Development Of Ph-dependent And Ultrasound-responsive O-carboxymethyl Chitosan Nanodroplets For Controlled Drug Release In Prostate Cancer Therapy

PART ? Preparation and characteristics of dual-responsive O-carboxymethyl chitosan nanodropletsObjective To prepare pH-dependent and ultrasound-responsive O-carboxymethyl chitosan nanodroplets as a drug delivery system,and to explore its physical properties,stabilities and the ultrasonic contrast imaging ability in vitro,and its interaction with the human prostate cancer cell line PC-3.Methods The nanodroplets were developed via nano-emulsion process with high-speed homogenizer.Mono-factor analysis was used to screen out the optimal combination of the main factors in the preparation process.Those important factors were the speed and time of the first and the second homogenizing,the concentration of O-CS as the shell,the concentration of PFH as a core,and the concentration of tween 20 and lecithin as surfactant and co-surfactant.The morphology and structure of O-CS NDs were observed using optical microscopy,scanning electron microscope(SEM)and transmission electron microscope(TEM).The average size distribution,polydispersity index(PDI)and zeta potential of the nanodroplets were measured through dynamic light scattering(DLS),Delsa Nano C Particle Size and Zeta Potential Analyzer.The surface charge of O-CS NDs at different pH values were measured by Zeta Potential Analyzer.The physical stability of O-CS NDs over 48h was evaluated by measuring their size and morphology over time.Hemolysis assay and protein absorption assay were used to investigate the stability of nanodroplets in blood circulation.CCK-8 assay was used to examine the cytotoxicity of the O-CS NDs.Fluorescent-activated cell sorting(FACS)and fluorescence microscopy were carried out to investigate the cellular interaction of O-CS NDs.In vitro ultrasound imaging experiments were performed to investigate the ultrasonic contrast imaging abilities of the nanodroplets with a clinical diagnosis ultrasound imaging system.Results The optimal factors in the preparation process of O-CS NDs are as follows:the first homogenization speed and time were 20000 rpm and lmin,the secondary homogenization speed and time were 14000 rpm and 2 min,the concentration of O-CS/CS,PFH,tween 20 and lecithin were 0.15%w/v,5%v/v,0.2%v/v and 0.15%w/v respectively.O-CS NDs developed here were spherical in shape with core-shell structure and with an average diameter of(182.8±9.4)nm with a narrow PDI(0.188-0.200).The zeta potential of O-CS NDs was(-9.14)mV.These negatively charged nanodroplets at physical pH value(7.4)showed good stabilities by resisting serum protein absorption.Moreover,the surface charge of the nanodroplets tuned to positive in weak acid tumor microenvironment(pH 6.3).The results of fluorescence microscopy and FACS showed O-CS NDs at pH 6.3 achieved higher cellular interaction abilities compared with O-CS NDs at pH 7.4 and CS NDs at both pH values.O-CS NDs exhibited superior ultrasound contrast imaging effect using a clinical ultrasound imaging system.Conclusion pH-dependent and ultrasound-responsive O-CS NDs developed as a nano-sized drug delivery system has stronger interaction ability with tumor cells,with a small particle size and uniform distribution,good physical stability and blood stability,excellent ultrasound contrast imaging ability.All these characteristics lay the foundation of further drug and gene delivery,indicating a promising and bright future of nanomedicine for cancer treatment.PART ? Preparation and characteristics of dual-responsive doxorubicin-loaded O-carboxymethyl chitosan nanodroplets for prostate cancer therapyObjective To prepare the doxorubicin-loaded O-carboxymethyl chitosan nanodroplets,and to investigate its characteristics,ultrasonic contrast imaging abilities in vitro and in vivo,drug release profile in vitro and the anti-cancer effect under ultrasound stimulation on human prostate cancer cell line PC-3 cell in vitro and nude mice of PC-3 model in vivo.Methods doxorubicin-loaded O-carboxymethyl chitosan nanodroplets(O-CS-DOX NDs)were developed using the same nano-emulsion method as that of O-CS NDs.Different initial concentrations of doxorubicin(0.5,1.0,1.5,2.0 mg/ml)were added during the preparation process to prepare the drug-loaded nanodroplet with optimal encapsulation efficiency(EE)and loading efficiency(LE).EE and LE were examined using a microplate spectrophotometer(480nm).The morphology,particle size,polydispersity index(PDI)and zeta potential of O-CS-DOX NDs were detected by fluorescence microscope,Delsa Nano C particle size analyzer and Zeta potential analyzer.The ultrasound contrast imaging effects in vitro and in vivo were investigated by a clinical diagnosis ultrasound imaging system.The drug release profile of O-CS-DOX NDs at pH 7.4,pH 6.3 and pH 5.4 in vitro were studied by dialysis method,and the effect of ultrasonic irradiation on drug release was preliminarily studied.The cytotoxic effects of O-CS-DOX NDs with and without ultrasound irradiation were examined by CCK-8 assay on PC-3 cells in vitro.Before studying the therapeutic effect of O-CS-DOX NDs in vivo,the safety and toxicity to major organs of O-CS-DOX NDs in nude mice were evaluated through blood test and organs' pathological performances.Then we studied the therapeutic effects of O-CS-DOX NDs combined with ultrasound irradiation on tumor-bearing nude mice.Results O-CS-DOX NDs were successfully synthesized with nano-emulsion method.EE of the nanodroplets increased with the increasing of doxorubicin concentration,while LE showed a different trend.It could be concluded that a concentration of 1.5 mg/ml was optimal,at which EE(92.2%)and LE(47.2%)were both satisfying.The typical mean diameter of O-CS-DOX NDs was(174.6±8.9)nm,with a narrow PDI(below 0.200).The zeta potential of O-CS-DOX NDs stayed about-(8-9)mV when the concentrations of doxorubicin increased.The fluorescence imaging of O-CS-DOX NDs showed discrete and spherical outlines,which were red because of the presence of doxorubicin.With a clinical ultrasound imaging system,O-CS-DOX NDs showed high echo and fine dots in the two-dimensional ultrasound mode,and a uniform enhanced high echo in the contrast ultrasound imaging mode,exhibiting superior ultrasound contrast imaging effect.There were no obvious changes in the EE and LE of O-CS-DOX NDs at both pH 7.4 and 6.3 in 2 h.The drug release profile of doxorubicin from O-CS-DOX NDs in vitro at different pH values showed a sustained and pH-dependent drug release profile.61.4%and 55.2%of doxorubicin were release from the nanodroplets at 72 h at pH 5.4 and 6.3,while only 16.8%of doxorubicin were released at pH 7.4.The effect of ultrasound exposure on the doxorubicin release was also investigated.The cumulative amount of doxorubicin released from O-CS-DOX NDs was only 0.59%within 10 min.With ultrasound exposure(1w/cm2)at 37? for 10 min,massive amount(73.6%)of drug released from the nanodroplets.The results of eradication effects of nanodroplets for PC-3 cells in different groups showed that the viability of PC-3 cells in the O-CS-DOX NDs group(75.0%)was significantly higher than that in the free DOX group(56.7%)without ultrasound irradiation.Contrarily,the viability of PC-3 cells in the O-CS-DOX NDs group was significantly lower than that in the free DOX group with ultrasonic irradiation.Among the free DOX samples with ultrasound stimulus,the viability of PC-3 cells showed slightly decreased with the intensity and time of ultrasound irradiation increased.Nonetheless,the cell viability in the DOX+US4 group(15.2%)was much lower than other DOX+US groups.The cell viability of PC-3 cells in the O-CS-DOX NDs groups decreased significantly as the ultrasound intensity and time increased.Moreover,the ratio of the viability was significantly decreased in cells treated with O-CS-DOX NDs+US compared to those treated with DOX+US,except that the cell viability in the O-CS-DOX NDs+US1(58%)was mildly higher than that in the DOX+US1 group(54.7%).There were no significant differences of the PC-3 cell viability between the O-CS-DOX NDs groups and the CS-DOX NDs groups.With the successfully establishment of PC-3 subcutaneous tumor model of nude mice,the ultrasonic imaging study showed the good ultrasonic contrast imaging capabilities of O-CS-DOX NDs in vivo,which was consistent with the results of ultrasound imaging experiments in vitro.The results of blood tests(blood routine and blood biochemistry)and pathological pictures of important organs(heart,liver,kidney and lung)showed high safety and no toxicity to organs of the O-CS-DOX NDs application in nude mice.Compared with the free drug group and the CS-DOX NDs+US group,the therapeutic effects of nude mice in the O-CS-DOX NDs combined with ultrasound irradiation group showed best with statistical differences.The results above indicated that O-CS-DOX NDs combined with ultrasound irradiation can enhance the killing effects on tumors in vivo,which were consistent with the results of experiments in vitro.O-CS-DOX NDs also were proved to be better in vivo than CS-DOX NDs,which strength the results of part ?.Conclusion O-CS-DOX NDs as a safe drug carrier have high stability in circulation,good ultrasonic contrast imaging performances in vitro and in vivo,and anticancer effect with the aid of ultrasound in vitro and in vivo.