We demonstrate formation of a hydrogel network by cross-linking
functionalized poly(ethylene glycol) (PEG) and a lysine-containing polypeptide through the
action of a natural tissue enzyme,
transglutaminase. The enzyme reaction rate using a PEG-modified
peptide substrate is the same as the
reaction rate for free substrate. Both the ratio and total
concentration of the two macromers determine
whether gelation will occur and the nature of the gel which forms.
Under suitable conditions, clear gels
form and swell to give a final composition which is 90% water.
Diffusion coefficients of small proteins
and albumin in the gel are comparable to those in free solution.
Gelation proceeds under mild conditions
and thus these gels hold potential for forming highly hydrated networks
around living cells.
We demonstrate control of gelation kinetics in formation of poly(ethylene glycol) (PEG)
hydrogels by enzymatic cross-linking. A predictive model for gelation kinetics based on macromer structure
and composition, stoichiometric ratios of reactants, cross-linking enzyme concentration, and the underlying
kinetics of enzyme reaction was developed on the basis of classical Flory−Stockmayer theory. Experiments
with substrate-functionalized multiarm comb PEG showed good agreement with theory upon variation
of the enzyme concentration, with a slight overprediction in the time to gelation. Experiments with a
substrate-functionalized difunctional PEG in conjunction with a polypeptide where macromer concentrations were varied were also consistent with theory, but with a slight underprediction of gelation time.
Matrix Attachment Therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(×6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in E.coli and purified by metal-chelation affinity chromatography. The purified LinkCD fusion protein exhibits a K m of 0.33 mM and V max of 15 μM/min/μg for the conversion of 5-FC to 5-fluorouracil (5-FU). The duration of the enzyme activity for LinkCD was longer than that of CD enzyme at 37 °C: the fusion protein retained 20% of its initial enzyme activity after 24 hr, and 12% after 48 hr. The LinkCD fusion protein can bind to a hyaluronan oligomer (12-mer) at a K D of 55 μM at pH 7.4 and a K D of 5.32 μM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the anti-tumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and the drinking water containing 10 mg/mL of 5-FC starting on day 11. To examine if the Link domain by itself was able to reduce tumor growth, we included treatment groups that received LinkCD without 5-FC and Link-mtCD (a functional mutant that lacks cytosine deaminase activity) with 5-FC. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results indicate that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC are sufficient to produce an anti-tumor effect. Thus, the LinkCD fusion protein is an alternative to antibody-directed prodrug enzyme therapy (ADEPT) approaches for cancer treatment.
A novel peptide DNase II inhibitor has been used to increase transfection. The level of enhancement was found to be significant in multiple cell types with multiple synthetic vectors.
Kinetic parameters have been measured for coupled nucleophilic and solvolytic reactions of 2,2,2-trifluoroethanesulfonyl (tresyl)-modified poly(ethylene glycol) based on a system of coupled differential equations implied by recently proposed elementary reaction mechanisms. Fitted kinetic parameters were found to be strong functions of pH, temperature, and steric factors. To maximize the total yield of coupled amine as well as the fraction of secondary amine linkages, our model predicts that it is desirable to run tresyl coupling reactions at low temperatures at pH approximately 8.0, depending on the amine pKa for primary, unhindered amines. For branched primary amines, our data favor room temperature at a slightly higher pH.
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