Study On Chondroitin Sulfate-based Redox-responsive Nanoparticles For Melanoma-targeted Drug Delivery

At present,malignant tumors are still a major disease that seriously threatens human life and health worldwide.In clinical practice,chemotherapy is the main treatment of tumors.However,the therapeutic efficacy of most chemotherapeutic drugs is hampered by low water solubility,poor stability,serious side effects and lack of tumor selectivity.Amphiphilic conjugates of polysaccharide polymers could self-assemble to form micellar nanoparticles in aqueous solution,avoiding the requirement of organic solution,and has gained widespread attention of scientists for tumor-targeted drug delivery.To remedy the problems riddled in cancer chemotherapy,novel chondroitin sulfate(CS)polysaccharide-based redox-sensitive,less toxic,biocompatible and actively tumor-targeting polymeric nanoparticles were developed.CS has been widely used as a backbone for fabrication of self-assembled nanoparticles.This system could not only respond to the reducing conditions in tumor cells,release drugs quickly and thoroughly,thereby improving the efficacy of chemotherapy.The innovation of this project was based on synthesizing novel polymer suitable for fabrication of nanoparticles that could realize redox-responsive release in tumor microenvironment and CD44-receptor mediated targeting of melanoma.This research was mainly divided into two parts:(a)CS-vitamin E succinate polymer(CS-CYS-TOS)with cystamine(CYS)as a linker was constructed,and then self-assembled to encapsulate the model drug docetaxel(DTX).(b)Adipic acid dihydrazide(ADH)was used as a linker to synthesize a non-reduction sensitive CS-adipate dihydrazide-vitamin E succinate(CS-ADH-TOS)polymer for fabrication of non-redox sensitive nanosystems.The rationally designed nanoformulations had excellent physiochemical properties with small size,narrow size distribution,negative zeta potential,high drug loading(DL)and encapsulation efficiency(EE),redox-release profile and biological safety.The biological performance of both redox-sensitive and non-redox sensitive nanosystems was compared,suggesting that the redox-sensitive property could enhance the curative effect.The nanoparticles had time-dependent cell-uptake and redox-sensitive nanoparticles had significantly higher cell-uptake than DTX solution.Studies found that CS-CYS-TOS nanoparticles had a good reduction response behavior,and killed tumor cells more effectively than non-sensitive CS-ADH-TOS nanoparticles.In situ tumor volume was reduced upon the administration of DTX-loaded CS-CYS-TOS nanoparticles via DTX-induced apoptosis.With only minor cytotoxicity,CS-CYS-TOS nanoparticles could be promising nano-drug delivery systems for successful management of melanoma.The intelligent nanoparticle construction method proposed in this research work was reasonable,could effectively enhance the effect of chemotherapy and provide experimental basis for the construction of multifunctional nanosystems.1.Synthesis and characterization of CS-TOS polymers(CS-CYS-TOS and CS-ADH-TOS)Reduction-sensitive polymer CS-CYS-TOS and non-reduction-sensitive polymer CS-ADH-TOS were synthesized by two-step amidation reaction using CYS and ADH as linking agents,respectively.Three kinds of CS-CYS-TOS polymers(1,2,3)were synthesized with different ratios of TOS and CS,and degree of substitution(DS)of TOS was 4.6,3.18,and 2.50,respectively.It was noted with the increase of hydrophobic segment TOS,the DS of TOS was increased.The successful synthesis of the polymer was confirmed by proton nuclear magnetic resonance(1H-NMR)and Fourier-transform infrared(FT-IR)spectra.Similarly,CS-ADH-TOS polymer was also synthesized and structure was confirmed by spectroscopic analysis.The DS of TOS to CS was 5.75.2.Preparation and in vitro evaluation of CS-CYS-TOS polymer nanoparticlesThe effects of DS of TOS on the properties of nanoparticles including particle size,zeta potential,DL,EE and critical aggregation concentration(CAC)were investigated.The results of polymer CAC experiments showed that the CAC value of CS-CYS-TOS polymers was decreased from 0.040 to 0.027 mg/mL with the increase of TOS DS.The CAC value of CS-ADH-TOS was found to be 0.050 mg/mL.Using CS-CYS-TOS 1,2,3 as a construction material,blank nanoparticles without drug were prepared by ultrasonication method.The particle size of CS-CYS-TOS nanoparticles was in the range of 161 nm to 220 nm and zeta potential was-11.2 mV to-16.5 mV.The transmission electron microscope(TEM)declared spherical nanoparticles with core-shell structure.The non-redox responsive CS-ADH-TOS nanoparticles had size of 208 run,polydispersity index(PDI)of 0.229 and zeta potential of-10.4 mV.The non-redox-sensitive nanoparticles also had spherical shape with core-shell structure.The redox-sensitive polymers CS-CYS-TOS 1,2,3 and non-redox sensitive polymer CS-ADH-TOS were selected as a carrier material,and DTX loaded nanoparticles were prepared by physically encapsulating the poorly soluble model drug DTX.It was found that both the drug to polymer mass ratio and water/organic phase volume ratio affected the DL of CS-CYS-TOS 1 nanoparticles;when the drug to polymer ratio was increased from 1:10 to 1:1,with the fixed volume ratio of methanol/phosphate-buffered saline(PBS)(3:0.375),the DL showed an increasing trend from 3.78%to 17.06%.The EE showed decreasing trend:when drug to polymer ratio increased from 1:10 to 1:1,the EE decreased from 50.83%to 23.46%.At fixed drug to polymer ratio(1:1),the CS-CYS-TOS 1 nanoparticles had shown highest DL than other formulations.The maximum DL capacity and EE of CS-ADH-TOS nanoparticles were also determined and were found to be 14.18%and 45.57%,respectively.Investigation of physical and chemical properties of DTX/CS-CYS-TOS 1 nanoparticles(optimal formulation):the particle size was 170.7 nm(PDI=0.130);the zeta potential was-26.5 mV;the TEM images showed that the particles were round and spherical with uniform size distribution.The DTX/CS-ADH-TOS nanoparticles had particle size of 240 nm(PDI=0.190)and zeta potential of-24.89 mV.The loading of drug resulted in increase of particle size and PDI and enhancement of negative charge on the surface of nanoparticles.Investigation of reduction sensitivity of CS-CYS-TOS nanoparticles:after incubating CS-CYS-TOS nanoparticles with 20 mM dithiothreitol(DTT),the particle size and PDI were increased significantly with the incubation time,the nanoparticles morphology changed significantly,particles became irregular in shape,and many fragments and particle aggregates were appeared.The drug release profile of redox-sensitive nanoparticles was compared with non-redox sensitive nanoparticle to ensure the redox-triggered release of drug from nanoparticles.Dialysis method was used to investigate the drug release behavior of drug-loaded nanoparticles in different release media,and it was found that in the release medium containing 20 mM DTT,the release of DTX significantly accelerated.The above results indicated that the nanosystem had good reduction-responsive behavior.Fluorescence microscope and flow cytometry(FCM)were used to determine the cell uptake.Cell uptake experiments showed that B16F10 cells efficiently taken up CS-CYS-TOS nanoparticles.The coumarin-6 florescence intensity was gradually increased with the increase of incubation time from 0.5 h to 4 h,proving that cell uptake was a time-dependent process.Despite of negative charge on the surface on the nanoparticle,they were still effectively taken up by cells.Thus,nanoparticles were expected to deliver the drugs to tumor cells.Similarly,CS-ADH-TOS nanoparticles also demonstrated time-dependent cell-uptake and were efficiently taken up by the B16F10 cells.The Sulforhodamine B(SRB)assay was used to investigate the cytotoxicity of blank and drug-loaded nanoparticles.CS-CYS-TOS and CS-ADH-TOS nanoparticles exhibited cell viability of more than 90%at all concentrations,indicating that CS-CY-TOS and CS-ADH-TOS polymers had no obvious toxic and side effects on B16F10 cells.For B16F10 cells,the IC50 value of reduction-sensitive DTX/CS-CYS-TOS nanoparticles(37.55 ?g/mL)was lower than that of non-reduction-sensitive DTX/CS-ADH-TOS nanoparticles(141.3 ?g/mL),indicating that the reduction-sensitive drug release mode was conducive to enhance the antitumor activity of the nanosystem in vitro.Apoptosis experiments were performed using the FITC-Annexin V/propidium iodide(PI)double staining method.The results showed that DTX/CS-ADH-TOS nanoparticles exhibited more late apoptosis rate,whereas,DTX/CS-CYS-TOS nanoparticles increased early apoptosis rate.The late apoptotic rate and total apoptotic rate further confirmed that the reduction-triggered drug release behavior was beneficial to induce apoptosis and enhance antitumor activity of the preparations.In vivo biodistribution and anti-tumor activity of nanoparticles were determined in tumor-bearing mice.A Xenogen IVIS Lumina System(Caliper Life Science,USA)was used to record the fluorescence images of tumor-bearing mice injected with CS-CYS-TOS nanoparticle solution or coumarin-6 solution.Coumarin-6 solution exhibited strong florescence signal in liver and weak florescence intensity in tumor site,indicating lack of tumor targeting ability of coumarin-6.Coumarin-6 intensity in the tumor site was gradually decreased and coumarin was rapidly eliminated in vivo.CS-CYS-TOS nanoparticles had shown significantly higher florescence intensity in tumor site than coumarin-6 solution.The nanoparticles facilitated accumulation and retention of coumarin-6 in tumor site.An ex-vivo imaging revealed strong florescence signal of comarin-6 in the liver and tumor site of mice treated with nanoparticles,whereas coumarin-6 group showed weak signals in tumor site.In vivo and ex vivo biodistribution studies demonstrated safety of nanoparticles towards major organs.In vivo antitumor activity was compared in four groups of tumor-bearing C57 mice:(1)normal saline(NS);(2)DTX solution;(3)DTX/CS-CYS-TOS nanoparticles;(4)DTX/CS-ADH-TOS nanoparticles.The above formulations were administered to the mice for three times after 4 days interval,and body weight and tumor volume were recorded.DTX/CS-CYS-TOS nanoparticles were superior in term of tumor volume inhibition than other formulations.In a summary,the stat-of-the-art multifunctional nanoparticles had unique physical and biological activities,especially redox-sensitivity and antitumor activity,exhibiting promising therapeutic potential for effective cancer treatment with little side effects.