Carboplation-loaded SMND To Reduce GSH-mediated Platinum Resistance For Enhancing Therapeutic Effect

Prostate cancer(PCa)poses a severe threat to health of men.Chemotherapy,a well-established paradigm in the treatment of cancer clinically,is one of the indispensable means in clinical prostate cancer treatment.As an effective anticancer medicine since decades for a large variety of cancers,Carboplatin(CBP)has been recommended by NCCN(National Comprehensive Cancer Network)to treat prostate cancer.However,glutathione(GSH)-mediated resistance in cancer cells has limited the applications of CBP.Due to abnormal metabolism,there was a strong reductive environment in tumor cells.The concentration of GSH in tumor cells is approximately 7-10 fold higher than in normal cells.Further,cytoplasmic GSH concentrations(2-10 mM)are substantially higher than extracellular GSH levels(2-20?M).There was a strong affinity between GSH and bivalent platinum.GSH will competitively bind with bivalent platinum and decrease the binding efficacy between CBP and DNA,thus weakening the antitumor activity of CBP.The solutions to solve GSH-mediated platinum resistance mainly including:1)Turning into reducing sensitive Pt(?)to competitively consume GSH;2)GSH inhibitors were applied to inhibit the synthesis of GSH;3)Glutathione S transferase(GST)inhibitor were applied to inhibit the binding between GSH and Pt(?).Redox sensitive Pt(?)has attracted much attention due to its easy modification,high reduction sensitivity and low toxicity.As with most of chemotherapy drugs,Pt(?)is also faced with the problems of poor selectivity in vivo distribution and side effects.It is still a challenge to effectively deliver Pt(?)in tumor sites,reduce Pt(?)into Pt(?)by consuming GSH in tumor cells,and take effectively antitumor activity.Nanodrug opens a new window for cancer therapy.Recently,therapeutic platforms based on a new type of small molecule based nanodrug(SMND)have gained great attention.SMND is a type of self-carried nanodrug,whose nanoparticles(NPs)are mainly composed of small molecules themselves with a little or without excipients as stabilizer.In addition to the advantages of traditional nanodrug,the SMND shows unique advantages in cancer treatment:(1)self-assemble into stable nanopar-ticles without durg leakage;(2)high drug loading,avoiding metabolic problems and toxic side effects caused by pharmaceutical excipients to some extent;(3)relative simple synthesis,defined structure and economical process for fabrication;(4)retaining advantages of nanodrug,such as improving the accumulation of drugs in the tumor site,enhancing the antitumor effect and reducing the toxic effects.Thus,SMND provided a good strategy to develop novel nanomedicine.Based on the SMND strategy,CBP(?)loaded SMND to reduce GSH-mediated platinum resistance for prostate cancer therapy was prepared.First,hydroxylated CBP(CBP-OH(?))was obtained by the oxidation of CBP.As the polar part,CBP-OH(?)was covalently linked with non-polar part lauric acid to obtain the amphiphilic conjugate CBP-LA.The amphiphilic conjugate can self-assemble into stable and uniform CBP-LA nanoparticles(CBP-LA NPs)in aqueous solution.CBP-LA NPs can effectively accumulate in the tumor site.After cellular internalization,CBP-LA NPs would competitively consume GSH and protect CBP from detoxification of GSH,which improved the binding efficiency between CBP with DNA,and ultimately improve the antitumor effect.The main contents and results were listed as follows:1 Synthesis of CBP-LA and determination of CBP-LA contentCBP-LA was successfully synthesized,and confirmed by IH-NMR and HRMS.Determination method of CBP-LA was determined by HPLC.The results showed that the linearity of CBP-LA in the concentration of 50?1250 ?g/mL was good(R=0.9996).The precision of within-day and between-days could meet the requirements(RSD<2%).The method recovery could meet the requirements(98%?102%,RSD<2%).2 The construction and primary evaluation of CBP loaded in SMND(CBP-LA NPs)CBP loaded in SMND(CBP-LA NPs)were prepared by nanoprecipitation.The morphology,average particle size,zeta potential,polydispersity index(PDI,critical aggregation concentration(CAC)and stability in serum of the resulting CBP-LA NPs were characterized.The amphiphilic conjugate of CBP-LA can self-assemble into stable nanoparticles.The average diameter of CBP-LA NPs was about(72.0±2.41)nm.The zeta potential of CBP-LA NPs was-(27.1±1.42)mV.When characterized by TEM,CBP-LA NPs showed a spherical morphology with an average diameter.The average size of CBP-LA NPs exhibited a minimal change in 24 h,which demonstrated that the excellent stability of CBP-LA NPs.The critical aggregation concentration(CAC)of CBP-LA NPs was around 1.4 ?g.mL-1.This low CAC of CBP-LA was crit-ical to maintain the micelle stability in an aqueous solution.The relative degradation of CBP-LA was monitored by HPLC.At pH = 7.4 or 6.5,the degradation of CBP-LA conjugates was slow in 48 h when no GSH was added;when 5 mM or 10 mM GSH was added,the degradation of CBP-LA was enhanced with an approximately 90%fast release at 48 h.At pH = 6.5,the degradation of CBP-LA was faster than that at pH 7.4.These results indicated that the CBP-LA conjugate was sensitive to reduction and acid hydrolysis,which could remotely control the drug release at tumor site in vivo.The consumption of GSH and cellular DNA-bound platinum concentration were used to evaluate the efficiency of overcoming platinum resistance.These results indicat cells treated with CBP-LA NPs exhibited a lower concentration of reduced GSH and improved bind efficiency between Pt(?)and DNA than that treated with CBP solution at equivalent concentration of Pt compared to CBP solution(p<0.05).These results suggested that CBP-LA NPs could reduce the intracellular GSH levels and protect the Pt(?)from detoxification of GSH under high concentrations of GSH in tumor cells,which improved the binding efficiency between Pt(?)with DNA.The in vitro cytotoxicity was evaluated using standard MTT assay.It was observed that the CBP-LA NPs showed excellent toxicity,similar to the CBP solution.The in vitro cellular uptake of CBP-LA NPs was studied by fluorescence microscopy and flow cytometry using coumarin-6.These results demonstrated that the CBP-LA NPs could be taken up effectively by the tumor cells,which was very beneficial for improving the therapeutic efficiency.3 The in vivo evaluation of CBP-LA NPsReal-time near infra-red fluorophore(NIRF)imaging was performed to investigate the targeted biodistribution of CBP-LA NPs in H22-bearing Kunming mice.The result that showed that CBP-LA NPs increased the accumulation of drugs in the tumor site and decreased the distribution in normal site.The RM-1(mouse prostate cancer cells)-bearing male C57BL/6 mice model was used to investigate the in vivo antitumor efficacy.The mice treated with CBP-LA NPs showed decreased tumor volume and tumor weight than those treated with CBP solution(p<0.05).These results indicated that the CBP-LA NPs group could significantly enhance the antitumor activity.It was attributed to that CBP-LA NPs could highly accumulate in the tumor site,competitively consume GSH,release active platinum,improve the binding efficacy between platinum and GSH,and thus enhance the antitumor effect.The primary safety of CBP-LA NPs was examined by haematoxylin and eosin(H&E)staining.These results indicated that there were no pathological change of organs of mice and CBP-LA NPs exhibited good biocompatibility and safety in vivo.In conclusion,CBP(?)loaded SMND was constructed in this project.The CBP-LA NPs with high drug loading can effectively accumulate in the tumor site,competitively consume GSH to overcome GSH mediated platinum resistance,improve the binding efficacy between platinum and GSH,and thus enhance the antitumor effect of CBP for prostate cancer treatment.