π-π Stacked Multifunctional Polypeptide Nanoparticles For Efficient And Targeted Cancer Theranostics

Biodegradable polypeptide nanovehicles with proven safety,excellent biodegradability,and versatile structures as well as functionalities have been increasingly employed for controlled delivery of anticancer drugs.The clinical outcomes display that polypeptide nanomedicines can significantly decrease the side effects and increase the therapeutic windows,however,still face several challenges such as low drug loading,poor stability,and low drug accumulation at tumor sites.In this thesis,we have designed and synthesized a series of poly(L-tyrosine)-based copolymers,and the obtained copolymers have been employed to fabricate π-π stacked multifunctional polypeptide nanovehicles for efficient encapsulation and tumor-targeted delivery of lipophilic anticancer agents such as doxorubicin(DOX),docetaxel(DTX),and paclitaxel(PTX).Meanwhile,a novel L-thyroxine-N-carboxylactamate(Thy-NCA)monomer and corresponding poly(L-thyroxine)-based copolymers with high iodine content have been developed.The copolymers can be easily self-assembled into polypeptide nanoparticles for achieving versatile SPECT/CT dual-modality imaging and targeted cancer radiotherapy in vivo.In Chapter 1,we have introduced various strategies on elevating drug loading and colloidal stability of nano-drugs,and summarized recent research on tumor-targeted polypeptide-based nanomedicines.Despite the great significance of clinically viable nanovehicles,very few of them exhibit high anticancer drug loading and stability with fast intracellular drug release.In Chapter 2,we report that polytyrosine nanoparticles(PTN)self-assembled from poly(ethylene glycol)-b-poly(L-tyrosine)block copolymer enable the ultra-high loading and rapid enzyme-responsive release of doxorubicin.Notably,PTN achieve a remarkably high DOX loading of 63.1 wt%likely owing to the existence of strong π-π stacking between DOX and polytyrosine,as shown by UV-vis analysis.Furthermore,PTN exhibit good colloidal stability in 10%FBS,but are quickly de-stabilized by proteinase K.Interestingly,ca.90%of DOX is released in RAW264.7 cells in 8 h or under 6 U/mL proteinase K in 24 h.The DOX-loaded PTN display efficient delivery and release of DOX in RAW264.7 cells,achieving a better in vitro antiproliferative effect than the clinically used liposomal DOX formulation(Lipo-DOX).Colorectal cancer remains one of the leading causes of cancer-related death worldwide.Nanomedicines used for colorectal cancer hold great promise in increasing therapeutic efficacy while decreasing adverse effects of chemical drugs,none of them,however,demonstrate decent colorectal cancer cell selectivity.In Chapter 3,we report that cRGD-decorated biodegradable polytyrosine nanoparticles(cRGD-PTN)boost encapsulation and targeted delivery of doxorubicin(DOX)to colorectal cancer in vivo.Flow cytometry,confocal microscopy and MTT assays displayed that cRGD-PTN-DOX was efficiently internalized into αvβ5 overexpressing HCT-116 colorectal cancer cells,rapidly released DOX into the nuclei,and induced several folds better antitumor activity than non-targeted PTN-DOX and Lipo-DOX.Notably,cRGD-PTN-DOX exhibited over 5 times better toleration than Lipo-DOX and significantly more effective inhibition of HCT-116 colorectal tumor than non-targeted PTN-DOX control,affording markedly improved survival rate in HCT-116 tumor-bearing mice with depleting side effects at 6 or 12 mg DOX equiv./kg.cRGD-PTN-DOX with great simplicity,robust drug encapsulation and efficient nucleic drug release appears promising for targeted chemotherapy of colorectal tumor.In Chapter 4,we have designed and constructed CD44-targeted and disulfide-crosslinked polypeptide nanovehicles(CLNPs)for efficient loading and active targeted delivery of DTX.CLNPs is formed by self-assembly of hyaluronic acid-b-polytyrosine-lipoic acid(HA-b-PTyr-LA),followed by reversible crosslinking in the presence of catalytic amount of dithiothreitol.Taking advantage of π-π stacking interactions between drug and PTyr segment,CLNPs presents high drug loading with a drug loading content of 11.2 wt.%.DTX-loaded CLNPs(DTX-CLNPs)displays a small size of around 68 nm,a narrow polydispersity,and high stability under physiological conditions.Under cytoplasm mimetic reduction environment(10 mM glutathione),DTX-CLNPs realizes triggered drug release due to the cleavage of disulfide bonds.Both confocal microscopy and flow cytometry measurement revealed CLNPs could be efficiently internalized into CD44 overexpressing A549 tumor cells via receptor-mediated endocytosis,which could be significantly suppressed by blocking the CD44 on the surface of cancer cells using free HA.MTT assay showed DTX-CLNPs could significantly inhibite the growth of A549 cells with an IC50 of 0.5 μg/mL,which was 2.2 and 5.8 fold lower than free DTX and HA blocking group,respectively.Furthermore,DTX-CLNPs displayed prolonged circulation time(ti/2p=4.71 h)and enhanced tumor accumulation of 14.1%ID/g.The in vivo therapeutic studies revealed that DTX-CLNPs could effectively suppress the growth of orthotopic A549 human lung tumor,resulting in long survival time with reduced side effects in nude mice.PTX is well known for its excellent anticancer efficacy,and often solubilized using Cremophor and ethanol in clinic.However,the Cremophor often causes hypersensitivity reactions,nephrotoxicity,neurotoxicity and cardiotoxicity.Besides,the formulation is lack of targeting ligands,and usually shows unsatisfactory cellular uptake in cancer cells.In Chapter 5,we designed and developed novel biocompatible and tumor-targeted polypeptide surfactant(A6-PTS)for PTX solubilization to achieve efficient therapy on A549 lung cancer cells.A6-PTS surfactant is consisted of PEG-b-PTyr and A6 functionalized PEG-b-PTyr(A6-PEG-b-PTyr)at a certain molar ratio.The results showed that the PTX could be efficiently solubilized by A6-PTS surfactant,and the solubilized PTX is up to 460 μg/mL,which was over 4-fold higher than a commercial surfactant,D-alpha-tocopheryl polyethylene glycol 1000 succinate(TPGS).Meanwhile,PTX-solubilized A6-PTyr(A6-PTS-PTX)exhibits small size and high stability.In comparison with non-targeting counterpart(PTS-PTX),A6-PTS-PTX displayed better cellular uptake and inhibition in CD44 overexpressing A549 tumor cells.Pharmacokinetics study demonstrated that A6-PTS-PTX had prolonged circulation time with an elimination half-life(t1/2p)of 2.13 h.Thus,the polypeptide surfactant(A6-PTS)with features like natural components,facile preparation,tumor selectivity holds great potential on efficient and targeted chemotherapy of different tumors.The future healthcare requires development of novel theranostic agents that are capable of not only enhancing diagnosis and monitoring therapeutic responses but also augmenting therapeutic outcomes.In Chapter 6,we report on a versatile and stable nano-agent based on poly(ethylene glycol)-b-poly(L-thyroxine)(PEG-PThy)block copolymer for enhanced SPECT/CT dual-modality imaging and targeted tumor radiotherapy in vivo.PThyN exhibited while comparable in vitro CT attenuation efficacy to iohexol greatly enhanced in vivo CT imaging of vascular systems and soft tissues(including heart,liver,spleen)within 8 hour post-injection.PThyN allows surface decoration with cRGD peptide achieving enhanced CT imaging of αvβ3 integrin-overexpressing tumors such as subcutaneous B16F10 melanoma and orthotopic A549 lung tumor.Taking advantages of facile iodine exchange reaction,we can readily obtain radioactive PThyN,in which 125I-labeled PThyN enabled SPECT/CT imaging of major organs and monitoring of PThyN biodistribution in vivo.Besides,131I-labeled and cRGD-functionalized PThyN displayed remarkable growth inhibition of B16F10 tumor in mice without causing significant adverse effects.These poly(L-thyroxine)nanoparticles provide a unique theranostic platform for tumor therapy.In Chapter 7,we overview the results of the thesis and present perspectives on the related research in the future.