Synthesis And Application Of Paclitaxel-terminated Peptide Brush Polymers

The loading of therapeutics to and within carriers is achieved by either physical encapsulation or covalent conjugation.The latter generally involves attaching drugs via chemical bonds,such as esters and disulfides,that facilitate release.Covalent conjugation has received increasing interest because it confers the drug delivery system with enhanced stability during blood circulation.This approach not only precludes premature release of therapeutics,but also allows for specific stimuli-triggered drug release in diseased tissues.In the case of polymeric carriers,drugs are typically tethered as side chains via direct polymerization of drug-modified monomers or via post-polymerization modifications.In each case,a statistical average of drugs is incorporated per polymer chain because of the inherent dispersity of the polymer.To overcome this issue and to prepare polymer-based drug delivery systems in a more precise and well-defined fashion,new approaches are needed.In ongoing work in our laboratory,we have sought to develop water soluble peptide-based polymers capable of cell internalization.These polymers can be tuned in terms of aggregated state and overall charge.Indeed,when dispersed as individual chains,we have shown that the polymers avoid macrophage uptake,a drawback inherent to nanoparticle formulations.Inspired by the concept of the chain end-modified polymers and our observations regarding the benefits of single-chain polymers,here we designed a polymer-drug conjugate with one drug residing on a water-soluble polymer chain end and an easily adjustable overall charge.The goal was to incorporate a drug to water soluble polymers via ring-opening metathesis polymerization(ROMP),where only a single drug per polymer chain was introduced.As such,the design employed herein avoids drug-containing norbornene monomers.We designed and synthesized a new paclitaxel-containing chain termination agent for ROMP,which was used to terminate the polymerization and concomitantly install one paclitaxel at the ω-terminus of polymers.Peptide based norbornene monomers easily confer hydrophilicity and enable the introduction of specific chemical functionality,including net charge.Since the cell membrane is negatively charged,we envision that positively charged peptide brush polymers would possess a higher performance in cell penetration than their neutral and negatively charged analogues.To verify our hypothesis,a series of peptides bearing different charges and charge numbers were synthesized,including GGSGSGS,GGSGSGE,GGSGSGK,GGSGSGR,and GGSGSGRR,respectively.Targeted polymers consisted of poly(GGSGSGS)-Dye-PTX,poly(GGSGSGE)-Dye-PTX,poly(GGSGSGK)-Dye-PTX,poly(GGSGSGR)-Dye-PTX and poly(GGSGSGRR)-Dye-PTX.Furthermore,the crude peptide brush polymer-drug conjugates could be purified via reverse-phase high performance liquid chromatography under normal peptide purification procedures,giving rise to well-defined and highly pure polymer-drug conjugates,consisting of one paclitaxel and one dye.The efficient purification process enabled us to directly compare the bioactivity of well-defined peptide-drug polymer conjugates,polymers not containing the drug,and the crude polymer mixtures.To evaluate the in vitro bioactivity of peptide brush polymer-drug conjugates carrying different charges,we performed flow cytometry assay to quantify their cellular uptake efficiency.Polymers bearing positive charges enter A549 cancer cells significantly more efficiently than those neutral and negative versions.Furthermore,cytotoxicity of polymer-drug conjugates to A549 cell line was assessed.All the peptide brush polymers without loading paclitaxel exhibited no toxicity,indicative of excellent cytocompatibility of peptide brush polymers.In the case of purified polymer-drug conjugates,cytotoxicity of positively charged polymer-drug conjugates was markedly higher than their neutral and anionic analogues,which is in good agreement with the cell-uptake results from flow cytometry.It is worthy to note that those pure polymer-drug conjugates showed higher cytotoxicity than that of their corresponding crude products,regardless of the nature of charges.This can be attributed to the higher drug loading of pure polymer-drug conjugates than crude product which contained polymer species lack of drugs.