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Final Report Summary - AMPHIDRUGS (Anticancer nanoparticles from amphiphilic macromolecular produgs)

Among the numerous classes of materials employed for drug delivery purposes, colloidal systems based on polymers have attracted much attention due to the flexibility offered by macromolecular synthesis methods and the great diversity of polymers in terms of nature, properties, composition and functionalization.1 Recently, polymer prodrug approach has been utilized to construct efficient drug delivery systems, which can alleviate the drawbacks of conventional polymeric nanoaggregates based on physical encapsulation, such as the “burst effect” and crystallization of the drugs upon encapsulation. For this approach, the standard method is to link hydrophobic drugs to the side chain of a preformed hydrophilic polymer, resulting in fully water-soluble conjugates or polymer aggregates.2 However, it only applies to hydrophobic drugs, leads to poor drug payloads, and doesn’t possess flexibility of the synthesis.

In order to develop an innovative and versatile nanoparticulate platform for efficient drug delivery, a general “drug-initiated” methodology was developed in this project to prepare well-defined amphiphilic polymer-drug conjugates that self-assemble into highly biologically active nanoassemblies. The main goal of the research project was to functionalize several anticancer drugs that would be further used as initiators to polymerize a monomer of opposite water-solubility by means of controlled/living radical polymerization (CLRP) techniques, such as nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP), or reversible addition-fragmentation chain transfer (RAFT) polymerization, resulting in well-defined amphiphilic polymer prodrugs. By playing with the hydrophilic-lipophilic balance of the drug/polymer couple, the idea was to synthesize two families of prodrug amphiphiles: one based on hydrophobic polyisoprene (PI) and one based on hydrophilic poly(oligo(ethylene glycol) methacrylate) (POEGMA), followed by the preparation of nanoparticles and their biological evaluation as anticancer polymer prodrug nanoparticles.

[See scheme 1 in the attached document]

In agreement with the research plan, 4 functionalized anticancer agents based on Cladribine (CdA-AMA-SG1, CdA-digly-AMA-SG1, CdA-CDP, and CdA-digly-CDP), 2 functionalized anticancer agent based on Paclitaxel (PTX-AMA-SG1 and Ptx-CDP), and 1 fluorescent NMP initiator based on naphthalimide (Napht-AMA-SG1) were successfully synthesized. The structures of the above agents were confirmed by 1H NMR, 13C NMR, and ESI-MS characterization.

[See scheme 2 in the attached document]

These functionalized initiators where then employed to prepare amphiphilic polymer prodrugs or fluorescent agents. Utilizing CdA-AMA-SG1 and CdA-digly-AMA-SG1 as initiators for NMP, a series of polyisoprene prodrugs (CdA-PI and CdA-digly-PI) with various molecular weights (1410-4980 g/mol), low dispersity (~1.1), and controllable drug payload (ranging from 5-20%) were successfully prepared.
Utilizing CdA-CDP and CdA-digly-CDP as chain transfer agents for RAFT polymerization, a series of poly(squalenyl methacrylate) prodrugs (CdA-PSqMA and CdA-digly-PSqMA) with various molecular weights (2710-4090 g/mol), low dispersity (~1.2), and controllable drug payload (ranging from 7-11%) were prepared.
Utilizing PTX-AMA-SG1 as initiator for NMP, a series of polyisoprene prodrugs (Ptx-PI) with various molecular weights (2640-4310 g/mol), low dispersity (~1.1), and controllable drug payload (ranging from 20-32%) were successfully prepared.
Utilizing PTX-CDP as initiator chain transfer agents for RAFT polymerization, a series of poly(oligo(ethylene glycol) methacrylate) prodrugs (Ptx-POEGMA) with various molecular weights (5270-8020 g/mol), low dispersity (~1.2), and controllable drug payload (ranging from 11-16%) were successfully prepared.
Utilizing Napht-AMA-SG1 as initiator for NMP, a series of fluorescent polyisoprene (Napht-PI) with various molecular weights (2070-3710 g/mol) with low dispersity (~1.2) were successfully prepared.

The polymer prodrugs were formed into nanoparticles by the nanoprecipitation technique. Their sizes (80-200 nm), distributions (~0.1), and zeta potentials (-30--70 mV) were measured by DLS, zetasizer, and CryoTEM. All of them exhibited high size stability in both aqueous solution and cell culture medium. In the case of polyisoprene and poly(squalenyl methacrylate)-based prodrugs, the size of the formed nanoparticles increased along with increasing the polymer chain length, resulted in tunable particle size by varying the molecular weight of the polymer prodrugs. In addition, the fluorescent polyisoprene was formed into fluorescent nanoparticles with the same method, which exhibited excellent aggregation-induced emission (AIE) properties in aqueous solution and potential fluorescence imaging ability in living cells.
The cytotoxicity of these polymer prodrug nanoparticles were investigated by the MTT test on cancer cells (L1210, A549 and MCF7 cell lines). With a labile linker (diglycolate), CdA-digly-PI and CdA-digly-PSqMA exhibited more significant anticancer activity than CdA-PI and CdA-PSqMA, respectively, which was further confirmed by the drug release experiment monitored by HPLC. Moreover, the cytotoxicity of the polymer prodrug nanoparticles increased along with increasing the polymer chain length, which provided a convenient and efficient way to tune the anticancer activity by varying the molecular weight of the polymers. In the case of Ptx-PI and Ptx-POEGMA, these formed polymer nanoparticles showed significant in vitro anticancer activity, the half maximal inhibitory concentration (IC50) of which is at the same level of the Ptx free drug or even lower. The further in vivo investigation is still under progress.

In summary, the “drug-initiated” controlled/living radical polymerization method has been successfully applied to prepare various drug-polymer conjugates with different groups of polymer chains and anticancer drugs, with tunable molecular weight, polymer nanoparticle size, and cytotoxicity. These results demonstrate the versatility of the drug-initiated method and opens new perspectives for developing significant polymer prodrugs with promising application in cancer therapy. This project lay the foundation of further preclinical evaluation of the different prodrug nanoparticles in vivo on tumor-bearing mice.

The project resulted in 2 submitted publications in high impact journals (Chem Mater and Chem Commun) and 2 under preparation manuscripts that will be submitted in the coming months to very reputed journals in the field. Also, a lecture has been given by the recruited researcher at the 251st American Chemical Society National Meeting & Exposition, San Diego, USA on this project.

1. Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Chem. Soc. Rev. 2013, 42, 1147
2. (a) Putnam, D.; Kopeček, J., Polymer conjugates with anticancer activity Biopolymers II. In Peppas, N.; Langer, R., Eds. Springer Berlin / Heidelberg: 1995; Vol. 122, pp 55; (b) Duncan, R. Nat. Rev. Cancer 2006, 6, 688.

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