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US7615593B2 - Bifunctional-modified hydrogels - Google Patents
US7615593B2
US7615593B2 US10128198 US12819802A US7615593B2 US 7615593 B2 US7615593 B2 US 7615593B2 US 10128198 US10128198 US 10128198 US 12819802 A US12819802 A US 12819802A US 7615593 B2 US7615593 B2 US 7615593B2
US10128198
US20030083389A1 (en )
Weiyun John Kao
Rathna Gundloori
Priority is hereby claimed to provisional application Ser. No. 60/285,782, filed 23 Apr. 2001, the entire contents of which is incorporated herein.
This invention was made with United States government support awarded by the following agencies: NIH HL63686. The United States has certain rights in this invention.
As noted above, the α- and/or ω-termini of the hydrogel may be substituted or unsubstituted. When substituted, it is preferred that the substitution is a moiety selected from the group consisting of halo, hydroxy, C1-C24-alkyl, C1-C24-alkenyl, C1-C24-alkynyl, C1-C24-alkoxy, C1-C24-heteroalkyl, C1-C24-heteroalkenyl, C1-C24-heteroalkynyl, cyano-C1-C24-alkyl, C3-C10-cycloalkyl, C3-C10-cycloalkenyl, C3-C10-cycloalkynyl, C3-C10-cycloheteroalkyl, C3-C10-cycloheteroalkenyl, C3-C10-cycloheteroalkynyl, acyl, acyl-C1-C24-alkyl, acyl-C1-C24-alkenyl, acyl-C1-C24-alkynyl, carboxy, C1-C24-alkylcarboxy, C1-C24-alkenylcarboxy, C1-C24-alkynylcarboxy, carboxy-C1-C24-alkyl, carboxy-C1-C24-alkenyl, carboxy-C1-C24-alkynyl, aryl, aryl-C1-C24-alkyl, aryl-C1-C24-alkenyl, aryl-C1-C24-alkynyl, heteroaryl, heteroaryl-C1-C24-alkyl, heteroaryl-C1-C24-alkenyl, heteroaryl-C1-C24-alkynyl, sulfonate, arylsulfonate, and heteroarylsulfonate.
Moreover, these moieties themselves may be further substituted. Thus, the moieties on the α-terminus and the ω-terminus when substituted bear a substituent selected from the group consisting of alkyl, aryl, acyl, halogen, hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, saturated cyclic hydrocabon, unsaturated cyclic hydrocarbon, heterocycle, aryl, and heteroaryl.
wherein at least one of the “A” or “Z” moieties is covalently bonded to the reactive amino moieties of the polymer matrix; and wherein “A” and “Z” are independently selected from the group consisting of hydrogen, halo, hydroxy, C1-C24-alkyl, C1-C24-alkenyl, C1-C24-alkynyl, C1-C24-alkoxy, C1-C24-heteroalkyl, C1-C24-heteroalkenyl, C1-C24-heteroalkynyl, cyano-C1-C24-alkyl, C3-C10-cycloalkyl, C3-C10-cycloalkenyl, C3-C10-cycloalkynyl, C3-C10-cycloheteroalkyl, C3-C10-cycloheteroalkenyl, C3-C10-cycloheteroalkynyl, acyl, acyl-C1-C24-alkyl, acyl-C1-C24-alkenyl, acyl-C1-C24-alkynyl, carboxy, C1-C24-alkylcarboxy, C1-C24-alkenylcarboxy, C1-C24-alkynylcarboxy, carboxy-C1-C24-alkyl, carboxy-C1-C24-alkenyl, carboxy-C1-C24-alkynyl, aryl, aryl-C1-C24-alkyl, aryl-C1-C24-alkenyl, aryl-C1-C24-alkynyl, heteroaryl, heteroaryl-C1-C24-alkyl, heteroaryl-C1-C24-alkenyl, heteroaryl-C1-C24-alkynyl, sulfonate, arylsulfonate, and heteroarylsulfonate; “m” is an integer of from 2 to 8; and “n” is an integer equal to or greater than 100. In the preferred embodiment, “m” equals 2 and “n” is greater than 2,000.
A third embodiment of the invention is directed to a method of making a hydrogel as described hereinabove. The method comprises reacting a polymer matrix with a bifunctional modifier comprising a poly(alkylene glycol) molecule having a substituted or unsubstituted α-terminus and a substituted or unsubstituted ω-terminus, whereby at least one of the α- or ω-termini is covalently bonded to the polymer matrix.
A fourth embodiment of the invention is directed to the method described in the previous paragraph, and further comprising contacting the first polymer matrix with a plurality of monomers and then polymerizing the monomers to yield a second polymer matrix, wherein the second polymer matrix interpenetrates with the first polymer matrix. This embodiment allows for the in situ formation of interpenetrating polymer networks.
FIGS. 3A and 3B. Surface hydrophilicity of the XPEGmA-co-Ac-co-TMPTA network containing XPEGmA of various concentration, terminal moiety, and molecular weight. (3A) 2 KDa XPEGmA and (3B) 5 KDa XPEGmA. Legend: ♦=M-PEG; ▪CN-PEG; ▴=COOH-PEG; and ●=PT-PEG.
Poly(alkylene glycols), such as poly(ethylene glycol) (PEG), are employed extensively in a number of medical and pharmaceutical fields due to their low toxicity, good biocompatibility, and excellent solubility (1-5). For sake of expository brevity, the following description shall be limited to gels modified by bifunctional poly(ethylene glycol) molecules. The invention, however, will function with equal success using any poly(alkylene glycol).
Using the hPEGs of the present invention, polymer networks having diverse physicochemical and surface properties were developed. These networks can be used to study cell-material interaction.(10-13)
In the Examples that follow, hPEGs were utilized to modify a polymer matrix to yield novel hydrogels. The effect of HPEG concentration, molecular weight, and terminal chemical functionality on the surface hydrophobicity and cell interaction with the hydrogels was investigated and is presented in the Examples. Multiple heterogeneous PEG modifications (e.g., carboxylic acids of the poly-acrylic acid backbone and the functional group at the dangling terminus of hPEG grafted at the pendent chain configuration) can be employed to bind several distinct types of biofunctional molecules such as peptides and pharmaceutics to the hydrogel.
“Ac”=acrylic acid
“AC”=acryloyl chloride (CAS No. 814-68-6)
“CHD”=chlorhexidine digluconate
“CN-PEG”=α-cyanoethyl-ω-acrylate-PEG
“COOH-PEG”=α-carboxyl-ω-acrylate-PEG
“EDTAD”=ethylene diaminetetracetic dianhydride
“hPEG”=heterobifunctional PEG
“IPN”=interpenetrating network hydrogels
“mPmA”=α-methyl-ω-aldehyde-PEG
“mPEG”=α-methoxy-PEG
“M-PEG”=α-methyl-ω-acrylate-PEG
“PEG” and “PEG diol”=polyethylene glycol
“PEGdA”=PEG-diacrylate
“PEG dial”=α-aldehyde-ω-aldehyde-PEG
“PT-PEG”=α-phthalimide-ω-acrylate-PEG
“TEA”=triethylamine
“THF”=tetrahydrofuran
“TMPTA”=trimethylolpropane triacrylate (i.e., 2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate, CAS No. 15625-89-5)
“XPEGmA”=hPEG with acrylate ω-terminal and α-terminal of different moiety
The term “acyl” is used to describe a ketone substituent, —C(O)R, where R is substituted or unsubstituted alkyl, alkenyl, alkynyl, or aryl as defined herein.
The term “carbonyl” is used to describe an aldehyde substituent. The term “carboxy” refers to an ester substituent or carboxylic acid, i.e., —C(O)O— or —C(O)—OH.
The PEG-diol can be directly converted, by simple halogenation of the hydroxy group to α-hydroxy-ω-halo-PEG. The PEG diol can also be tosylated and acrylated to thereby yield α-acrylate-ω-tosylated-PEG. The tosyl group can be exchanged for a succinimidyl or phthalimidyl or other nitrogen-containing heterocycle group. α-Hydroxy-ω-methoxy-PEG can be converted directly into α-acrylate-ω-methoxy-PEG. See FIG. 1. (See also Hem & Hubbell,(1998) J. Biomed. Mater. Res. 39:266-276; Morpurgo et al. (1996) App. Biochem. Biotech. 56:59-72; and Abuchowski et al. (1984) Cancer Biochem. Biophys. 7:175-186.)
The PEG molecules may also be modified to introduce other amide bonds into the molecule. The formation of an amide bond is, of course, extremely useful in modifying the PEG molecule to contain an amino acid, peptide, or protein terminus. Thus, for example α-succinimidylglutarate-ω-tryptophanylglutarate PEG can be synthesized by dissolving the peptide or amino acid in 0.1 M 2-(N-morpholino)-ethanesulfonic acid (MES) at 0° C. α,ω-Bis-N-succinimidylglutarate-PEG is added dropwise to the solution with constant stirring. The reaction is allowed to continue at 0° C. for 1 hour and then allowed to come to room temperature with constant stirring for 4 hours. The reaction solution is then dialyzed against 50 volumes of deionized water and the resulting solution lyophilized. This yields the desired α-N-succinimidylglutarate-ω-tryptophanylglutarate in rougly 40% yield.
2.12. To synthesize α-hydroxy, ω-tryptophanylglutrate PEG, a-hydroxy, w-succinimidylglutrate PEG (1 eq.) from series 2.11 was dissolved in DMF followed by addition of tryptophan (1.5 eq.). The solution was stirred under argon for 24 hrs, dialyzed in deionized water and dried by lyophilizer for 3 days.
3.9. To synthesize α-acylate, ω-triethoxysilane PEG, α-hydroxy, ω-triethoxysilane PEG (1 eq.) from series 2.7 was dissolved in dry THF followed by the addition of acryloyl chloride (1.2 eq.) and triethylamine (1.5 eq.). The solution was stirred at room temperature for 2 hr, filtered and the filtrate was precipitated in cold hexane, collected by filtration and dried in vacuum oven for 24 hr.
Thus, according to the present invention, a polymer matrix, preferably gelatin, is modified to contain one or more of the modified PEG molecules awrydisclosed herein. The PEG molecule may be bis-modified, using the same type of moiety. Or, the α-terminus of the PEG may have a different moiety than the ω-terminus. Both versions of the modified PEG molecules, as incorporated into a hydrogel, fall within the scope of the present invention.
FIG. 6 is a schematic representation of interpenetrating network hydrogels according to the present invention. The gels can contain living cells or pharmcalogically-active agents, or both.
Example 1 Synthesis and Characterization of Heterobifunctional PEGs
To synthesize α-cyanoethyl-ω-acrylate-PEGs (CN-PEG), PEG-diols (2 kDa or 5 kDa) (1 eq.) were dissolved in dry CH2Cl2 solution followed by the addition of fine sodium metal (2 eq.) stirred for 12 hr at room temperature. An excess amount of acrylonitrile was added into the solution(15,16), stirred for 12 hr, filtered, and dried by rotary evaporation. The product thus formed (i.e., α-nitrile-ω-hydroxy-PEG) was dissolved in dry THF, followed by the addition of TEA (2 eq.) and AC (4 eq.). The solution was stirred under Ar for 10 min at room temperature. Triethylammonium chloride was removed by filtration and the solvent was removed by rotary evaporation. The final product was re-dissolved into CH2Cl2, precipitated in cold hexane, filtered, and stored in vacuo at room temperature.
To synthesize α-phthalimide-ω-acrylate-PEGs (PT-PEG), PEG-diols (2 kDa or 5 kDa) (1 eq.) were dissolved in dry CH2Cl2 solution followed by the addition of TEA (4 eq.) and p-toluenesulfonyl chloride (2 eq.) (19) and stirred under Ar for 8 hr at room temperature. Solvent was removed by rotary evaporation to obtain yellowish white solids. This mixture of PEG-diols, α-hydroxyl-ω-tosyl-PEGs, and bis-tosyl-PEG (1 eq.) was dissolved in dry THF, followed by the addition of TEA (2 eq.) and AC (4 eq.), stirred at room temperature under Ar for 10 min, filtered to remove triethylammonium chloride, dried via rotary evaporation to remove solvents, re-dissolved into CH2Cl2, and precipitated in cold hexane. The solid product (mainly α-tosyl-ω-acrylate-PEG) was filtered, dried in vacuo, dissolved (1 eq.) in CH2Cl2, followed by the addition of potassium phthalimide (3 eq.)(20) and refluxed for 18 hr. The solution was filtered, dried via rotary evaporation to remove solvents, re-dissolved into CH2Cl2, precipitated in cold hexane, filtered, dried, and stored in vacuo at room temperature.
Specifically, XPEGmAs were grafted to a gelatin polymer matrix with various dangling terminal functional groups and incorporated throughout the polymer matrix by copolymerizing the acrylate terminal into a randomly polymerized network of Ac and TMPTA.(10-13) This type of polymer network containing M-PEG is nonionic, low swelling, glassy when dry, optically transparent, and colorless.(10-13) In spite of the relatively high mass fraction of M-PEGs present, minimal swelling was observed for the polymer due to the highly cross-linked and hydrophobic nature of the TMPTA network. Differential scanning calorimetry analysis showed that these materials are completely amorphous and the M-PEG component is completely phase-mixed in the cross-linked TMPTA matrix.(10)
The surface hydrophilicity of XPEGmA-co-Ac-co-TMPTA networks was quantified with an underwater air bubble captive system. The hydrogel was completely suspended in water that was maintained at a physiologically-relevant temperature of 37.5° C. An air bubble was placed at the down side of the gel and the contact angle was measured using a modified computer-assisted video contact angle system (AST Inc). Measurement was made at six randomly selected areas, averaged, and repeated three times on three different polymer samples (n=3). Because the air bubble contact angle was measured through the aqueous phase and performed under water, the value obtained is essentially the water-receding contact angle; furthermore, the higher the contact angle, the higher is the hydrophilicity of the film.
Compounds with the Following General Structure Chemical Group of
Y-C(α1)H2C(β1)H2O(CH2CH2O)nC(β2)H2C(α2)H2OH
70.4 61.3 72.4 61.3 72.4 — — — — — — — —OH
70.5 30.3 71.2 62.9 72.3 — — — — — — — —Br
70.4 19.8 66.2 62.3 72.4 119.0 — — — — — — —C(1)N
70.4 68.2 70.2 61.3 72.4 53.7 171.2 — — — — — —OC(1)H2C(2)OOH
70.4 64.4 71.8 61.6 69.7 58.2 — — — — — — —OC(1)H3
70.5 68.6 69.2 61.6 72.5 163.6 114.7 131.6 130.0 21.8 — —
Y-C(α1)H2C(β1)H2O(CH2CH2O)nC(β2)H2C(α2)H2OC(1)OC(2)HC(3)H2
70.5 64.6 71.1 63.9 68.2 165.3 130.6 128.2 58.2 — — — —OC(4)H3
70.4 18.6 66.5 64.3 68.7 165.9 130.6 128.0 117.7 — — — —C(4)N
70.5 68.5 70.8 64.6 68.4 166.0 130.9 128.2 53.6 170.3 — — —OC(4)H2C(5)OOH
70.5 37.2 67.8 170.6 63.8 68.8 167.8 130.9 128.2 168.0 132.1 123.1 133.9
Comparison of HPLC retention time, normalized peak area, and percent
conversion for M-PEG, CN-PEG, COOH-PEG, and PT-PEG synthesized
from 2K Da PEG-diol precursors
PEG Retention Normalized Conversion UV PEG Derivative
Product Time (min) Peak Area Factor (%) Signal Identification
PEG-diol 16 1.0 1 no α-methyl-ω-hydroxyl
M-PEG 21 1.0 100 strong α-methyl-ω-acrylate
CN-PEG 21 1.1 13 strong bis-ethylcyano
23 5.2 63 strong α-ethylcyano-ω-acrylate
24 2.0 24 no α-nitrile-ω-hydroxy
COOH-PEG 11 2.5 14 no bis-carboxyl
13 2.3 13 no α-carboxyl-ω-hydroxyl
15 10.1 57 weak α-carboxyl-ω-acrylate
16 1 6 no bis-hydroxyl
19 1.2 7 weak α-hydroxyl-ω-acrylate
23 0.6 3 weak bis-acrylate
PT-PEG 22 2.0 7 weak α-tosyl-ω-acrylate
24 8.1 26 strong bis-phthalimide
26 19.5 64 strong α-phthalimide-ω-acrylate
These heterobifunctional intermediates and final products of XPEGmA are stable under storage in vacuo at room temperature and can be modified further by a broad range of chemical methods for various applications. For example, the phthalimide group is a good protecting group that can be hydrolyzed to form
Surface hydrophilicity of the XPEGmA-co-Ac-co-TMPTA network
containing XPEGmA of various concentration, molecular weight, and
terminal moiety
XPEGmA XPEGmA concentration in the network formulation (g/ml)
type 0.2 0.4 0.8 1.25 2.5
2 K (Da)
M-PEG 37 ± 8 34 ± 6 37 ± 4 34 ± 2 29 ± 5
CN-PEG 46 ± 4 32 ± 2† 36 ± 5† 37 ± 2† 39 ± 2†§
COOH-PEG 44 ± 3 42 ± 6 38 ± 6† 46 ± 7§ 43 ± 4§
PT-PEG 23 ± 4§ 45 ± 4†§ 40 ± 6† 38 ± 2† 41 ± 2†§
5 K (Da)
M-PEG 41 ± 6 45 ± 6‡ 51 ± 7‡ 42 ± 5‡ 47 ± 1‡
CN-PEG 46 ± 5 32 ± 2†§ 36 ± 7†§ 37 ± 1†§ 39 ± 3†§
COOH-PEG 51 ± 3‡§ 42 ± 2† 39 ± 1†§ 43 ± 4† 44 ± 4†
PT-PEG 46 ± 4‡ 46 ± 1 51 ± 7 40 ± 3† 39 ± 2†§
Adherent human dermal fibroblast density on the XPEGmA-co-Ac-co-TMPTA
network containing XPEGmA of various concentration, molecular weight, and terminal moiety
2 hr 24 hr 48 hr
type 0.2 0.4 0.8 1.25 2.5 0.2 0.4 0.8 1.25 2.5 0.2 0.4 0.8 1.25 2.5
M-PEG 3 ± 2 3 ± 2 0 0 0 5 ± 2 2 ± 1 0 0 0 3 ± 2 4 ± 2 1 ± 1 0 0
CN-PEG 5 ± 4 2 ± 1 1 ± 1 0 0 3 ± 2 4 ± 3 1 ± 1 1 ± 1 0 5 ± 34 4 ± 3 3 ± 1 5 ± 2 0
COOH-PEG 2 ± 1 1 ± 0 1 ± 1 0 0 3 ± 1 2 ± 1 6 ± 4 0 ± 0 0 3 ± 2 1 ± 1 1 ± 1 1 ± 1 0
PT-PEG 2 ± 1 2 ± 1 1 ± 1 1 ± 0 0 1 ± 0 3 ± 2 3 ± 2 0 0 3 ± 1 2 ± 1 3 ± 3 0 ± 0 0
M-PEG 3 ± 1 3 ± 2 0 0 0 3 ± 3 3 ± 2 1 ± 1 0 0 2 ± 1 3 ± 2 1 ± 1 0 0
CN-PEG 2 ± 2 1 ± 1 1 ± 0 0 0 4 ± 3 5 ± 4 0 ± 0 2 ± 1 0 3 ± 2 3 ± 2 0 ± 0 2 ± 2 0
PT-PEG 3 ± 1 1 ± 0 0 ± 0 0 0 2 ± 1 2 ± 2 0 ± 0 0 0 3 ± 1 3 ± 2 1 ± 1 0 0
COOH-PEG 4 ± 0 1 ± 1 1 ± 1 0 0 4 ± 2 2 ± 1 1 ± 0 1 ± 0 0 2 ± 1 3 ± 1 1 ± 0 0 ± 0 0
Example 2 Drug Release Kinetics
The lysyl amino groups of gelatin samples (Sigma, St. Louis, Mo.; Type A, from porcine skin, 300 bloom, cell culture tested) were modified by PEGdial to form PEG-modified gelatin (PG). Gelatin samples were also modified using EDTAD (Aldrich) to form EDTAD-modified gelatin (EG). Still further gelatin samples were modified with PEGdial and EDTAD to yield PEG-modified-EDTAD-modified gelatin (P/EG). PG or P/EG was created by adding PEGdial dissolved in 10 ml of H2O (Milli-Q synthesis, 18.2 MΩ-cm, Millipore) and NaCNBH3 dissolved in 10 ml of H2O separately and simultaneously to a 5% (w.v) gelatin or EG solution at 50 to 60° C. for 24 hours in a wt ratio of gelatin/EG: PEGdial: NaCNBH3 of (1:0.66:0.186). The theoretical maximum percent modification using this method is 100% modification of gelatin lysyl residues, based on an average 300 bloom gelatin molecular weight and average lysine content of the gelatin. See, e.g., Merck Index, 12th Ed. (1996) #4388, p. 742. EG was created by adding EDTAD to a 1% (w/v) gelatin solution at pH 10, 40° C. for 3 hours in a wt ratio of gelatin:EDTAD of 1:0.034. The theoretical maximum percent modification of gelatin lysyl residues using this method is 38%. Thus, modifications larger than this indicate that both functional groups of the added EDTAD have bonded to lysyl residues in the gelatin, thereby cross-linking the gelatin chains. The level of gelatin modification was quantified using the 2,4,6-trinitrobenzene sulfonic acid spectrophotometric method. See Hwang & Damodaran, supra, and Offner & Bubnis (1996) Pharm. Res. 13:1821-1827.
To evaluate swelling and degradation kinetics, dried hydrogels were placed in 5 ml of aqueous solutions of pH 4.5, pH 7.0 or pH 7.4 in a water bath at 37° C. Aqueous solutions were created by adjusting the pH of H2O with dilute HCl and NaOH. Hydrogels were transferred to fresh aqueous solutions at approximately 3 and 6 wks. Swollen hydrogels were weighed at 2, 4, and 6 hours, 1, 2, 3, 4, and 5 days, and 1, 2, 3, 4, 5, 6, 7, and 8 weeks to characterize the swelling/degradation kinetics. Extreme care was taken to preserve the integrity of the hydrogels at every step in the weighing process. The swelling weight ratio at each time point for each hydrogel was calculated as: (Ws−Wd)/Wd, where Ws is the weight of the swollen gel and Wd is the weight of the dry gel (in grams). The maximum swelling weight ratio that occurred over 8 weeks and the time it occurred was also calculated (Rmax & Tmax, respectively). The last attainable swelling weight ratio (due to hydrogel dissolution) and the time it occurred was also calculated (Rfail & Tfail, respectively). Statistical analysis was performed using ANOVA and Tukey multiple comparisons tests (p<0.05). Individual sample solutions from the swelling study were collected for ongoing GPC analysis of degradation products (results not shown) (20% (v/v) acetonitrile: 0.1 M NaNO3 at a flow rate of 0.7 ml/min, 60 min., using three Ultrahydrogel columns in series, Ultrahydrogel 250, 1000 and Linear, on a Waters system).
RMAX, TMAX, RFAIL, AND TFAIL FOR ALL LEVELS OF
GLUTERALDEHYDE/HEAT TREATMENT, PH AND GELATIN
fixation/heat G
treatment pH Modc R-max T-max R-fail T-fail
0.1% 4.5 G 6.30 108 4.11 >1344
PG 6.98 1344b 6.98 >1344
EG 8.77 720 7.71 >1344
7.0 G 5.94 108 2.88 >1344
PG 6.64 1092 4.55 >1344
EG 12.04 1008 6.24 >1344
7.4 G 4.68 96 1.45 1092
PG 6.60 1092 5.35 >1344
EG 894.17 924 892.52 >1344
0.01% 4.5 G 35.48 36 7.25 132
PG 11.54b 24 5.80 420
EG 31.53 2 14.07 84
7.0 G 40.23 48 8.49 84
PG 10.63 96 8.36 336
EG 26.96 2 7.71 168
7.4 G 26.29 36 8.21 72
PG 10.48 96 5.06 336
EG 30.88 12 6.47 168
0.001% 4.5 G 0.10 1 −0.01 2
EG — — — —
7.0 G 0.33 1 0.17 2
7.4 G 0.36 1 0.36 1
LN2-baked G 4.5 G 3.96 24 2.06 252b
7.0 G 4.76 24 0.40 72
7.4 G 4.05 15 72 96
TOTAL AND DIFFERENTIAL LEUCOCYTE CONCENTRATION IN
THE INFLAMMATORY EXUDATES OF GELATIN HYDROGELS
tation Cell concentration (×cells/μL)a
Sample time (day) Total Lymphocyte Monocyte PMN
Empty 4 184 ± 25 168 ± 23 16 ± 7 1 ± 1
cage (no 7 57 ± 12c 49 ± 10c 7 ± 2 0 ± 0
sample) 14 55 ± 7 36 ± 3 12 ± 4 7 ± 5
21 91 ± 69 98 ± 54 20 ± 16 0 ± 0
0.1% 4 597 ± 392 255 ± 116 126 ± 113 217 ± 21
7 183 ± 129 78 ± 40 26 ± 14 2
14 235 ± 65b 118 ± 30b 40 ± 16 79 ± 74b
21 200 167 33 77 ± 75
0.01% 4 477 ± 195 412 ± 172 57 ± 28 8 ± 5
7 178 ± 78b 157 ± 80 17 ± 1b 4 ± 3
14 72 ± 36 60 ± 29 10 ± 7 2 ± 1
21 93 ± 3 72 ± 5 9 ± 4 12 ± 11
Example 3 In vivo Modulation of Host Response Using Gels Grafted with Fibronectin-Derived Biomimetic Oligopeptides
Oligopeptides were designed based on the primary and tertiary structure of human plasma fibronectin to study the structure-functional relationship of RGD and PHSRN regions of fibronectin in regulating the host inflammatory response and macrophage behavior in vivo. Peptides included RGD and PHSRN sequences alone or in combination. The tertiary structure of fibronectin was utilized as a guide in the formulation of peptides. The distance between the PHSRN sequence and the RGD sequence within the natural fibronectin molecule in solution was approximated using the structural coordinates archived in the SwissProt Databases® (sequence FINC_HUMAN P02751). Based on the measurement, a hexamer of glycine (G6) of approximately the same length was used to link the two bioactive sequences in both possible orientations. A terminal trimeric glycine domain (G3) was employed as a spacer in all peptides. Oligopeptides were synthesized using solid-resin methods on an automated peptide synthesizer (Milipore) using conventional 9-fluorenylmethyloxycarbonyl chemistry without further purification and with a final coupling efficiency of approximately≦85% purity. Peptides were characterized and analyzed using mass spectroscopy and reverse phase HPLC coupled to photodiode array, evaporative light scatter, and UV/Vis detectors. The following oligopeptides were synthesized: G3RGDG (SEQ. ID. NO: 3), G3PHSRNG (SEQ. ID. NO: 4), G3RGDG6PHSRNG (SEQ. ID. NO: 5), G3PHSRNG6RGDG (SEQ. ID. NO: 6), and G3RDGG (SEQ. ID. NO: 7) as a nonspecific control. Peptides were covalently grafted onto hydrogels as described in Example 1 to investigate the influence of peptides on the host response and macrophage behavior in vivo.
A previously developed mathematical model describing the in vivo kinetics of macrophage fusion on various biomaterials was employed to provide insights into the effect of materials and peptides on foreign body giant cell (FBGC) formation. The model was formulated based on Flory's most-probable molecular weight distribution of polymer chains. In the analysis, each adherent macrophage is analogous to a monomer and the process of cell fusion is analogous to the polymerization process. Two initial premises are necessary: (1) the FBGC size is directly proportional to the number of nuclei in a given FBGC; and (2) the ability for each cell to fuse is constant and independent of the cell size. The FBGC size-distribution equation (Nx=pax−3(1−p)) was applied to the measured FBGC size-distribution result of each sample at each retrieval time. Nx is the cell size number-fraction of FBGCs with area x; p is the probability of cell fusion or the ratio of the number of cell fusion to the initial adherent macrophage density; a is a constant relating to the number of nuclei per FBGC to the cell area (FBGC/mm2) and has been found to be constant for various clinically relevant biomaterials under different mechanical stress conditions. See Kao et al. (1994) J. Biomed. Mater. Res. 28:73-79; Kao et al. (1995) J. Biomed. Mater. Res. 29(10); 1267-75; and Kao et al. (1994) J. Biomed. Mater. Res. 2:819:829. Values for p and a were obtained through a curve-fit iteration until r2>0.98. The resulting values of p for each sample at each retrieval time were utilized to calculate two kinetic parameters that characterize the process of cell fusion: the density of adherent macrophages that participate in the FBGC formation (d0=df/[p2(1−p)] and the rate constant of cell fusion (1/(1−p)=d0tk+1). d0 is the calculated density of adherent macrophages that participate in the FBGC formation process (macrophages/mm2), df the measured adherent macrophage density at 4 days post-implantation (macrophages1 mm−2), t the implantation time (week), and k the inverse rate constant of cell fusion (mm2cell−1 week−1).
This Example shows that the hydrogels of the present invention can be used to support peptide, proteins, and the like, within a modified, three-dimentional hydrogel matrix.
Total and different leukocyte concentration in the inflammatory exudate of
mPEGmA-co-AC-co-TMPTA networks grafted with various fibronectin-derived
oligopeptidesa
Implantation Cell concentration (×10 cells/μl)
Peptide (days) Total Lymphocyte Monocyte PMN
G3RGDG 4 127 ± 25 71 ± 22 56 ± 5b 0 ± 0
7 67 ± 13 24 ± 4c 43 ± 9b 0 ± 0
14 74 ± 18 21 ± 4 53 ± 25 0 ± 0
21 31 ± 27 ± 8c,b 5 ± 1d 0 ± 0
8c,d,b
G3PHSRNG 4 63 ± 32 25 ± 22b 38 ± 17b 0 ± 0
7 61 ± 9 25 ± 6 36 ± 3b 0 ± 0
14 56 ± 19 33 ± 15 24 ± 4 0 ± 0
21 77 ± 2c 69 ± 2c,d 7 ± 3 0 ± 0
G3RGDG6PHSRNG 4 129 ± 29 ± 10b 99 ± 62b 1 ± 1
7 52 24 ± 6 44 ± 17b 0 ± 0
14 68 ± 23 21 ± 9 36 ± 10 0 ± 0
21 57 ± 12 67 ± 3c,d 7 ± 3 0 ± 0
74 ± 2c
G3PHSRNG6RGDG 4 109 ± 53 ± 14b 56 ± 5b 0 ± 0
7 16 21 ± 8 28 ± 3b,d 0 ± 0
14 49 ± 11d 38 ± 12 49 ± 29 0 ± 0
21 87 ± 23 55 ± 8 5 ± 3d 0 ± 0
G3RDGG 4 91 ± 11 51 ± 1b 40 ± 10b 0 ± 0
7 66 ± 16 30 ± 11 36 ± 6b 0 ± 0
14 48 ± 9d 23 ± 6d 25 ± 6 0 ± 0
21 35 ± 32 ± 9c,b 4 ± 2d 0 ± 0
10c,d,b
No grafted peptide 4 94 ± 32 42 ± 27b 52 ± 16b 0 ± 0
7 41 ± 10 11 ± 2b 30 ± 6b 0 ± 0
14 89 ± 21 56 ± 18 33 ± 15 0 ± 0
21 63 ± 4 55 ± 4 7 ± 2d 0 ± 0
Empty cage 4 135 ± 129 ± 22 6 ± 1 0 ± 0
7 22 38 ± 8d 4 ± 1 0 ± 0
14 42 ± 8d 35 ± 6d 15 ± 10 0 ± 0
21 51 ± 10d 80 ± 25d 2 ± 2 0 ± 0
82 ± 22d
aAll values expressed in (mean ± s.e.m., n = 3).
networks grafted with various fibronectin-derived oligopeptidesa
Adherent macrophage density (×10 macrophages/mm2) at
various post-implantation time (days)
Peptide 4 7 14 21 35 70
G3RGDG 138 ± 22b 85 ± 12b,c 33 ± 12b,c 15 ± 3c 14 ± 2c 4 ± 2c
G3PHSRNG 124 ± 12b 57 ± 10b,c 31 ± 11b,c 10 ± 0c 9 ± 1c 4 ± 2c
G3EGDG6PHSRNG 126 ± 8b 58 ± 12b,c 23 ± 4b,c 14 ± 4c 6 ± 5c 0 ± 0c
G3PHSRNG6RGDG 183 ± 27b 69 ± 6b,c 30 ± 5b,c 16 ± 4c 12 ± 5c 3 ± 1c
G3RDGG 75 ± 16 36 ± 5c 15 ± 3c 15 ± 6c 9 ± 3c 3 ± 2c
No grafted peptide 74 ± 26 37 ± 4c 14 ± 2c 19 ± 3c 6 ± 3c 1 ± 1c
Example 4 Interpenetrating Membranes Comprising Modified Hydrogels
IPNs were created using modified and unmodified gelatin, PEGdA (2, 4.6, or 8 kDa molecular weight), initiator (2,2-dimethoxy-2-phenylacetophenone, DMPA), and a long wavelength UV source. Gelatin was dissolved in deionized water with heat (80° C.) to form a 20 wt % gelatin solution. PEGdA was dissolved in deionized water, without heat, in an aluminum foil wrapped glass vial to form a 100 wt % PEGdA solution. The gelatin solution was then added to the PEGdA solution and the mixture was agitated thoroughly. DMPA was then added to the gelatin/PEGdA mixture and this final mixture was again agitated and then heated (80° C.) throughout the rest of the procedure. IPNs were created through injection molding. The final gelatin/PEGdA/DMPA mixture was injected with a Pasteur pipette into a Teflon mold that was clamped between 2 glass slides. The mold has the approximate dimensions of 20 mm long by 10 mm wide by 1.6 mm thick. The mold/IPN mixture was then irradiated with Uw light from the top and bottom for approximately 3 minutes. During this time, the UV light initiates the cross-linking G of PEGdA, entrapping the gelatin within the PEGdA cross-links. The mold/IPN was allowed to cool before the IPN was removed from the mold.
4=40 wt %
6=60 wt %
Y=type of gelatin
G=gelatin
EG=EDTAD-modified gelatin
mPMaG=mPmA-modified gelatin
mPmAEG=mPmA/EDTAD-modified gelatin
Z=wt % PEGdA
k=molecular weight PEGdA
2 k=2000 Da
4.6 k=4,600 Da
8 k=8,000 Da
The swelling/degradation kinetics of the IPNs were characterized by weighing swollen IPNs at predetermined times (up to 8 weeks). The IPNs were added to test tubes containing 5 ml deionized water with environmental pHs of 4.5, 7.0, and 7.4. The test tubes were then placed in water baths at 37° C. At the qLpredetermined times, the samples were removed with extreme caution from the test tubes using a bent spatula, blotted dry, weighed, and then placed back in the same test tube. This was done until the sample had degraded completely or until the sample had degraded into too many pieces and they could no longer be removed from the test tube. The swelling weight ratio at each time point for each IPN was calculated as: (Ws−Wo/Wo), where Ws is the weight of the swollen IPN and Wo is the original weight of the IPN. The maximum swelling weight ratio that occurred over 8 weeks and the time it occurred was calculated (Rmax, Tmax). The last attainable swelling weight ratio (due to IPN degradation) and the time it occurred was also calculated (Rfail, Tfail).
Rmax, Tmax, Rfail, and Tfail FOR VARIOUS IPN FORMULATIONS
Formulation Rmax Tmax Rfail Tfail
6G4P2K 0 0 −0.736 9
4G6P2K 0.754 1 <0.444 >1344
6G4P4.6K 1.733 225.667 <0.505 >1344
4G6P4.6K 2.2 4.333 <1.538 >1344
6G4P8K 1.646 225 0.758 451.333
4G6P8K 3.911 227.333 <1.542 >1344
6EG4P2K 0.128 1 −0.214 336.33
4EG6P2K 0.712 27 <0.532 >1344
6EG4P4.6K 2.288 35.333 <0.818 >1344
4EG6P4.6K 1.452 17.667 <0.773 >1344
6EG4P8K 2.639 5 0.794 960
4EG6P8K 3.026 1 −0.252 1097.3
6mPmAG4P2K 0.469 1.667 <−0.23 >1344
4mPmAG6P2K 0.891 672 <0.827 >1344
6mPmAG4P4.6K 2.467 226.667 <0.621 >1344
4mPmAG6P4.6K 2.578 616 <2.445 >1344
6mPmAG4P8K 3.854 336.667 2.717 944
4mPmAG6P8K 6.075 337 <2.626 >1344
6mPmAEG4P8K 2.715 2.333 <1.265 >1344
4mPmAEG6P8K 4.224 192.333 <1.472 >1344
17. H. Houben-Weyl, E. Muller, und T. Verlag. “Methoden der Organischen Chemie.” Stuttgart, XIII, 377 (1970).
a first polymer matrix containing reactive amino moieties;
a heterobifunctional modifier comprising a compound of formula:
wherein at least one of the “A” or “Z” moieties is covalently bonded to the reactive amino moieties of the first polymer matrix; and wherein “A” and “Z” are independently a monovalent or divalent organic moiety; wherein at least one of A and Z is a divalent moiety; if A or Z is a monovalent moiety, then A or Z is selected from the group consisting of halo, hydroxy, C1-C24-alkyl, C1-C24-alkenyl, C1-C24-alkynyl, C1-C24-alkoxy, C1-C24-heteroalkyl, C1-C24-heteroalkenyl, C1-C24-heteroalkynyl, cyano-C1-C24-alkyl, a C3-C10-cycloalkyl, C3-C10-cycloalkenyl, C3-C10-cycloalkynyl, C3-C10-cycloheteroalkyl, C3-C10-cycloheteroalkenyl, C3-C10-cycloheteroalkynyl, acyl, acyl-C1-C24-alkyl, acyl-C1-C24-alkenyl, acyl-C1-C24-alkynyl, carboxy, C1-C24-alkylcarboxy, C1-C24-alkenylcarboxy, C1-C24-alkynylcarboxy, carboxy-C1-C24-alkyl, carboxy-C1-C24-alkenyl, carboxy-C1-C24-alkynyl, aryl, aryl-C1-C24-alkyl, aryl-C1-C24-alkenyl, aryl-C1-C24-alkynyl, heteroaryl, heteroaryl-C1-C24-alkyl, heteroaryl-C1-C24-alkenyl, heteroaryl-C1-C24-alkynyl, sulfonate, arylsulfonate, and heteroarylsulfonate;
if A or Z is a divalent moiety, then A or Z is independently selected from a divalent equivalent of a monovalent moiety selected from the group consisting of C1-C24-alkyl, C1-C24-alkenyl C1-C24-alkynyl, C1-C24-alkoxy, C1-C24-heteroalkyl, C1-C24-heteroalkenyl, C1-C24-heteroalkynyl, cyano-C1-C24-alkyl, a C3-C10-cycloalkyl, C3-C10-cycloalkenyl, C3-C10-cycloalkynyl, C3-C10-cycloheteroalkyl, C3-C10-cycloheteroalkenyl, C3-C10-cycloheteroalkynyl, acyl, acyl-C1-C24-alkyl, acyl-C1-C24-alkenyl, acyl-C1-C24-alkynyl, carboxy, C1-C24-alkylcarboxy, C1-C24-alkenylcarboxy, C1-C24-alkynylcarboxy, carboxy-C1-C24-alkyl, carboxy-C1-C24-alkenyl, carboxy-C1-C24-alkynyl, aryl, aryl-C1-C24-alkyl, aryl-C1-C24-alkenyl, aryl-C1-C24-alkynyl, heteroaryl, heteroaryl-C1-C24-alkyl, heteroaryl-C1-C24-alkenyl, heteroaryl-C1-C24-alkynyl, sulfonate, arylsulfonate, and heteroarylsulfonate;
wherein the “A” moiety and the “Z” moiety are different from one another;
wherein only one of A and Z is bound to the first polymer matrix;
wherein “m” is an integer of from 2 to 8 and “n” is an integer equal to or greater than 100; and
a second polymer matrix that interpenetrates with the first polymer matrix.
2. The hydrogel of claim 1, further comprising a pharmacologically-active agent covalently bonded to one of the “A” or “Z” moieties that is not bonded to the first polymer matrix.
3. The hydrogel of claim 1, where the first polymer matrix is proteinaceous.
4. The hydrogel of claim 1, wherein the first polymer matrix is selected from the group consisting of gelatin, calcium alginate, calcium/sodium alginate, collagen, oxidized regenerated cellulose, carboxymethylcellulose, amino-modified cellulose, and whey protein.
5. The hydrogel of claim 1, wherein the first polymer matrix is selected from the group consisting of gelatin and collagen.
6. The hydrogel of claim 1, wherein the first polymer matrix is cross-linked with a cross-linking reagent.
7. The hydrogel of claim 1, wherein the first polymer matrix is cross-linked with glutaraldehyde.
8. The hydrogel of claim 1, wherein the first polymer matrix further comprises EDTAD moieties bonded to it.
9. The hydrogel of claim 1, wherein “n” is equal to or greater than 200.
10. The hydrogel of claim 1, wherein “n” is equal to or greater than 2,000.
11. The hydrogel of claim 1, wherein “n” is equal to or greater than 20,000.
12. The hydrogel of claim 1, further comprising a pharmacologically-active agent entrained within the hydrogel.
13. The hydrogel of claim 1, further comprising living cells entrained within the hydrogel.
14. The hydrogen of claim 2, wherein the pharmacologically active agent is selected from the group consisting of vulnerary agents, hemostatic agents, antibiotics, antithelmintics, antifungal agents, hormones, anti-inflammatory agents, proteins, polypeptides, oligonucleotides, cytokines, and enzymes.
15. The hydrogel of claim 14, wherein the pharmacologically active agent is a vulnerary agent.
16. The hydrogel of claim 12, wherein the pharmacologically active agent is selected from the group consisting of vulnerary agents, hemostatic agents, antibiotics, antithelmintics antifungal agents, hormones, anti-inflammatory agents, proteins, polypeptides, oligonucleotides, cytokines, and enzymes.
17. The hydrogel of claim 16, wherein the pharmacologically active agent is a vulnerary agent.
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WO2011014594A1 (en) 2009-07-29 2011-02-03 Corning Incorporated Peptide-polymer cell culture articles and methods of making
WO2011014605A1 (en) 2009-07-29 2011-02-03 Corning Incorporated Functionalized cell binding peptides and cell culture articles
US8025901B2 (en) 2001-04-23 2011-09-27 Wisconsin Alumni Research Foundation Bifunctional-modified hydrogels
US7605231B2 (en) * 2002-04-26 2009-10-20 Yasuhiko Tabata Gelatin derivatives and high-molecular micelle comprising the derivatives
WO2006085653A1 (en) * 2005-02-14 2006-08-17 Medgel Corporation Hydrogel for medical use
WO2007005754A3 (en) * 2005-07-01 2007-04-05 Alza Corp Liposomal delivery vehicle for hydrophobic drugs
EP2091998A2 (en) * 2006-12-19 2009-08-26 Sicit Chemitech S.p.A. Biodegradable polymeric derivatives
CN101220090B (en) * 2007-01-09 2010-10-13 上海百瑞吉生物医药有限公司 Multi-modification derivant of glutin and crosslinked material thereof
US9181404B2 (en) * 2007-06-20 2015-11-10 Sicit Chemitech S.P.A. Manufacture of leather and fabric from materials containing protein hydrolysates and gelatins
WO2010120361A3 (en) * 2009-04-14 2011-03-24 The Regents Of The University Of California Method of creating colored materials by fixing ordered structures of magnetite nanoparticles within a solid media
US20120003192A1 (en) * 2010-07-02 2012-01-05 Zimmer Orthobiologics, Inc. Hydrogel-forming composition comprising protein and non-protein segments
WO2016204258A1 (en) * 2015-06-19 2016-12-22 日産化学工業株式会社 Polymer for gel formation and dermatological adhesive material
JPH08231435A (en) 1995-02-27 1996-09-10 Res Dev Corp Of Japan In vivo degradable polymeric hydrogel
EP0747066A2 (en) 1995-06-07 1996-12-11 Collagen Corporation Biocompatible adhesive compositions
WO1996040817A1 (en) 1995-06-07 1996-12-19 Wisconsin Alumni Research Foundation Carboxyl-modified superabsorbent protein hydrogel
WO1997003106A1 (en) 1995-07-07 1997-01-30 Shearwater Polymers, Inc. Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications
JPH1085318A (en) 1996-09-18 1998-04-07 Terumo Corp Medical material and skin ulcer filling/recovering material
WO2001005443A1 (en) 1999-07-21 2001-01-25 Imedex Biomateriaux Adhesive protein foam for surgical and/or therapeutic uses
US6310105B1 (en) * 2000-02-15 2001-10-30 Wisconsin Alumni Research Foundation Carboxyl-modified superabsorbent protein hydrogel
WO2002085419A2 (en) 2001-04-23 2002-10-31 Wisconsin Alumni Research Foundation Bifunctional-modified hydrogels
US20050276858A1 (en) 2001-04-23 2005-12-15 Kao Weiyuan J Bifunctional-modified hydrogels
JPH06503840A (en) 1991-07-09 1994-04-28
JPH0999052A (en) 1995-06-07 1997-04-15 Collagn Corp Bioadaptable tacky adhesive composition
US5847089A (en) * 1995-06-07 1998-12-08 Wisconsin Alumni Research Foundation Carboxyl-modified superabsorbent protein hydrogel
US20060100369A1 (en) 2001-04-23 2006-05-11 Kao Weiyuan J Bifunctional-modified hydrogels
Abuchowski et al., "Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol-asparaginase conjugates," Cancer Biochem. Biophys. (1984) 7:175-186.
Andreopoulos, F.M. et al., "Photoimmobilization of organophosphorus hydrolase within a PEG-based hydrogel," Biotechnol. Bioeng. (1999) 65:579-588.
Brown et al., the Structure of Propadienone, J. Am. Chem. Soc., 107:4109 (1985).
Bruson, Cyanoethylation, Organic Reactions, 5:79 (1949).
Buckmann et al., Functionalization of Poly(ethylene glycol) and Monomethoxy-Poly(ethylene glycol), Makromol. Chem., 182:1379-1384 (1981).
Burmania et al., Protein-based Interpenetrating networks (IPN) for tissue scaffolds/drug release, May 2, 2002.
Delgado et al., The Uses and Properties of PEG-Linked Proteins, Crit. Rev. Ther. Drug Carrier Syst. 9:249 (1992).
Drumheller et al., Densely crosslinked polymer networks of poly(ethylene glycol) in trimethylolpropane triacrylate for cell-adhesion-resistant surfaces, J. Biomed. Mater. Res. 29:207 (1995).
D'Urso, E.M. et al., "Albumin-poly(ethylene glycol) hydrogel as matrix for enzyme immobilization: biochemical characterization of crosslinked acid phosphatase," Enz, Micro. Tech. (1996) 18:482-488.
Einerson et al., Synthesis and Physiochemical analysis of Gelatin-based hydrogels for cell/drug carrier matrices, May 2, 2002.
Fortier, New Polyethylene Glycols for Biomedical Applications, Biotechnol. Genet. Eng. Rev. 12:329 (1994).
Harris et al., "Poly(ethylene glycol) Chemistry and Biological Applications," American Chemical Society, Washington, D.C. (1997).
Harris et al., Introduction to Chemistry and Biological Applications of Poly(ethylene glycol) Am. Chem. Soc., Polymer Preprints, 30:356 (1989).
Hern et al., "Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing," J. Biomed. Mater. Res. (1998) 39:2666-2676.
Inada et al., Biomedical and biotechnological applications of PEG- and PM-modified proteins, Trends Biotechnol. 13:86 (1995).
Kao et al., "Role for interleukin-4 in foreign-body giant cell formation on a poly(etherurethane urea) in vivo," J. Biomed. Mater. Res. (1995) 29(10):1267-1275.
Kao et al., "Theoretical analysis of in vivo macrophage adhesion and foreign body giant cell formation on polydimethylsiloxane, low density polyethylene and polytheruretanes," J. Biomed. Mater. Res. (1994) 28:73-79.
Kao et al., "Theoretical analysis of in vivo macrophage and foreign body giant cell formation on strained poly(etherurethane urea) elastomers," J. Biomed. Mater. Res. (1994) 2:819-829.
Kao et al., Biotech. Bioengrn., Murine Macrophage Behavior on Peptide-grafted Polyehtylenegoycol-containing Networks, 59:2 (1998).
Kao et al., Engineering endogenous inflammatory cells as delivery vehicles, Journal of Controlled Release, 78:219-233 (2002).
Kao et al., Evaluation of protein-modulated macrophage behavior on biomaterials, designing biomimetic materials for cellular engineering, Biomaterials, 20:2213-2221 (1999).
Kao et al., Fibronectin modulates macrophage adhesion and FBGC formation: The role of RGD, PHSRN, and PRRARV domains, 2000, published online Jan. 4, 2001.
Kao et al., Handbook of Biomaterial Evaluation, 2nd Edition, Taylor & Frances Publishing, Philadelphia, PA (1999) 659-669.
Kao et al., In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: the role of RGD and PHSRN domains, Biomaterials, 22:2901-2909 (2001).
Kao et al., Utilizing Biomimetic Oligopeptides to probe fibronectin-integrin Binding and Signaling in regulating macrophage function in vitro and in vivo, Frontiers in Biosciences 6, D992-999 (2001).
Kao et al.,Preparation of heterodifunctional polyethyleneglycols: Network formation, characterization, and cell culture analysis, J. Biomataer, Sci. Polymer Edn, 12(6) 599-611 (2001).
Liu et al., Human macrophage adhesion on fibronectin: The role of substratum and intracellular signalling kinases, Cellular Signalling, 14:145-152 (2002).
Llanos, G.R. et al., "Heparin-poly(ethylene glycol)-poly(vinyl alcohol) hydrogel: preparation and assessment of thrombogenicity," Biomaterials (1992) 13(7):421-424.
Llanos, Gerard R., et al., (1991), Immobilization of Poly(ethylene glycol) onto a Poly(vinyl alcohol) Hydrogel. 1. Synthesis and Characterization, Macromolecules, vol. 24, 6065-6072.
Llanos, Gerard R., et al., (1993), Immobilization of Poly(ethylene glycol) onto a Poly(vinyl alcohol) Hydrogel: 2. Evaluation of thrombogenicity, Journal of Biomedical Research, vol. 27, 1383-1381.
Mehvar, R., Modulation of the Pharmacokinetics and Pharmacodynamics of Proteins by Polyehtylene Glycol Conjugation, J. Pharm. Pharm. Sci. 3:125-136 (2000).
Morpurgo et al., "Covalent modification of mushroom tyrosinase with different amphibic polymers for pharmaceutical and biocatalysis applications," App. Biochem. Biotech. (1996) 56:59-72.
Nagasaki et al., Synthesis of Heterotelechelic Poly(ethylene glycol) Macromonomers. Preparatio of Poly(ethylene glycol) Possessing a methacryloyl Group at One End and a Formyl Group at the Other End, Macromolecules, 30:6489-6493 (1997).
Nakamura et al., Synthesis of Heterobifunctinal Poly(ethylene glycol) with a Reducing Monosaccharide Residue at One End, Bioconjugate Chem. 9:300-303 (1998).
Offner et al., "Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices," Pharm. Res. (1996) 13:1821-1827.
Yokoyama et al., Synthesis of Poly(ethylene oxide) with Heterobifunctional Reactive Groups at Its Terminals by an Anionic Intitiator, Bioconjugate Chem. 3:275-276 (1992).
Zalipsky et al., Facile synthesis of alpha-Hydroxy-omega-Carboxymethylpolyethylene Oxide, J. Bioact. Biocompatible Polym. 5:227-231 (1990).
Zalipsky, Samuel Functionalized Poly(ethylene glycol) for Preparation of Biologically Relevant Conjugates, Bioconjugate Chem., 1995, 6, 150-165. *
US20110183418A1 (en) * 2009-07-29 2011-07-28 Arthur Winston Martin Peptide-Polymer Cell Culture Articles and Methods of Making
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Nie et al. 2007 Production of heparin-functionalized hydrogels for the development of responsive and controlled growth factor delivery systems
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