Polymer Masked−Unmasked Protein Therapy. 1. Bioresponsive Dextrin−Trypsin and −Melanocyte Stimulating Hormone Conjugates Designed for α-Amylase Activation

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• 作者：Ruth Duncan ; Helena R. P. Gilbert ; Rodrigo J. Carbajo ; Marí ; a J. Vicent
• 刊名：Biomacromolecules
• 出版年：2008
• 出版时间：April 2008
• 年：2008
• 卷：9
• 期：4
• 页码：1146 - 1154
• 全文大小：1743K
• 年卷期：v.9,no.4(April 2008)
• ISSN：1526-4602

文摘

Polymer−protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer−protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (α-1,4 polyglucose, a natural polymer degraded by α-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor−binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9–32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34–69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with α-amylase led to reinstatement of activity to a maximum of 92–115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking−unmasking. This intermediate was used to synthesize a dextrin−MSH conjugate (dextrin M_w = 47200 g/mol; MSH content 37 wt %), and its biological activity (±α-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of α-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeutic application.