Application Of Targeted Hydroxyethyl Starch Conjugates For The Treatment Of Prostate Cancer

Background:Worldwide prostate cancer is the fourth most common cancer and the second most frequently diagnosed cancer in men.It is the most frequently diagnosed cancer among men in developed countries.According to the American Cancer Society estimates for 2017 in the United States,prostate cancer will account for nearly 19%of newly diagnosed cancers and 8%of cancer-related deaths among men.Meanwhile,the prostate cancer incidence rate in our country has been gradually increasing in the past ten years.As prostate cancer progresses,it can metastasize to the liver,lung,bone and lymph node.At the same time,some patients may be diagnosed advanced prostate cancer at first diagnose.For these patients,androgen deprivation therapy(ADT)is the standard first-line therapy.However,most patients with metastatic prostate cancer can only benefit from ADT for less than 24 months.Moreover,metastatic castration-resistant prostate cancer(mCRPC)produces painful osteoblastic bone metastases that severely impair the patient's quality of life,and patients with mCRPC are expected to survive less than 19 months.Although chemotherapeutic treatment of mCRPC has been shown to improve the patient's quality of life and lengthen the period of survival to a certain extent,some defects of small-molecule drugs such as brief half-life and circulation time,poor targeting,low utilization,serious undesired side effects,susceptibility to drug resistance and requiring the dose of drug to be increased gradually,have hampered its further application.In resent years,various polymeric nanoparticles including micelles,vesicles and conjugates have been intensively researched to enhance the anti-tumor efficacy,reduce the toxicity and improve the drug resistance of small-molecule drugs.Among these,conjugate is considered as an ideal drug carrier due to its favourable biocompatibility,controlled biodegradation and release,easy surface modification and structure of a hydrophilic shell and a hydrophobic core after the self-assembly in aqueous solution.These characteristics make the chemotherapeutic drugs escape from the prompt uptake by the reticuloendothelial system(RES)and glomerular filtration.At the same time,selective intratumoral accumulation via the enhanced permeability and retention(EPR)effect reduces systemic toxicity and improves the anti-tumor efficacy of conventional antitumor drugs.In this work,the conjugates were prepared via the reaction between the aldehyde group of oxidized hydroxyethyl starch(HES-CHO)and the amino groups of doxorubicin(DOX)and luteinizing hormone-releasing hormone(LHRH)analog.We would give a comprehensive illustration about the advantages of conjugates for treating the prostate cancer through the preparations and characterizations of conjugates,in vitro cell test and in vivo animal experiment.Materials and methods:?pH-responsive non-targeted HES-DOX and targeted HES-DOX/LHRH were prepared via the Schiff base reaction between the aldehyde group of HES-CHO and the amino groups of DOX and LHRH analog.The drug binding content(DBC)and drug binding efficiency(DBE)were determined by the ultraviolet-visible spectrophotometry(UV-1800).The characterizations and drug release profiles of conjugates and micelles were detected by the spectra of proton nuclear magnetic resonance(~1H NMR),spectra of Fourier-transform infrared(FT-IR),critical micelle concentration(CMC),dynamic laser scattering(DLS),transmission electron microscopy(TEM),X-ray photoelectron spectroscopic(XPS)and in vitro drug release test.?The cellular uptake and intracellular DOX release behaviors of conjugates were estimated by using confocal laser scanning microscopy(CLSM)and flow cytometry(FCM)on mouse prostate carcinoma(RM-1)cells.The cytotoxicities of conjugates toward RM-1 cells were assessed by a MTT assay.?Wistar rats administered drugs via the lateral tail vein were used to detect the pharmacokinetics by High Performance Liquid Chromatography(HPLC).The tumor-bearing mouse model was prepared by subcutaneously injecting RM-1 cells into the armpits of the right anterior limbs with six-week-old male C57BL/6 mice weighing from 18.0 to 20.0 g.The tissue distribution of DOX in the tumors and internal organs excised from the tumor-bearing mouse model which had been injected drugs was determined by In-Vivo Imaging System.At the same time,treatments were carried out with drugs via intravenous injection for 3 times every 4 days.PBS served as the control.The tumor volumes and body weights of mice were monitored in real-time.In the end,the histopathological and immunohistochemical analyses of the tumors,major organs,lymph nodes and sternums were used to identify the necrosis,apoptosis of tumors and metastasis,and to evaluate the systemic toxicity.Results:?Non-targeted HES-DOX and targeted HES-DOX/LHRH were facilely synthesized via the Schiff base reaction between the aldehyde group of HES-CHO and the amino groups of DOX and LHRH analog.The chemical structures of conjugates were confirmed via the above methods.The DBC and DBE of HES-DOX were 6.03 and 79.7 wt.%,respectively,and those of HES-DOX/LHRH were 4.92 and 81.5 wt.%,respectively.The HES-DOX conjugates could self-assemble into nanoscale micelles in aqueous solution.The CMC of HES-DOX and HES-DOX/LHRH were 8.5 and 11.0?g/m L,respectively.In addition,the micelles signified excellent stability under physiological conditions.The TEM and DLS revealed that the micelles exhibited clear spherical morphologies with a radius of~55 nm.The in vitro DOX release behaviors indicated that conjugates possessed excellent controlled drug release,and as the p H decreased,the cumulative drug release from conjugates was significantly accelerated.?The results of CLSM and FCM assays revealed that micelles possessed great cellular endocytosis and intracellular sustained DOX release.Meanwhile,as time went on,the amount of DOX release increased.After 24 h,the intracellular DOX amount of targeted HES-DOX/LHRH group significantly exceeded that of the free DOX·HCl group.MTT assay showed that the cell viability of every group was significantly inhibited in a dose-and time-dependent manner.As time extended and drug dose increased,the cell inhibition efficacy of conjugate groups obviously approached that of the free DOX·HCl group.?The result of pharmacokinetics revealed that the conjugates reduced blood clearance and prolonged blood circulation time of DOX.The result of in vivo tissue distribution showed that conjugates enhanced the accumulation of DOX in tumor site.The tumor volumes,histopathological and immunohistochemical assays indicated that the conjugates,especially targeted HES-DOX/LHRH,possessed greater anti-tumor and anti-metastasis effects.In vivo safety assessments performed through evaluation of body weight changes,organ index detection,and histopathological analyses of visceral organs showed that conjugates significantly reduced the systemic toxicity of DOX.Conclusions:In this work,the two conjugates possessed high DBE,and the radius of micelles was a suitable size which facilitated the selective accumulation of micelles in tumor tissue via the EPR effect.The conjugates,especially targeted HES-DOX/LHRH,possessed greater anti-tumor and anti-metastasis effects with lower systemic toxicity.In summary,the targeted conjugates have great potential for clinical chemotherapeutic treatment of metastatic prostate cancer.