which the micronized protein powder is dispersed into
an organic solvent phase containing the dissolved polymer. However,
many proteins are irreversibly denatured by contact with organic
solvents necessary for dissolving the polymer. Thus, one of the
main problems of such methods is the partial or complete loss of
biological activity. There are some additives, such as albumin and
polysaccharides, which can be used to stabilize the proteins to a
certain degree and affect the release behavior, Baldwin et al.,
(1998) Adv. Drug. Deliv. Rev., 33: 71-86. The alternative, but less
frequently reported method is loading the proteins or peptides into
a microgel by swelling the microgels in an aqueous solution and
subsequent drying. However, this method provides only a low level
of encapsulation efficiency. Thus, a further object of the present
invention is to provide a process of manufacturing a microgel and
loading a protein drug into the microgel with a relatively high
loading level.
[0023] Compared to in situ encapsulation of the drug, absorption of
the drug AFTER the preparation of the gel or hydrogel provides
several striking advantages: (1) The biocompatibility of the
polymeric material may be enhanced because gel without protein
loaded can be easily cleaned to wash out any residual monomers,
initiators etc.; (2) the steps of preparing the microgel followed
by encapsulation of the protein are separate steps, making it much
easier to control each of the two steps independently. On its face,
it has been considered impractical to adsorb the proteins after the
preparation of the gel or microgel. The reasoning was that if a
drug can penetrate the gel easily, it would not be released slowly;
whereas, if drug release can be controlled well, it would be hard
to adsorb the drug into the gel after gel formation and would lead
to a very low loading level.
[0024] In accordance with the present invention, the hydrogels of
the present invention have been designed to be "intelligent" to
overcome the difficulties encountered previously. The "intelligent"
hydrogels of our invention comes from the temperature sensitivity
of the polymeric material in a particular solvent media. The gel
swells at a low temperature and is ready for drug absorption at
this low temperature. However, the gel contracts or gelates at a
higher temperature, such as the body temperature, and provides a
unique way for controlling the release of the drug after injection
into the body.
[0025] The object and features of the present invention will be
made apparent from the following description.
SUMMARY OF THE INVENTION
[0026] In accordance with a preferred embodiment of the present
invention, the patent provides a thermosensitive, biocompatible and
biodegradable microgel for potential use in drug delivery and other
biomedical applications such as tissue engineering. The microgel is
obtained by polymerizing a macromer through inverse suspension
polymerization. Suitable thermosensitive, biocompatible,
polymerizable and basically water soluble macromers are disclosed
herein. The term "macromer" refers to the macro-monomer which is a
macromolecule or an oligomer and is itself polymerizable. The
macromer according to the present invention comprises a water
soluble and thermosensitive region as the core with hydrolyzable
biodegradable oligomeric extensions, and free-radical-polymerizable
moiety(es) at terminals.
[0027] The microgels resulting from polymerization of such
macromers are particularly useful for controlled drug delivery. The
pore size of the microgel is decreased greatly when the temperature
is higher than its phase transition temperature, namely, the
physical gellation temperature, which is preferably between
4.degree. C. and 37.degree. C. The chemically cross-linked microgel
network is able to entrap and homogeneously disperse a protein drug
throughout the network at a lower temperature, usually at 4.degree.
C., and release the protein drug at a controlled rate at a higher
temperature, usually at 37.degree. C.
[0028] Organic solvents and toxic substances can be removed
completely before drug loading, and the loaded drug would not leach
out from the microgel at body temperature, if the size of the drug,
such as a protein drug, is between the pore sizes of microgel
before and after phase transition temperature, usually between
4.degree. C. and 37.degree. C.; or if the entrapped substance has
been held in the microgel network due to further gelation within
the microgel over the phase transition temperature. In this manner,
protein activity is maintained with a relatively high level of
encapsulation efficiency. Drug release is also effected by
diffusion of the drug and the degradation of the microgels. The
rate of degradation can be controlled by custom designing the
structure of the polymer or polymers used to make the microgel.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 schematically illustrates the internal chemical
structure of the microgels in the present invention (a) and
associated macromer (b), where represents chemical cross-linked
bonds; F(DC).sub.2 represents a microgel block co-polymer; wherein
F is a temperature sensitive polymer or oligomer; D is a
biodegradable moiety, such as a polyglycolide, C* is a
cross-linkable moiety provided at the end of the block co-polymer
chain of F and D to form a macromer, F(DC*).sub.2, and C is a
crosslinked moiety after cross-linking of the macromers
F(DC*).sub.2.
[0030] FIG. 2a shows the morphology of a microgel resulting from
(PE0.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA macromers at
4.degree. C. observed in an inverted optical microscope.
[0031] FIG. 2b shows the morphology of a microgel resulting from
(PE0.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA macromers, at
37.degree. C. observed in an inverted optical microscope.
[0032] FIG. 3 shows the effect of temperature on the particle size
of a microgel. The volume of each microparticle was calculated from
the diameter measured using the optical micrograph.
[0033] FIG. 4 shows the rate of degradation of the microgel
resulting from (PE0.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.4-DA
macromers and (PEO.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA
macromers in PBS at 37.degree. C.
[0034] FIG. 5 shows the release of BSA from
(PE0.sub.100-PPO.sub.65-PEO.sub.100-LA.sub.4 microgel into PBS at
37.degree. C.
[0035] FIG. 6 shows the release of insulin from
(PE0.sub.100-PPO.sub.65-PEO.sub.100)-CL.sub.4 microgel into PBS at
37.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Disclosed herein is a biodegradable and thermosensitive
microgel formed via polymerization of macromers. The macromers are
obtained as follows: a thermosensitive oligomer or polymer (F)
polymerized with a biodegradable moiety (D), and provided with a
polymerizable moiety (C*). The microgel is formed by free radical
initiation in an inverse suspension polymerization process. In
order to clarify the terms, "catalyst" herein denotes an additive
in the chemical reaction between F and D in one of the steps in the
preparation of the macromers, whereas the term "initiator" denotes
an additive in the crosslinking or polymerization of the macromers
to form the microgel of the present invention.
The Macromers and Microgels
[0037] The macro-monomer ("macromer") is a high molecular weight
polymerizable monomer and comprises a biocompatible and
thermosensitive polymer or an oligomer. (F) preferably as a central
part of the system. A block copolymer comprising DFD, wherein D is
a biodegradable moiety, is synthesized by a ring opening
polymerization process in the presence of an aliphatic internal
ester. The block copolymer formed provided with crosslinkable ends
(the macromer F(DC*).sub.2) is then crosslinked to form a microgel.
The crosslinkable moiety C* may comprise a double bond end group
provided at each terminus of the block copolymer DFD.
[0038] The resultant microgels are chemically cross-linked networks
comprising at least one thermosensitive region and one
biodegradable region. In particular, the microgel comprises a
thermosensitive core region F extended at both ends by a
biodegradable moiety D, and provided with a crosslinked moiety C at
each terminus. The chemical structure of the microgel may be
represented by the structure shown in FIG. 1.
[0039] In addition to a thermosensitive region, a biodegradable
component and two cross-linkable moieties at two ends, the block
copolymer may comprise different molecular structures and may be
combined in ways that are obvious to those skilled in the art in
the pertinent field. For instance, the block copolymer may have a
biodegradable polymer as the central portion flanked by
thermosensitive extensions and provided with cross-linkable
moieties at both ends. Alternatively, the block copolymer may be
branched with varying branching structures.
Thermosensitive Regions
[0040] In a preferred embodiment, the core thermosensitive oligomer
or polymer can be a block copolymer of poly(ethylene oxide) (PEO)
and poly(propylene oxide) (PPO). Most preferably, the oligomer or
polymer may be a symmetrical block copolymer with three blockes
with alternating PEO-PPO-PEO. The molecular formula can be written
as ##STR1## wherein X ranges from 1 to 300 and Y ranges from 1 to
300 Biodegradable Regions
[0041] "Biodegradable" means that the block copolymer can break
down or degrade within the body of human or warm-blood animals to
release the entrapped drug.
[0042] The biodegradable block co-polymer D is an oligerester or
polyester, or is a random or block co-polyester or co-oligoester.
The biodegradable moiety, a block co-polyester may comprise
(R.sub.1).sub.m(R.sub.2).sub.n or (R.sub.3).sub.l, wherein R.sub.1,
R.sub.2 and R.sub.3 are respectively ##STR2## wherein
m=0.about.100, n=0.about.100, l=0.about.100 with all of m, n and l
not being zero simultaneously, although one or two of the three may
be zero. The aliphatic polyester R.sub.1 (or R.sub.2 or R.sub.3) or
their copolymers are selected from the group(s) consisting of
polymers or oligomers of d,l-lactic acid, l-lactic acid, glycolide,
.epsilon.-caprolactone and alkyl substituted
.epsilon.-caprolactone, and copolymers thereof. Polymerizable
Regions
[0043] Cross linking of the copolymer with crosslinkable ends are
preferably formed via free radical polymerization, most preferably
via a chemical initiator. The crosslinked moiety C can be expressed
as ##STR3## wherein R can be --H, --CH.sub.3 or an alkyl. The
preferred moiety with a crosslinkable group C* for further
polymerization and is selected from the group consisting of
acrylates, diacrylates, methacrylates, alkyl substituted acrylated,
or other acrylic acid derivatives. Macromer Synthesis
[0044] The ring-opening copolymerization of F and a lactone or an
internal ester may be carried out under 0.1 mm Hg vacuum in the
presence of a catalyst. The reaction temperature is in a range
between 80.about.180.degree. C., preferably 120.about.180.degree.
C., and most preferably 140.about.160.degree. C. The reaction time
is over 1 hour, usually in the range of 3.about.50 hours,
preferably 15.about.24 hours. The catalyst is generally stannous
octoate in an amount that is above 0.01 mole % per hydroxyl end
group of F. Preferably, the amount of the catalyst is in the range
of between 0.1 mole % to 5 mole %, most preferably from 0.5 mole
%.about.1.5 mole %. The catalyst may also be selected from the
group consisting of calcium hydride or zinc powder in a molar ratio
of catalyst to hydroxyl end groups of F in the range of from
0.2/0.8 to 0.8/0.2, preferably ranging from 0.4/0.6 to 0.6/0.4.
When the catalyst is calcium hydride or zinc powder, the reaction
temperature is in the range of between 50.about.250.degree. C.,
preferably 120.about.160.degree. C. The reaction time is above 1
hour, preferably 3.about.50 hours. The molar ratio of F to the
internal ester is in the range of from 0.1.about.99.9 to
99.9.about.0.1.
[0045] The resultant viscous material is dissolved with an organic
solvent, such as dichloromethane, and then precipitated with an
excess of anhydrous ether at a temperature in the range of
-10.about.4.degree. C. to remove the unreacted themosensitive
oligomer or polymer F, the biodegradable moiety R.sub.1 (R.sub.2 or
R.sub.3 or their copolymers) and the residual catalyst. The product
collected by filtration is then dried and preserved in a vacuum
oven. The copolymer formed is then end-capped with acryloyl groups
to form a polymerizable macromolecule. The molar ratio of acryloyl
chloride or methyl acryloyl chloride or acryloyl chloride
derivative to the hydroxyl end groups of F, or the hydroxyl end
group of the copolymer of F and D, is generally in the range of
from 1:1 to 100:1. preferably from 2:1 to 20:1.
[0046] A typical synthesis procedure is illustrated as follows: the
copolymers of F and D are dissolved in dichloromethane or
trichloromethane in a round-bottomed flask, and cooled to 0.degree.
C. in an ice bath. Triethylamine at the same molar amount as
acryloyl chloride or its derivative is then added to the flask with
stirring and a protective layer of dried nitrogen. Acryloyl
chloride, methyl acryloyl chloride or a derivative of acryloyl
chloride, is added dropwise to the mixture The reaction was carried
out for 4.about.12 hours in the ice bath at a temperature in the
range of 0.about.5.degree. C., and then was maintained for
10.about.12 hours at room temperature of about 20.degree. C. The
reaction mixture was filtered to remove triethanolamine
hydrochloride and then precipitated in excessive anhydrous ether.
Finally, the macromer F(DC*).sub.2 is obtained by drying the
precipitate collected by filtration in a vacuum oven.
Formation of Microgel
[0047] In one embodiment, the thermosensitive and biodegradable
macromers (F(DC*).sub.2) are covalently cross-linked to form a
microgel through inverse suspension polymerization. During the
polymerization process, the macromer or a 1 wt %.about.49 wt %
solution of the macromer is the dispersed or continuous medium. The
concentration of the macromer solution ranges from 3 wt %.about.98
wt %. The solvent can be water, an aqueous solution, a hydrophilic
solvent or a hydrophilic solution. The polymerization may be
initiated using a water-soluble redox system with an oxidant, such
as persulfate potassium or persulfate ammonium, or a reductant,
such as a sulphite, hyposulphite or tetramethylethylenediamine
(TEMED), where the latter is also used as an accelerator in the
reaction. The amount of the initiating agent is above 0.001 wt % of
the macromer, generally in the range of 0.01 wt %.about.8 wt %. The
"initiating agent" refers to a single initiator, or a mixture of
initiators, a co-catalyst or an accelerator. In some cases, it is
advantageous to use a redox system for polymerization, because the
associated free radical initiation may be triggered at a reasonable
rate over a wide range of temperatures, and may even be triggered
at low temperature of between 0-20.degree. C.
[0048] Alternatively, polymerization may also be performed using
thermal initiation, where the initiator is azoisobutyronitrile
(AIBN) or benzoperoxide (BPO), etc.
[0049] During the process of polymerization, the continuous phase
is generally a water-immiscible organic solvent selected from the
group consisting of heptane, octane, cyclohexane, toluene,
dimethylbenzene etc., and a mixture thereof. The dispersion phase
is generally a hydrophilic solvent selected from the group
consisting of water, an aqueous solution, a hydrophilic solvent, a
hydrophilic solution, and a mixture thereof.
[0050] During the process of polymerization, the W/O nonionic
emulsifier is added to the organic solvent. The emulsifier is
selected from the group consisting of Span, Tween or their
mixtures, at a weight ratio of between 100.about.50/0.about.50,
preferably 100.about.80/0.about.20. Other nonionic emulsifier which
can form a W/O emulsion may also be used.
[0051] During the polymerization process, the amount of emulsifier
used ranges from 1 wt % to 40 wt %, preferably from 5 wt % to 15 wt
%. The amount of the macromer in solution ranges from 1 wt % to 49
wt %, preferable from 7 wt % to 35 wt %. The reaction temperature
is in the range of 20-100.degree. C., generally 45.about.80.degree.
C. with a stirring speed is in the range from 60 to 2000 rpm. The
speed of stirring may be constant or varied during the process. The
reaction time is usually above 30 minutes, generally in the range
of about 0.5.about.8 hours. The microgel formed is collected by
filtration, washed several times with acetone and water and then
freeze dried or stored at a low temperature. The particle sizes of
the microgel prepared in accordance with the present invention is
from 5 nanometers to 5 millimeters.
[0052] During the process of polymerization, an initiator can be
added to the flask of the macromer solution held in the bath set at
the polymerization temperature. Since the polymerization
temperature is usually above the phase transition temperature, the
macromer solution might have physically gelated while the microgel
particles are formed. This may result in the lack of well formed
dispersed particles. In order to avoid physical gelation in the
macromolecule solution before microparticle formation, the
initiating agent can also be added into the flask together with
macromolecule solution but at a lower temperature. The flask may
then be heated to trigger polymerization only after the macromer
solution has been well dispersed. The initiator can also be added
in a different manner, for instance, as an initiator or accelerator
solution into the continuous phase after the disperse phase of the
macromer solution has formed.
[0053] The cross-linking may take place with a mixture of different
macromers of the said (FDC*) structure.
[0054] The "particles" of "microgel" provided in accordance with
the present invention can be stored in bulk or as a solvent
mixture. The suitable solvent may be an aqueous or an organic
solvent. Generally, water or an aqueous solution is used as the
medium to swell the particles to form a hydrogel. The microgel
particles formed in accordance with the present invention may be a
xerogel, without a solvent or in a solvent. When the particles are
in a hydrogel state, the solvent used is selected from the group
consisting of distilled water, a buffer solution, a body fluid, a
cell culture fluid, a tissue culture solution, or any other aqueous
solution or solvent. The solvent used should not comprise an
organic solvent as a major or principal part. The solvent may be a
mixture.
[0055] It is understood that there might be some free ends in a
chemically cross-linked network, wherein only one end of the chain
is connected with an infinite cross-linked network. The
macromolecule provided in the present invention can comprise
0.about.49 wt % of a block co-polymer with a double-bond at only
one end of the molecule. Because of the possibility of the presence
of some macromers with only a single crosslinkable end double bond,
or less than 100% conversion of the double double-bond-ended
macromers, it is inevitable and allowable for the microgel to
comprise some dangling unpolymerized ends within the chemical gel
network synthesized in accordance with the present invention. The
chemically cross-linked network of the microgel of the present
invention may comprise 0-49 wt % of structures with a dangling
unpolymerizable end.
[0056] In the microgel provided by the present invention, the
amount of the temperature-sensitive polymer or oligomer F
comprising the principal part is in the range of 51 wt % to 99.9 wt
%, while the biodegradable moiety or oligomer R.sub.1 (or R.sub.2
or R.sub.3) or their copolymer is present in an amount in the range
of 0.1 wt % to 49 wt %.
Drug Loading in the "Intelligent" Microgel
[0057] It is particularly advantageous that "intelligent" microgels
are used for the controlled delivery of drugs, especially for
bio-active agents such as proteins, including growth factors, etc.
The term "intelligent" herein refers to a microgel that has a
negative temperature sensitivity, i.e., the gel swells at a low
temperature and contracts at a temperature that is above the "phase
transition temperature" of the microgel. Such a temperature
sensitivity at high temperature leads to "physical gellation" in
this invention.
[0058] Conventional protein loading by in situ polymerization
suffers from being conducted at a high temperature in the presence
of organic solvents. These two conditions (organic solvent and high
temperature) detrimentally affect the activity of the drug and
sometimes even denature or degrade the protein drug. Moreover, when
a drug solution is adsorbed into a conventional, known hydrogel,
the amount of drug loaded is a serious limitation, in that a low
loading level of less than 0.1 wt % is achieved according to
Bromberg et al., (1998) Adv. Drug. Deliv. 31: 197-221).
[0059] A further problem is the difficulty of controlling the pore
sizes in the microgel network, to permit the proteins to penetrate
easily into the microgels and at the same time prevent the protein
from leaching out from the microgel. To overcome these incompatible
requirements, the principles governing chemical gelation and
physical gelation have been taken into consideration together to
develop the `intelligent` microgels of the present invention. The
incompatible requirements were solved by taking advantage of the
intelligent design of a suitable material that has the desirable
properties of a chemical gel and a physical gel.
[0060] Chemical gels are known to be permanent gels with covalently
cross-linked infinite networks. The gelation process is not
reversible in most of cases. Whereas, physical gels are formed due
to a change in a physical parameter, such as a temperature change.
In such cases, the process of gelation is usually reversible.
[0061] The microgels provided in the present invention are
thermosensitive. They are chemical gels at a low temperature but
are both chemical and physical gels simultaneously at a higher
temperature, such as the body temperature. The particle sizes of
the microgel change remarkably, i.e., the microgel particles
shrink, when the temperature is higher than the phase transition
temperature. In a gel with negative or reverse temperature
sensitivity, a protein may be absorbed into the gel below the phase
transition temperature.
[0062] When the temperature is raised over the phase transition
temperature, the effective pore size of the gel network decreases
and the protein absorbed therein are entrapped or kept in the
microgel. In this case the network further gelates in response to a
change in temperature. This is physical gelation causing the
substance to be entrapped in the network.
[0063] Straightforward drying after absorbing the substance such as
a drug is, of course, an alternative approach to keep the drug
within the microgel. Drying after raising temperature following
absorbing the drug at a low temperature is also necessary in
storage of the drug-loaded microgels, as usual.
[0064] If the size of the protein is below the pore size of the
microgel, the protein can enter relatively easily into microgel at
a low temperature. When the microgel is heated to body temperature,
the protein is entrapped in the microgel and is prevented from
easily leaching out from the gel. It may be said that the protein
molecules are entrapped within the microgel at the body temperature
and thus may be released gradually.
[0065] The microgels of the present invention are biodegradable.
The rate of degradation of the microgels of the present invention
can be adjusted by selecting a suitable polyester or copolyester.
The loaded protein or other adsorbed substance is thus released
through diffusion and the degradation of the microgel.
[0066] The loaded substance may be any molecule or molecular
mixture which does not react chemically with the microgel. A
biologically active molecule is preferred, and may be any
biomacromolecule or a derivative thereof. The solvent for
biologically active molecules is usually water or an aqueous
solution such as a PBS solution. The microgels of the present
invention are particularly suitable for the controlled release of a
protein, such as a growth factor.
[0067] In the present invention, the temperature sensitive macromer
constitutes the principal part of the biodegradable microgel
particles which might be used for loading the protein. It is
contemplated that the microgels of the present invention are to be
used not only for the controlled release of cell growth factors in
tissue engineering, but may be a non-protein, a macromolecular drug
or other material for which controlled release is desired.
[0068] The loaded microgels may be preserved at a low temperature
or freeze dried and preserved at a low temperature. The activity of
the loaded substance, particularly of a protein, can therefore be
maintained easily and conveniently.
[0069] Compared with other carriers used in the controlled release
of a drug, the biodegradable temperature-sensitive microgels
provided in the present invention exhibit the following
characteristics: [0070] 1. The hydrogel is in the form of an
intelligent microgel and is biodegradable. [0071] 2. The microgel
basically is composed of a synthetic polymer, principally a block
copolymer with good biocompatibility. [0072] 3. The microgel
provided in the present invention is prepared by inverse suspension
polymerization from macromers comprising a thermosensitive central
part linked with a biodegradable moiety, and polymerizable end(s).
[0073] 4. The microgel provided in the present invention is
temperature-sensitive. Because of this unique characteristic, a
drug or an entrapped material, usually another macromolecular
material, such as a protein, can be encapsulated AFTER the microgel
has formed. So, during the drug loading process, the protein drug
is not exposed to any organic solvents or a temperature that is
higher than the human body temperature or that of a warm-blood
animal. [0074] 5. In addition, because drug loading is performed
after the microgel has formed, any residual starting material
and/or initiator within the formed microgel can be thoroughly
removed by washing to ensure the biocompatibility of the microgels.
[0075] 6. The microgel is uncharged and is biodegradable primarily
by hydrolysis. Thus, aside from pore size, there are no stringent
selection requirements for the substance that is desired to be
loaded. The encapsulation and release mechanism is universally
applicable to any macromolecular drugs and may even be potentially
useful for other substances that are to be applied in other areas
beyond Pharmacology where the controlled release of biologically
active macromolecules and their derivatives is desired. [0076] 7.
The sizes of the microgel particles of the present invention can be
controlled by adjusting the stirring speed and the concentration of
the emulsifier. [0077] 8. The microgels provided in the present
invention possess the characteristics of both a chemically
cross-linked gel and a physical gel. The phase transition
temperature associated to physical gelation of the microgels of the
present invention is adjustable by changing the ratio of the
various macromers in the block co-polymer or other properties of
the macromers, such as chain length, etc. [0078] 9. The effective
pore size of the microgel network provided in accordance with the
present invention can be controlled by selecting the molecular
weight of the central block copolymer and by varying the amounts of
the various components of the macromers or varying the macromer
mixtures. [0079] 10. The degradation rate of microgel provided in
the present invention is also controllable by selecting different
polyesters to be co-polymerized with the temperature sensitive
macromer, and by controlling their ratio in the copolymer. The
degradation rate is generally not affected by the rate of gelation.
[0080] 11. The microgels provided in accordance with the present
invention are intelligent and biodegradable. Furthermore, the
characteristics of the microgel, such as degradation rate, pore
size, etc, can be adjusted independently. Consequently, the
microgels of the present invention can be easily manipulated for
desirable loading and release of various macromolecules. The
technology is highly flexible and versatile.
[0081] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLES
[0082] Materials
[0083] PEO, PPO tri-block copolymers and stannous octoate were
obtained from Sigma Aldrich; lactide, glycolide and caprolactone
were obtained from PURAC and ACROS Organics respectively.
Purification of ammonium persulphate and sodium bisulfite was
carried out by the method of recrystallization. Dichloromethane was
distilled prior to use.
[0084] Characterization
[0085] FTIR was carried out using Magna-550 instrument and 500 MHz
proton was carried out using SF-500 instrument.
Example 1
Preparation of a Macromer
[0086] A 50 ml ampoule was flame dried under vacuum. 20 g of
PEO.sub.100-PPO.sub.65-PEO.sub.100 (the number in the subscript
denotes the associated averaged degree of polymerization), 0.92 g
of l-lactide and 1.3 ml of 10 mg/ml stannous octoate toluene
solution were charged into the tube. After mixing the contents
well, the tube was preheated to 60.degree. C. for 6 hours under
repeated cycles of vacuum and drying in argon or nitrogen to remove
any volatile materials. The tube was then sealed off under vacuum
(0.1 mmHg) and the mixture was copolymerized at 140.degree. C. for
24 hours. The resultant copolymer was dissolved with
dichloromethane, then precipitated with excess anhydrous ether
(-10.about.0.degree. C.), and recovered by filtration. The dried
copolymer was then used for subsequent reactions. Other polymers
were similarly synthesized using d,l-lactide or
.epsilon.-caprolactone and calcium hydroxide in place of glycolide
and stannous octoate and different PEO-PPO tri-block copolymers
with different ratios of three components.
[0087] The above dried PEO.sub.100-PPO.sub.65-PEO.sub.100-l-lactide
copolymer was dissolved in dichloromethane in 250 ml round bottom
flask. A ten fold molar excess, based on the molecular weight of
the copolymer, of Triethylamine was added to the flask under the
protection of dried nitrogen. Acryloyl chlorine in the same amount
as triethylamine was added dropwise to the flask. The reaction
mixture was stirred hours in an ice bath, followed by stirring for
12 hours or more at room temperature. After the reaction mixture
was filtered to remove any insoluble triethylamine salt, the
macromolecule was obtained by pouring the filtrate into a large
excess of anhydrous ether (-10.about.0.degree. C.) and recovering
the precipitate by further filtration. Finally, the precipitate was
dried in vacuum oven for two or three days. The macromer obtained
was called (PEO.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.4-DA
(LA.sub.4 denotes that there are, on average, about 4 lactic-acid
repeating units on each end of the block copolymer based upon the
feed molar ratio, DA denotes di-acryloyl groups, namely, each
acryloyl group at one end).
Production of Microgels Using a REDOX Initiating System
[0088] Inverse suspension polymerization reactions were performed
in a 250 ml four-neck flask, fitted with a reflux condenser and a
mechanical Teflon stirrer and under an inert atmosphere of
nitrogen. The continuous phase comprised 200 ml of heptane and an
emulsifier Span 60, in an amount of 4 wt % of heptane. The disperse
phase was 10 ml of 20% w/w of the macromolecule, with ammonium
persulphate and TEMED. After the flask was heated to 60.degree. C.
using an oil bath, the aqueous disperse phase was added dropwise at
a rate of 1-2 drops per second into the flask. (3 wt % ammonium
persulphate based on the amount of the macromolecule and 15 .mu.l
of the accelerator, TEMED were dissolved in the macromolecule
solution 30 sec. before adding the disperse phase into the
flask.)
[0089] The reaction was allowed to proceed for one hour at a
stirring speed of 450 rpm. The resultant microgel was separated
from the solvent with a standard sieve. The sieved microgel was
washed firstly with acetone, followed by distilled water. After
freeze drying, the particles sizes of the microgel obtained were in
the range of 10-150 .mu.m. When the microgel was allowed to swell
in distilled water at 37.degree. C., the particle sizes increased
to 10-200 .mu.m. The particle sizes further increased upon lowering
of the temperature to 4.degree. C. The morphology of the swollen
microgel is shown in FIG. 2. The influence of temperature on the
sizes of microgel particles was also investigated using a
microscope provided with a device for controlling the temperature.
The result is shown in FIG. 3, where the average volume or size of
the microgel particles at 4.degree. C. was taken as a control. The
sizes of the microgel particles dropped sharply between 15.degree.
C. to 20.degree. C.
Characterization of Macromers
[0090] An FTIR spectrum of the macromolecule was measured and
calibrated. The strong absorption at 1730 cm.sup.-1 demonstrates
the presence of the lactide ester, the weak absorption at 1560
cm.sup.-1 shows the presence of acrylic double bonds at the end
groups.
[0091] A 500 MHz proton spectrum was recorded on an SF-500
instrument. The peaks at 1.13 ppm and 3.6 ppm reflect the presence
of --CH.sub.3 and --CH.sub.2 in PPO and PEO respectively, and the
peaks at 5.2 ppm and 1.4 ppm are attributable to the presence of
--CH and --CH.sub.3 in the lactic acid.
Example 2
[0092] The procedure employed is similar to that in Example 1.
After a 50 ml ampoule was flame dried under vacuum,
PEO.sub.129-PPO.sub.56-PEO.sub.129, .epsilon.-carprolactone and
stannous octoate were charged into the tube (the molar ratio of
three components is 1:8:0.02). After the contents were well-mixed,
the tube was preheated to 60.degree. C. for 6 hours under repeated
cycles of vacuuming and drying with argon or nitrogen to remove
volatile materials. The tube was then sealed under vacuum (0.1
mmHg), and the mixture was copolymerized at 150.degree. C. for 24
hours. Then the copolymer in dichloromethane was allowed to react
with methylacryloyl chlorine and triethylamine. After filtration to
remove solid materials, the filtrate was precipitated with excess
anhydrous ether (-10.about.0.degree. C.) and the precipitate was
recovered by filtration and the dried macromolecular polymer
obtained was the (PEO.sub.129-PPO.sub.56-PEO.sub.129)-CL.sub.4-DMA
(CL.sub.4 denotes that there are, on average, about 4
.epsilon.-carprolactone repeating units on each end of the block
copolymer based upon the feed molar ratio; DMA denotes
di-methylacryloyl groups, namely one methylacryloyl group at each
end).
[0093] For the inverse suspension polymerization process, the
continuous phase comprised dimethylbenzene and an emulsifier
composed of Span 60 and Tween 80 in a weight ratio of 80:20 After
the flask was heated to 80.degree. C., a disperse phase of an
aqueous solution of the
(PEO.sub.129-PPO.sub.56-PEO.sub.129)-CL.sub.4-DMA macromers was
added dropwise at a rate of 1-2 drops per second into the flask.
The amount of the disperse phase was 6 wt % of the continuous
phase. The reaction was carried out for half an hour at a stirring
speed of 550 rpm. The microgel was separated from the liquid phase
with a sieve and washed firstly with acetone, followed by distilled
water. The sizes of the microgel particles ranged from 20-100
.mu.m.
Example 3
[0094] The procedure used is similar to that described in Example 1
The macromer (PEO.sub.103-PPO.sub.39-PEO.sub.103)-LA.sub.16-DA was
obtained by feeding PEO.sub.103-PPO.sub.39-PEO.sub.103, l-lactide
and stannous octoate in a molar ratio of 1:16:0.02 before
di-acryloylated. For the inverse suspension polymerization process,
the continuous phase comprises heptane and an emulsifier composed
of Span 80 and Tween 80 in a weight ratio of 84:16. The amount of
the emulsifier was 8 wt % of heptane. While the solution was
stirred under nitrogen for 30 min in an ice bath, the aqueous
disperse phase containing 15% w/w macromers was added dropwise at a
rate of 1-2 drops per second into the flask. Then 3 wt % ammonium
persulphate based on the macromer and 10 wt % sodium bisulfite
based on the macromer were added into the flask. The amount of the
disperse phase was 8 wt % of the continuous phase. After 30 min,
the flask was heated to 50.degree. C., and then held at this
temperature for 1 hour at a stirring speed of 450 rpm. The microgel
was separated from the mixture with a sieve. The sieved microgel
was washed firstly with acetone, followed by distilled water. The
resultant microgel particles were in the range of 20-150 .mu.m.
Example 4
[0095] The procedure is similar to that in Example 1. The macromer
(PEO.sub.103-PPO.sub.39-PEO.sub.103)-(CL.sub.4-LA.sub.4)-DA was
obtained by feeding PEO.sub.103-PPO.sub.39-PEO.sub.103,
.epsilon.-carprolactone, l-lactide and calcium hydroxide in a molar
ratio of 1:8:4:2 followed by di-acryloylation. During the process
of inverse suspension polymerization, the continuous phase
comprised toluene and a mixed emulsifier (Span 80 and Tween 80 in
weight ratio of 90:10). The amount of the emulsifier was 12 wt % of
toluene. Then, 20 wt % of
(PEO.sub.103-PPO.sub.39-PEO.sub.103)-(CL.sub.4-LA.sub.4)-DA
macromer in an aqueous solution was added dropwise at a rate of 1-2
drops per second into the flask. The suspension was stirred under
nitrogen in an ice bath at a speed of 1000 rpm. The amount of the
disperse phase was 10 wt % of the continuous phase. After 30
minutes, the flask was heated to 70.degree. C. at a stirring speed
of 360 rpm. Then a 3 wt % ammonium persulphate based on the
macromolecule in 400 .mu.l TEMED was added into the flask. The
reaction was allowed to proceed for 1 hour. The microgel was
separated from the reaction mixture with a sieve and washed with
acetone and then by distilled water. The sizes of the microgel
particles ranged from 30-150 .mu.m.
Example 5
[0096] The continuous phase comprised heptane and an emulsifier
Span 60, in an amount of 10 wt % heptane. After the flask was
heated to 60.degree. C. using a thermostatted oil bath, a mixture
of 10 wt % of (PEO.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA and
13 wt % of (PEO.sub.129-PPO.sub.56-PEO.sub.129)-CL.sub.4-DA in
aqueous solution was added dropwise at a rate of 1-2 drops per
second into the flask. The disperse phase was 30 wt % of the
continuous phase. The reaction was allowed to proceed for one hour
with a stirring speed of 240 rpm. The microgel was separated with a
sieve and washed firstly with acetone, followed by distilled water.
After freeze drying, the sizes of the prepared microgel particles
ranged from 20-150 .mu.m.
Example 6
[0097] The procedure is similar to that in example 1. The macromer
(PEO.sub.100-PPO.sub.65-PEO.sub.100)-CL.sub.8-DMA was obtained by
feeding PEO.sub.100-PPO.sub.65-PEO.sub.100, .epsilon.-carprolactone
and stannous octoate in molar ratio of 1:16:0.2 followed by
di-methyl acryloylation. During the inverse suspension
polymerization process, the continuous phase comprised hexane and a
mixed emulsifier (Span 80 and Tween 80 in weight ratio of 75:25)
and AIBN (1 wt % of the macromer). The amount of the emulsifier was
8 wt % of hexane. Then, 20 wt % of the macromer
(PEO.sub.100-PPO.sub.65-PE.sub.100)-CL.sub.8-DMA in an aqueous
solution was added dropwise at a rate of 1-2 drops per second into
the flask. The amount of the disperse phase was 10 wt % of the
continuous phase. The solution was stirred under nitrogen for 30
min in an ice bath, then the flask was heated to 60.degree. C. with
a stirring speed 400 rpm. The reaction was allowed to proceed for 4
hours. The microgel was separated with a sieve and washed with
acetone and then distilled water. The sizes of the microgel
particles ranged from 30-250 .mu.m.
Biodegradability
[0098] Biodegradation of the microgels is essential for its use as
a biomedical material. The rate of degradation also determines to a
large extent the release rate of the loaded material. The
biodegradation of the microgels in accordance with the present
invention mainly takes place through the hydrolysis of the ester
bond. FIG. 4 presents the hydrolysis data for two microgels in PBS
at 37.degree. C.
Example 7
Encapsulation and Release of a Protein (Bovine Serum Albumin)
[0099] 2.8 ml of a 20 mg/ml Bovine serum albumin (BSA) aqueous
solution was slowly added at 4.degree. C. dropwise into 200 mg of a
dried microgel prepared from the
(PEO.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA macromer. Swelling
took place for 48 hours at 4.degree. C. followed by 24 hours at
37.degree. C. The resultant loaded microgel was freeze dried.
[0100] The microgel loaded with the BSA protein was placed in a
flask containing 40 ml of a phosphate-buffer solution (PBS, 0.1M,
pH 7.4) and incubated at 37.degree. C. At appropriate intervals
shown in FIG. 5, 1.0 ml of the aqueous solution was collected and
1.0 ml of fresh PBS was added to the flask. The amount of BSA
released was assayed by the Lowry method using an ultraviolet
spectrophotometer. The release profile for BSA is shown in FIG. 5.
The cumulative BSA released was 73% after 120 hr, from a drug
loading level of 20 wt % of BSA in the microgel.
Example 8
Insulin Release Assay
[0101] Insulin was dissolved in PBS at a concentration of 20 mg/ml.
Dry microgel was loaded by equilibration with an excess of the
buffered solutions of insulin. The loaded microgels were placed in
a flask containing 40 ml of PBS and incubated at 37.degree. C. At
appropriate intervals shown in FIG. 6, 1.0 ml of the aqueous PBS
solution was collected and 1.0 ml of fresh PBS was added to the
flask. The amount of insulin released was assayed by a Bradford
method using an ultraviolet spectrophotometer. The release profile
for Insulin is shown in FIG. 6.
Example 9
Toxicity of Microgels
[0102] The toxicity of the microgels described herein was evaluated
by intramuscular injection of the
(PEO.sub.100-PPO.sub.65-PEO.sub.100)-LA.sub.8-DA microgel in the
skeletal muscle of SD rats.
[0103] The rats were purchased from the College of Medicine of
Fudan University. The procedure used was as follows. Fifteen SD
rats having an average weight of 250 g were divided into five
groups. Each group consists of three rats. The test microgels were
sterilized using alcohol aqueous solution in cell-culture room.
After a sterilized microgel suspension of the microgel in
physiological saline (20 mg/ml) was prepared, 0.5 ml of the
suspension was injected into the skeletal muscle of each rat. Each
rat received two injections. The injected microgels along with
their surrounding tissues were retrieved by surgical incision after
1, 3, 5, 7, 9 days. The retrieved samples were processed for
histological examination by paraffin embedment using hematoxylin
and eosin (H & E) dye.
[0104] One-day after injection some inflammatory cells surrounding
the tissues at the injection site were observed. However, there was
no striking acute inflammatory reaction observed. After three days,
the inflammatory cells almost disappeared. The microgel completely
degraded and disappeared after 7 days.
[0105] It is obvious to those of skill in the art that
modifications and variations can be made to the composition, the
structure of the macromers, the methods of preparing the macromer
and/or microgel, the resultant external shape and internal
structure of the microgel, the ways to encapsulate and release the
entrapped substances in the microparticles, based on the forgoing
description. Such modifications and variations are intended to be
within the scope of the following claims.
LIST OF REFERENCES
[0106] U.S. Patent Documents TABLE-US-00001 1. 4,352,883 October
1982 Lim 435/178 2. 4,716,203 December 1987 Casey et al. 525/408 3.
5,986,043 November 1999 Hubbell et al. 528/354 4. 6,030,634
February 2000 Wu et al. 424/423 5. 6,060,582 September 2000 Hubbell
et al. 528/354 6. 6,129,761 October 2000 Hubbell et al. 623/11 7.
6,306,922 October 2003 Hubbell et al. 522/71 8. 6,541,033 April
2003 Shah 424/486 9. 0,099,709 May 2003 Shah et al. 424/469
[0107] Other Publications [0108] 10. Baldwin S P., Saltzman W M.,
"Materials for Protein Delivery in Tissue Engineering," Adv. Drug.
Deliv. Rev. 33: 71-86 (1998) [0109] 11. Blanco D., Alonso M J.,
"Protein Encapsulation and Release from Poly(lactide-co-glycolide)
Microspheres: Effect of the Protein and Polymer Properties and of
the Co-encapsulation of Surfactants," Eur. J. Pharm. Biopharm. 45:
285-294 (1998) [0110] 12. Bromberg L E., Ron E S.,
"Temperature-responsive Gels and Thermogelling Polymeric Matrices
for Protein and Peptide Delivery," Adv. Drug. Deliv. Rev. 31:
197-221 (1998) [0111] 13. Cho K Y., Choi S H., Kim C H., Nam Y S.,
Park T G., Park J K., "Protein Release Microparticles Based on the
Blend of Poly(d,l-lactic-co-glycolic acid) and Oligo-ethylene
glycol Grafted Poly(l-lactide)," J. Control. Release 76: 275-284
(2001) [0112] 14. Cohen S., Yoshioka T., Lucarelli M., Hwang L H.,
Langer R., "Controlled Delivery Systems for Proteins Based on
Poly(lactic/glycolic acid) Microspheres," Pharm. Res. 8: 713-720
(1991) [0113] 15. Fu J., Zhou R X., Fan C L., "Studies on the
Syntheses and Properties of Poly(ester-anhydride)s for DDS,"
Chemical Journal of Chinese Universities 19(5): 813-816 (1998)
[0114] 16. Fu J., Zhou R X., Fan C L., "Studies on the Syntheses
and Drug Release Properties of Polyanhydrides Containing
Phosphonoformic (or Acetic) Acid Ethyl Ester in the Main Chain."
Chemical Journal of Chinese Universities 18(10): 1706-1710 (1997)
[0115] 17. Jain R A., Rhodes C T., Railkar A M., Malick A W., Shal
N H., "Controlled Release of Drugs from Injectable in Situ Formed
Biodegradable PLGA Microspheres. Effect of Various Formulation
Variables," Eur. J. Pharm. Biopharm. 50: 257-262 (2000) [0116] 18.
Jeong B., Bae Y H., Lee D S., Kim S W., "Biodegradable Block
Copolymers as Injectable Drug-delivery Systems," Nature 388:
860-862 (1997) [0117] 19. Langer R., "Drug Delivery and Targeting,"
Nature 392: 5-10 (1998) [0118] 20. Lanza R P., Langer R., Vacanti
J., "Principles of Tissue Engineering (2.sup.nd Ed.). Academic
Press, New York, 2000 [0119] 21. Li M X., Zhuo R X., Qu F Q.,
"Synthesis and Characterization of Novel Biodegradable Poly(ester
amide) with Ether Linkage in the Backbone Chain," J. Polym. Sci.
part A: Polym. Chem. 40(24): 4550-4555 (2002) [0120] 22. Sawhney A
S., Hubbell J A., "Rapidly Degraded Teroplymers of dl-lactide,
glycolide, and .epsilon.-caprolactone with Increased Hydrophilicity
and Copolymerization with Polyethers," J Biomed. Mater. Res.
24(10): 1397-1411 (1990) [0121] 23. Sawhney A S., Pathak C P.,
Hubbell J A., "Bioerodible Hydrogels Based on Photopolymerized
Poly(ethyleneglycol)-co-poly(o-hydroxy acid) Diacrylate Macromers,"
Macromolecules 26: 581-587 (1993) [0122] 24. Singh M., Shirley B.,
Bajwa K., Samara E., Hora M., O'Hagan D., "Controlled Release of
Recombinant Insulin-like Growth Factor from a Novel Formulation of
Polylactide-co-glycolide Microparticles," J. Control. Release 70:
21-28 (2001) [0123] 25. Wu C., Zhou S Q., Thermodynamically Stable
Globule State of a Single Poly(N-isopropylacrylamide) Chain in
Water," Macromolecules 28(15): 5388-5390 (1995) [0124] 26. Wu C.,
Zhou S Q., "Internal Motions of Both Poly(N-isopropylacrylamide)
Linear Chains and Spherical Microgel Particles in Water,"
Macromolecules 29(5):1574-1578 (1996) [0125] 27. Yasuhiko T., "The
Importance of Drug Delivery Systems in Tissue Engineering,"
Pharmaceutical Science & Technology Today 3: 80-89 (2000)
[0126] 28. Zentner G M., Rathi R., Shin C., McRea J C., Seo M H.,
Oh H., Rhee B G., Mestecky J., Moldoveanu Z., Morgan M., and
Weitman S., "Biodegradable Block Copolymers for Delivery of
Proteins and Water-insoluble Drugs," J. Control. Release 72:
203-215 (2001) [0127] 29. Zhuo R X., Li W., "Preparation and
Characterization of Macroporous Poly(N-isopropylacrylamide)
Hydrogels for the Controlled Release of Proteins," J. Polym. Sci.
part A: Polym. Chem. 41(1): 152-159 (2003) [0128] 30. Zhu Z X.,
Xiong C D., Zhang L L., Yuan M L., Deng X M., "Preparation of
Biodegradable Polylactide-co-poly(ethylene glycol) Copolymer by
Lactide Reacted Poly(ethylene glycol)", Eur. Polym. J. 35:
1821-1828 (1999)
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