The Prostate Cancer Targeted Drug Delivery Systems

Prostate cancer is the second most frequently diagnosed malignancy in men worldwide and the fifth leading cause of cancer-related death.According to statistics released by the American cancer society in 2021,the incidence of prostate cancer ranks first among male tumors and the mortality rate is second.The early stage of prostate cancer is mostly androgen dependent,and then it usually becomes androgen independent prostate cancer after endocrine therapy for a period of time about 14～30 months,and finally turns to hormone refractory prostate cancer(HRPC).HRPC is low differentiation,high malignancy,no response to androgen removal,and high mortality.Targeted therapy,with its highly specific intervention mode,plays an important role in HRPC therapy to improve tumor therapeutic effect and reduce adverse reactions caused by radiotherapy and chemotherapy,and has become an important option of HRPC therapy and even the whole field of tumor therapy.In the first part of research,we selected overexpressed α-enolase as the target on human advanced prostate carcinoma cells for the first time.α-Enolase is a glycolytic enzyme which plays an important role in catalysing 2-phosphoglyceric acid to phosphoenolpyruvic acid in the cytoplasm.It has been reported that glycolysis and glucose transport are upregulated generally in various cancer cells,and indeed αenolase is increased at the mRNA and/or protein level in a variety of cancers,including prostate cancer.Additionally,the expression level of α-enolase is positively correlated with the status of cancer pathology classification.High expression of α-enolase is found in patients with a larger tumor size,and a shorter disease-free interval.All these pieces of evidence indicate that α-enolase could be chose as an effective therapeutic target for human advanced prostate cancer.For the targeting ligand,we chose the peptide pHCT74(SSMDIVLRAPLM)which was recently reported to specifically bindα-enolase with high affinity,in order to target α-enolase in prostate cancer.In this study,the prostate cancer targeted liposome using pHCT74 as the targeting ligand(pHCT74-lipo)was prepared and characterized.The size,distribution,zeta potential,drug encapsulation efficiency,and the release property of drug in vitro were studied.Then we confirmed the level of α-enolase expression by PC-3 cells,REWP-1 cells and B16F10 cells.Through in vitro cell assay and in vivo distribution assay,we explored prostate cancer targeting capability of pHCT74-lipo.Afterwards,doxorubicin was incorporated into pHCT74-lipo to achieve the targeted delivery to the prostate tumor.We further evaluated therapeutic efficacy of pHCT74-lipo-Dox in mice bearing PC-3 xenograft tumors as well as safety.pHCT74-lipo was prepared with the thin film hydration method,displaying an average size of 111.7±4.15 nm and zeta potential of 29.4±0.67 mV.Compared with pHCT74-lipo,pHCT74-lipo-Dox had no significant difference in particle size and zeta potential after loading doxorubicin.The encapsulation efficiency of pHCT74-lipo-Dox was about 97.0±1.34%.Through monitoring absorbance variation,we confirmed the stability of liposomes in vitro.Furthermore,both PEG-lip-Dox and pHCT74-lipo-Dox showed the sustained release pattern up to 72 h and no burst release was observed before 24 h.Afterwards,we evaluated the α-enolase expression in RWPE-1(normal prostate cell line,as a negative control),PC-3(prostate cancer cells)and B16F10(murine melanoma cell line,as a positive control)cell lines.The α-enolase expression in PC-3 and B16F10 cell lines was significantly high compared with that of RWPE-1 cell line.In the cellular uptake studies,the ability of pHCT74-lipo to get into PC-3 cells was much stronger than PEG-lipo.The mechanism study proved that the cellular uptake was mediated by the interaction between pHCT74 and α-enolase,which helped liposomes into cells.To investigate the prostate tumor targeting ability of DiD labeled pHCT74-lipo in vivo and ex vivo,near-infrared fluorescence imaging technique was used.Strong fluorescence signal in the tumor of pHCT74-lipo treated mice was detected after intravenous administration,peaking at 12 h.To further confirm that pHCT74-lipo could help deliver doxorubicin to the prostate tumor,doxorubicin distribution was also analyzed by semiquantitative fluorescent intensity of ex vivo tumors at 24 h.Results showed that the level of doxorubicin in the tumor of pHCT74-lipo treated mice was significantly higher(P<0.001)than other groups.Through laser confocal scanning microscope,we found pHCT74-lipo group showed the strongest fluorescence signal in every tumor slide,which would benefit the treatment of prostate cancer.We then investigated the therapeutic effects of pHCT74-lipo-Dox on mice bearing PC-3 xenograft tumor model.The results showed that pHCT74-lipo-Dox could effectively inhibit the tumor growth,improve the survival period of PC-3 tumor bearing mice.These results suggested pHCT74-lipo-Dox had the preferable therapeutic efficacy for prostate cancer.No obvious pathology and abnormal values of various biomarkers were observed in mice treated with pHCT74-lipo-Dox.Thus pHCT74-lipo-Dox could be a safe targeted drug delivery system for the treatment of prostate cancer.In summary,the pHCT74 peptide,as a targeting ligand,could be utilized to target the prostate cancer cells,and pHCT74-lipo-Dox is an effective and safe tumor targeting drug delivery system against prostate cancer.However,we found that these ligand modified conventional spherical liposomes can improve the tumor targeting and accumulation ability,thus improving the efficacy of anti-PCa,but there may still be possibility to further improve tumor accumulation and permeability of drug delivery system.In the second part of this project,encouraged by recent researches showing possible drug retention and tissue penetration advantages of non-spherical nanoparticles over spherical nanoparticles,we aim to design and construct a novel non-spherical liposomal nanodisk drug delivery system for treating prostate cancer,and compared with liposomal spherical nanoparticles.In this study,we attempted to use liposomal nanodisks as drug carrier.Liposomal nanodisks have both large length to diameter ratio like nanorods and overall shape closer to nanospheres,hence possibly possess advantages of both types of nanoparticles.Besides,liposomal nanodisks may also exert bionic-related effects like good biocompatibility,since there are natural disk-like structures in blood like platelets.Though there are only a few published literatures on using liposomal nanodisks for drug delivery,we speculated that liposomal nanodisks could improve tumor penetration and retention of loaded drugs.Besides,conventional tumor targeting strategies often try to directly guide drug delivery system to tumor sites by using ligands that specifically binds to unique overexpressed receptors on tumor cell surface.However,due to the complex tumor microenvironment,tumor cells are often protected by dense layers of extracellular matrix,making it difficult to contact tumor cells directly and hence reduce targeting efficiency of tumor cell-binding ligands.Moreover,a small population of cancer cells in tumor microenvironment,often competes for survival and quickly evolves.This leads to the genetical heterogeneity and possible variances of surface receptors of cancer cells,which may lower binding capability of the designed targeting ligand to tumor cells.Because of these drawbacks,we searched for targeting ligands that bind to tumor stroma cells instead,as they generally have more constant biomolecular properties,making them easier to penetrate and more stable to anchor.Here,the CysArg-Glu-Lys-Ala peptide with N-methylated Glu(CR(NMe)EKA)was chose as the targeting ligand.The original unmodified Cys-ArgGlu-Lys-Ala(CREKA)peptide binds to clotted plasma proteins including fibrins and its complex,thus accumulates at tumor stroma and vessel walls as there are abundant fibrins complex in these sites.The N-methylation of Glu in CREKA enhances its stability in vivo without altering its tumor homing ability,which further improves the applicability of CREKA.Based on these two design strategies,we successfully constructed paclitaxelloaded liposomal nanodisks with surface CR(NMe)EKA modification(ND-P/PTX).Preparation of ND-P/PTX was as simple and stable as conventional CR(NMe)EKA modified liposomal nanospheres(NS-P/PTX),and paclitaxel loading efficiency with ND-P/PTX was also close to that of NS-P/PTX.ND-P/PTX have a reasonable encapsulated efficiency(87.63±2.33%),good stability and no rapid release of PTX from ND-P/PTX in vitro.Transmission electron microscopy revealed that liposomal nanodisks were indeed platelet-like particles with disk diameter of～100 nm and thickness of 5 nm,while liposomal nanospheres were uniformly spherical of～100nm.To investigate the fibrin targeting ability of CR(NMe)EKA in vitro,the binding of DiD-labeled liposomal nanodisks or nanospheres to fibrin clots was conducted.Subsequently,the fibrin targeting ability of CR(NMe)EKA in vivo was further studied,DiD-labeled liposomal nanodisks or nanospheres were administered intravenously to ectopic prostate tumor mice,and near-infrared fluorescence images were acquired at different time points.The fluorescence signal of ND-P/DiD was significantly stronger than other groups at every time points examined.Ex vivo fluorescent images of isolated organs harvested at 48 h after injection further displayed that ND-P/DiD treated mice had strongest tumor fluorescence.In fact,fluorescence intensity of NDP/DiD and ND/DiD were significantly higher than NS/DiD or NS-P/DiD at 12 h,24 h,and 48 h in tumors.Immunofluorescence experiments showed that CR(NMe)EKA modified nanoparticles were well-colocalized with vessel marker CD31 and fibrinfibronectin complexes in vivo.Encouraged by results using DiD fluorescence marker labelled liposomes,we further examined drug biodistribution using paclitaxel loaded liposomal nanoparticles.Then,paclitaxel in organs,tumors and blood from tumor-bearing mice treated with paclitaxel loading liposomal nanoparticles 24 h before were measured using liquid chromatography-tandem mass chromatography(LC-MS/MS).Again,ND-P/PTX treatment showed the highest paclitaxel accumulation at tumor sites,which was consistent with DiD fluorescent results.These visual evidences indicated that liposomal nanodisks had better tumor targeting and accumulation capability than that of liposomal nanospheres,both with and without CR(NMe)EKA modification.Also,these results displayed that CR(NMe)EKA peptide modification enhanced tumor targeting ability of both liposomal nanodisks and nanosphere in vivo.Having verified the targeting ability of ND-P,we loaded ND-P with paclitaxel for prostate cancer treatment.As expected,ND-P/PTX showed the strongest anti-tumor effect at the same dose in in vivo prostate tumor xenograft mice model.Further examination revealed that ND-P/PTX and NS-P/PTX were indeed both strongly targeted to tumor stroma and vessels walls,where CR(NMe)EKA played vital roles.ND-P/PTX also displayed robust systemic safety both in vitro and in vivo,fulfilling basic requirements for possible clinical usage.The second part of research,on the basis of first part study,we successfully designed and constructed a novel liposomal nanodisk based targeted drug delivery system that had significantly better tumor targeting and anti-tumor efficiency than spherical counterparts.These results provide a theoretical and data supplement for the study of different shapes of liposomal drug delivery system to realize the treatment of prostate cancer,and also provide valuable data for further understanding of the biological effects of non-spherical nanodisks.In summary,aiming at the prostate cancer targeted therapy,we continuously optimized targeted design strategies and prepared different liposomal targeted drug delivery systems in this project.Firstly,we designed and constructed the traditional liposomes modified with the pHCT74 targeting ligand.The modification of pHCT74 peptide ligand effectively enhanced the targeting effect of liposomes.However,tumortargeting and deep tumor penetration capabilities of this traditional spherical liposomes are still far from satisfactory.Therefore,in order to further improve prostate cancer targeting and penetration capability of the drug delivery system,we further constructed liposomal nanodisks,and the results showed that liposomal nanodisks had better tumor targeting and penetration ability,also showed better anti-PCa effect.Therefore,this research provides valuable data and ideas for the development of prostate cancer targeted liposomal drug delivery system.The nanodisks drug delivery system has great clinical potential and is highly adaptable for delivering other drugs/agents.Additionally,our data highlight the advantages of liposomal nanodisks in drug delivery against prostate cancer,and underline the potential for developing more nonspherical nanoparticles for biomedical applications in the future.