Multifunctional Polylipopeptide-based Nanomedicines For Targeted Cancer Therapy

Poly?ethylene glycol?-b-polypeptide copolymers with excellent safety,are one of the most clinically studied materials for anticancer drug delivery.For example,paclitaxel?PTX?,doxorubicin?Dox?,and cisplatin-loaded polypeptide-based nanotherapeutics have proceeded into phase II and III clinical trials and achieved enhanced therapeutic efficacy against intractable tumors and reduced side effects in comparison with free drugs.It should be noted,however,that these nanoformulations,mostly based on hydrophilic polypeptides,usually involve multistep synthesis,including protection and deprotection of functional groups and drug conjugation.Nanosystems based on hydrophobic polypeptide often plagued with low drug loading and insufficient in vivo stability.To this end,we designed and prepared a series of poly??-aminopalmitic acid?-based nanoplatforms,including micelles and polymersomes for targeted delivery of small anti-cancer drugs,protein,and siRNA.In chapter 1,we present a brief literature overview on the current research status,challenges and perspectives of polypeptide-based anti-cancer nanomedicines.In chapter 2,we designed and prepared a robust and versatile polylipopeptide nanoplatform based on poly?ethylene glycol?-b-poly??-aminopalmitic acid??PEG-b-PAPA?block copolymers for the delivery of hydrophobic anticancer drugs like docetaxel?DTX?to B16F10 melanoma.PEG-b-PAPA copolymers with tailored M_n were easily prepared through ring-opening polymerization of?-aminopalmitic acid N-carboxyanhydride?APA-NCA?.Interestingly,PEG-b-PAPA copolymers exhibit superb solubility in common organic solvents?e.g.CHCl₃,CH₂Cl₂,and THF?and form stable polylipopeptide micelles?Lipep-Ms?that show efficient loading and intracellular triggered release of potent anticancer drugs like DTX.Notably,cRGD peptide-functionalized Lipep-Ms?cRGD-Lipep-Ms?were also obtained with similar biophysical characteristics.DTX-loaded cRGD-Lipep-Ms?DTX-cRGD-Lipep-Ms?exhibited superior tumor growth inhibition of B16F10 melanoma with markedly improved survival rate with a median survival time of 36 d,which was significantly longer than that?9 d?observed for PBS and free DTX groups and caused little side effects as compared to free DTX.In chapter 3,polylipopeptide-based micelles were explored for enhanced loading and targeted delivery of peptide drugs like Monomethyl auristatin E?MMAE?to HCT-116 colorectal tumor xenografts in mice.MMAE is an extremely potent peptide drug that is currently used and investigated in the form of antibody drug conjugates?ADCs?for treating different cancers.ADCs are,however,associated with low drug conjugation,immunogenicity,small scale production,and high costs.Interestingly,cRGD-Lipep-Ms achieved a MMAE loading contentof5.5wt.%,whichwas55-foldhigherthanpoly?ethylene glycol?-b-poly?D,L-lactide?micelles.MMAE-loaded cRGD-Lipep-Ms?MMAE-cRGD-Lipep-Ms?showed a small hydrodynamic size of 59 nm,minimal drug leakage in 10%FBS,and efficient uptake and superb antiproliferative activity in?_v?₅–overexpressing HCT-116 tumor cells with an IC₅₀ of 7.10 nM,which was about 4.2 and 1.9-fold lower than that of free MMAE and non-targeted MMAE-Lipep-Ms,respectively.Remarkably,MMAE-cRGD-Lipep-Ms displayed over 10-fold better toleration?MTD=2 mg MMAE equiv./kg?than free MMAE in mice and completely suppressed growth of HCT-116colorectal tumor xenografts at a dosage of 0.2 mg MMAE equiv./kg.It is known that Doxil^??PEGylated liposomal doxorubicin formulation?is the first FDA-approved anti-cancer nano-drug.Notably,no targeted liposomal formulation has advanced to clinical stage despite tremendous work undertaken,partly due to a low stability of liposomes.To achieve high loading and targeted delivery of hydrophilic anticancer drugs,in chapter 4,we further developed a robust lipopepsomes from PEG-b-PAPA with longer PAPA chains for targeted delivery of doxorubicin hydrochloride?Dox?HCl?.Interestingly,lipopepsomes could be easily decorated with 20 mol.%cRGD peptide and loaded with 17.4 wt.%Dox?HCl,giving cRGD-LPP-Dox with a small size of⁸0 nm.cRGD-LPP-Dox exhibited a high stability against 10%FBS and restrained drug release under physiological conditions.Flow cytometry,confocal microscopy and MTT assays using?_v?₃–overexpressing A549 tumor cells showed obviously more efficient uptake and higher anticancer activity for cRGD-LPP-Dox than for non-targeted LPP-Dox and clinically used liposomal Dox?Lipo-Dox?controls.Notably,cRGD-LPP-Dox exhibited markedly enhanced toleration and tumor accumulation than Lipo-Dox.The therapeutic studies demonstrated that cRGD-LPP-Dox achieved effective suppression of orthotopic A549 human lung tumor in nude mice,resulting in significantly improved survival rate as compared to LPP-Dox and Lipo-Dox groups.How to efficiently load and systemically deliver protein drugs into target cancer cells is a big challenge.In chapter 5,we further developed a small-sized and robust chimaeric vesicles?lipopepsomes?self-assembledfromasymmetricpoly?ethylene glycol?-b-poly??-aminopalmitic acid?-b-poly?L-aspartic acid?triblock copolymer as a simple and functional platform for high loading and targeted intracellular delivery of protein drugs in vivo.Cyclic RGD peptide-decorated chimaeric lipopepsomes?cRGD-CLP?following the loading of FITC-labeled cytochrome C?1.99–9.42 wt.%?showed small hydrodynamic sizes of 81–86 nm,enhanced internalization by?_v?₃-overexpressing A549lung cancer cells in vitro,and remarkable accumulation of 7.73%ID/g in the cancerous lung in an orthotopic A549 human lung tumor model.Saporin-loaded cRGD-CLP displayed a low half-inhibitory concentration of 16.3 nM to A549 cancer cells.Intriguingly,saporin-loaded cRGD-CLP at a low dose of 16.7 nmol saporin equiv./kg caused complete suppression of tumor growth and significantly improved survival rate in an orthotopic A549 human lung tumor model.These chimaeric lipopepsomes are versatile and provide a potential means for targeted protein therapy of various malignancies.Small interfering RNA?siRNA?is considered a highly specific and effective biotherapeutic that holds tremendous potential for the treatment of various diseases,including cancer.The clinical translation of siRNA is,however,greatly hampered by the lack of safe and efficient systemic siRNA delivery vehicles in vivo.In chapter 6,we report on the development of selective cell penetrating peptide?CPP33?-functionalized chimaeric lipopepsomes?CPP33-CLP?self-assembledfromasymmetricpoly?ethylene glycol?-b-poly??-aminopalmitic acid?-b-poly?L-lysine?triblock copolymer for efficient encapsulation and targeted delivery of polo-like kinase 1 specific siRNA?siPLK1?to orthotopic A549 human lung tumor in nude mice.Interestingly,siRNA was tightly loaded into CPP33-CLP with decent siRNA encapsulation efficiency?>95%?.siPLK1-loaded CPP33-CLP?siPLK1-CPP33-CLP?were selectively internalized by A549 human lung cancer cells and efficiently escape from endosomes and rapidly release siRNA into the cytoplasm,leading to a significant sequence-specific gene silencing in vitro.The in vivo studies demonstrated that siPLK1-CPP33-CLP exhibited prolonged blood circulation,enhanced tumor accumulation,effective suppression of tumor growth,and significantly improved survival time of orthotopic A549 human lung tumor-bearing nude mice.These chimaeric lipopepsomes appear as an attractive and potent nanoplatform for safe and targeted siRNA delivery.Chapter 7 summarizes the results of the thesis and presents perspectives for the future application of these nanoplatforms.