Enzymatically-responsive poly(ethylene glycol) hydrogels for the controlled delivery of therapeutic peptides

Van Hove Amy,Antonienko Erin,Benoit Danielle

doi:10.3389/conf.fbioe.2016.01.01371

Abstract

Therapeutic angiogenesis holds great potential for treatment of ischemic tissues and in tissue engineering, where insufficient vascularization limits construct size, complexity, and anastomosis with host vasculature. However, no FDA approved treatments exist to robustly enhance vascularization within ischemic tissue. Many proangiogenic approaches have been developed, often via delivery of angiogenic proteins or peptides. Peptides typically mimic the bioactivity of larger proteins or growth factors, and offer advantages over traditional protein delivery, including more versatile synthesis methods and increased stability in vivo. However, both proteins and peptides suffer from rapid clearance and poor pharmacokinetics when delivered systemically, motivating the development of controlled release systems. Enzymatically-responsive systems where delivery of protein drugs is controlled by the local tissue microenvironment have shown improved tissue healing over bolus protein delivery. Therefore, a hydrogel-based platform technology was developed to control and sustain peptide drug release via matrix metalloproteinase (MMP) activity. Poly(ethylene glycol) (PEG) hydrogels were formed by crosslinking norbornene functionalized multi-arm PEG with peptide drugs flanked by MMP substrates and terminal cysteine residues. In vitro bioactivity testing identified three peptides (Qk (from Vascular Endothelial Growth Factor), SPARC113, and SPARC118 (from Secreted Protein Acidic and Rich in Cysteine)) that retained bioactivity in their expected released forms (e.g., with residual amino acids left by MMP substrates after cleavage). Incorporation of these peptides into hydrogels flanked by MMPdegradable substrates successfully produced hydrogels with enzymatically-responsive hydrogel degradation and peptide release behaviors. Hydrogels degraded via bulk degradation, and peptide release was in close agreement with mass loss. Qk, SPARC113, and SPARC118-releasing hydrogels were confirmed to release bioactive components in vitro after MMP-mediated degradation. Further investigation revealed key peptide drug properties, specifically size and hydrophobicity, control the rate of hydrogel degradation and peptide release. When implanted subcutaneously, SPARC113 and SPARC118-releasing hydrogels both significantly increased vascular ingrowth compared to controls one week after implantation without significantly affecting vessel size. As the longitudinal availability of VEGF has been shown critical for bioactivity, alternate hydrogels were formed to provide temporal control over enzymatically-responsive release of the VEGF peptide mimic, Qk. MMP-degradable linkers with a variety of cleavage kinetics were used to tether Qk to hydrogels formed with non-degradable peptide crosslinks. Hydrogel degradation was successfully uncoupled from peptide release, and three of the linkers provided temporal control over enzymatically-responsive peptide release kinetics in vitro and in vivo. Qk was confirmed to be bioactive as released,…

Highlights

Amino acids (AAPPTec) were prepared at 0.2 mass pre- (Ms) in N-methylpyrrolidone (NMP). 5% piperazine (Alfa Aesar) in dimethylformamide (DMF, Fisher Scientific) was used for deprotection, 0.5 M O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU, AnaSpec) in DMF was used as the activator, and 2 M diisopropylethylamine (DIEA, Alfa Aesar) in NMP was used as the activator base, except for Qk(DL), SPARC113(DL), Scrambled(DL), and SPARC3X(DL) where 0.5 M diisopropylcarbodiimide (DIC, Chem-Impex International) in DMF was used on the activator position and 1 M hydroxybenzotriazole (HOBt, Advanced ChemTech) in DMF used on the activator base position
In vitro testing of degraded SPARC118(DL), SPARC113(DL), and Qk(DL) hydrogels demonstrated that enzymatically-responsive hydrogels release bioactive constituents upon matrix metalloproteinase (MMP)-mediated degradation
While many drugs have been shown to be more potent in vivo with sustained delivery [4, 13, 27], to the best of our knowledge the same has not been shown for the SPARC113 and SPARC118 peptides

Summary

Introduction

Peptides do not require complex tertiary structures for bioactivity, resulting in increased stability in vivo [5] Both proteins and peptides suffer from poor targeting, short circulation time, and rapid clearance when delivered by direct injection [8, 9], motivating the development of controlled release systems. Peptides offer advantages over proteins as they do not require complex tertiary structure for bioactivity, and due to small molecular weights, can be produced synthetically and delivered at higher concentrations to target tissues [5]. Poly(lactic–co-glycolic acid) [12, 13] and alginate [3, 4] materials have successfully extended the duration of encapsulated VEGF delivery through hindered diffusion or upon hydrolytic degradation, increasing tissue vascularization These approaches deliver VEGF over pre-dictated, and not necessarily therapeutically-relevant, time frames. These previously developed materials are unable to investigate the role of temporally controlled enzymatically-responsive growth factor delivery in angiogenesis

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