Nanoparticles Based On Polypeptide For Efficient Intracellular Delivery And Targeting Release Of Anticancer Drugs

Biodegradable polymeric nanocarriers have emerged as one of the most promising platforms for targeted tumor therapy owing to their excellent biocompatibility, prolonged circulation time, enhanced accumulation in tumors, and in vivo biodegradability. Poly(amino acid)s inherit many intriguing properties of proteins such as controllable hydrophilicity/hydrophobicity, biodegradability in vivo against specific enzymes, unique hierarchical assembly property and versatile functionalities, thus have been widely employed for polymeric nanocarriers. Especially, several poly(amino acid)-based nanomedicines(NK105, NK911, NK012, NC4016, NC6300, etc) have been advanced into clinic and clinical trials. In chapter one, we have summarized various nanomedicines for cancer therapy currently used in clinic and clinical trials, discussed various responsive and targeting poly(amino acid)-based nanocarriers recently developed to improve the therapeutic effect and their applications on drug(small molecular drug, protein, gene, etc) controlled delivery and tumor imaging.In the second chapter, p H-responsive chimaeric poly(amino acid)-based polymersomes(refer to as pepsomes) were designed and developed from asymmetric poly(ethylene glycol)-b-poly(L-leucine)-b-poly(L-glutamic acid)(PEG-PLeu-PGA, PEG is longer than PGA) triblock copolymers for efficient encapsulation and triggered intracellular delivery of doxorubicin hydrochloride(DOX·HCl). PEG-PLeu-PGA was conveniently prepared by sequential ring-opening polymerization of L-leucine N-carboxyanhydride(Leu-NCA) and γ–benzyl-L-glutamate N-carboxyanhydride(BLG-NCA) using PEG-NH2 as an initiator followed by deprotection of benzyl groups. Pepsomes formed from PEG-PLeu-PGA had unimodal distribution and small sizes of 54-71 nm depending on PLeu block lengths. Interestingly, these chimaeric pepsomes while stable at p H 7.4 were quickly disrupted at p H 5.0, likely due to alternation of ionization state of the carboxylic groups in PGA that shifts PGA blocks from hydrophilic and random coil structure into hydrophobic and α–helical structure. DOX·HCl could be actively loaded into the watery core of pepsomes via the electrostatic interaction with the negative charged PGA segments at p H 7.4. Remarkably, the in vitro release studies revealed that release of DOX·HCl was highly dependent on p H, in which about 24.0% and 75.7% of drug was released at p H 7.4 and p H 5.0, respectively, at 37 oC in 24 h. MTT assays demonstrated that DOX·HCl-loaded pepsomes exhibited high antitumor activity, similar to free DOX·HCl in RAW 264.7 cells. Moreover, they were also potent toward drug-resistant MCF-7 cancer cells(MCF-7/ADR). Confocal microscopy studies showed that DOX·HCl-loaded pepsomes delivered and released drug into the cell nuclei of MCF-7/ADR cells in 4 h, while little DOX·HCl fluorescence was observed in MCF-7/ADR cells treated with free drug under otherwise the same conditions. These chimaeric pepsomes with facile synthesis, efficient loading of water soluble anticancer drug, and p H-triggered drug release behavior are an attractive alternative to liposomes for targeted anticancer drug delivery.In the third chapter, p H and reduction dual-responsive reversibly crosslinked hyaluronic acid-graft-poly(amino acid)s nanoparticles were developed for intracellular triggered release of hydrophobic anticancer drugs and tumor targeted therapy. Hyaluronic acid-graft-poly(β-(diisopropylamino)ethylamine)-aspartic acid)-co-L-leucine)-lipoic acid(HA-g-P(Asp(DIP)-co-Leu)-LA) was prepared by the ring opening polymerization of Leu-NCA and β–benzyl-L-aspartate N-carboxyanhydride(BLA-NCA) using N-lipoyl-1,3-diaminopropane(LA-NH2) as an initiator, followed by grafting the obtained poly(amino acid)s onto HA with the help of DCC/NHS and then aminolysis of PBLA segment with DIP. HA-g-P(Asp(DIP)-co-Leu)-LA could readily self-assemble into nanoparticles in water, which were able to be crosslinked through the formation of a linear polydisulfide in the presence of a catalytic amount of dithiothreitol(DTT). Dynamic light scattering(DLS) measurements showed that the average hydrodynamic diameters of uncrosslinked and crosslinked nanoparticles were 108 nm and 92 nm, respectively, and both nanoparticles had a relatively narrow polydispersities(PDI) of around 0.16. The crosslinked nanoparticles while exhibiting high stability against 100-fold dilution and 2 M Na Cl salt concentration were quickly swollen and dissociated under intracellular reduction environment containing 10 m M glutathione(GSH). DOX loading experiments showed that HA-g-P(Asp(DIP)-co-Leu)-LA nanoparticles generally had high drug loading content(6.8-20.1%) and drug loading efficiency(58.7-65.3%). The in vitro release results exhibited that DOX release from crosslinked nanoparticles at physiological environment(p H 7.4, 10 m M PB and 37oC) was greatly suppressed with less than 20% release in 24 h, while the drug released was higher than 30% under endosomal p H of 5.0 mainly due to protonation of PAsp(DIP) segements. More interestingly, much faster drug release was observed under a reductive condition(10 m M GSH), in which 80.0%(p H 7.4) and 92.7%(p H 5.0) of DOX were released in 24 h under otherwise the same conditions. MTT assay exhibited that these crosslinked nanoparticles were practically non-toxic up to a tested concentration of 0.5 mg/m L, indicating these nanoparticles had excellent biocompatibility. However, the DOX-loaded nanoparticles caused pronounced cytotoxic effects to MCF-7 tumor cells with CD44 receptor(IC50 = 5.5 μg DOX equiv/m L), much less cytotoxic effects to U87 tumor cell without CD44 receptor, suggesting that the HA-CD44 interaction could significantly improve the cellular uptake of DOX-loaded nanoparticles. Confocal microscopy observations displayed that DOX-loaded HA-g-P(Asp(DIP)-co- Leu)-LA nanoparticles could release drug to the perinuclei region and nuclei of MCF-7 cells following 2 and 8 h incubation, respectively. These dual-bioresponsive reversibly crosslinked HA-graft-poly(amino acid)s nanoparticles have great potential for targeted cancer chemotherapy.