| Chemotherapy is the major method for cancer treatment especially for melanoma.The application of chemotherapeutic agents frequently leads to side effects due to systemic administration.It also increases the risk of multi-drug resistance(MDR).Currently,polymeric nanoparticles own the excellent potential as drug delivery carriers due to their nano-structure and physico-chemical properties.Hydrophobic molecular modified polysaccharides can form amphiphilic conjugates with advantages of biocompatibility,biodegradation and low toxicity.Instead of introducing organic solvents,these conjugates are able to self-assemble to nanoparticles via simple preparation under aqueous environment.Therefore,polysaccharides based nanoparticles have attracted tremendous attention in drug delivery field of cancer therapy.However,challenges emerged in the design of structure of polymeric nanoparticles to improve the in vivo stability,achieve stimuli-responsive release of drugs within tumor microenvironment(TEM)and combine multiple theraptical functions in one nanoparticle.In this paper,chondroitin sulfate(CS)was used as the hydrophilic backbone to synthesize amphiphilic conjugates via grafting hydrophobic small molecules to CS backbone.On this basis,novel self-assembled drug delivery systems were constructed with excellent stability and controlled drug release.The correlation between conjugates pattern and nanoparticles structure were investigated in detail.The impact of degree of substitution(DS)of hydrophobic segments on the properties of nanoparticles was deeply studied.These research provided theoretical basis for the drug delivery application of nanoparticles.We further designed multiple stimuli-responsive,multifunctional"All in one" nanoparticles,which could rapidly release drugs in response to the stimuli in TEM.These works were expected to provide guidelines for the design of theraptical nanocarriers.In this work,to investigate the structure relation of conjugates and nanoparticles,pectinate CS-adipic dihydrazide-deoxycholic acid(CS-ADH-DOCA,CSAD)conjugates were synthesized via grafting DOCA to CS backbone using ADH as the linker.CSAD nanoparticles were self-assembled then for the delivery of docetaxel(DTX).A series of CSAD nanoparticles were synthesized with different DS of DOCA(the amount of DOCA in 100 sugar units)by changing the ratio of DOCA to CS.We investigated the influence of DS on the properties of nanoparticles including self-assembly,drug release,tumor cell internalization and cell killing ability.On the basis of characteristics of CS based nanoparticles,redox/enzyme responsiveness nanoparticles(CS-CYS-DOCA,CSCD)were designed as delievery carriers of DTX.CSCD nanoparticles featured redox/enzyme responsiveness were prepared by synthesizing CSCD conjugates through grafting DOCA to CS via cystamine(CYS)containing sulfate bonds.CS could be degraded by hyaluronidase-1(Hyal-1)in tumor cells,which contributed to enzyme sensitivity.The stimuli responsive disassembly,drug release,cell uptake,tissue distribution and anticancer abilities were assessed.To further improve the stability in vivo and optimize the release rate in TEM,a novel type of redox/enzyme responsive crosslinked nanoparticles(X-NPs)were developed for DTX delivery as synergetic chemo-sonodynamic therapical carriers.In detail,hydrophobic chlorin e6(Ce6),an ultrasound sensitive agent,was grafted to CS backbone via ADH,followed by the graft of lipoic acid(LA)which was the crosslinking agent with sulfate bonds.To acheve this design,CS-Ce6-LA conjugate were synthesized,which self-assembled to non-crosslinked nanoparticles(NX-NPs).X-NPs were then formed via crosslinking intermolecular disulfide bonds.X-NPs were expected to maintain stable when they encountered severe dilution in blood circulation.After they were internalized into tumor cells,they could degrade with rapid drug release,leading to effective chemotherapy.Meanwhile,Ce6 and ultrasound contributed to sonodynamic therapy(SDT),inducing tumor cell apoptosis.Based on this design,the stability,responsive drug release and internalization of X-NPs were assessed,the mechanism of SDT and the anticancer effect of chemo-sonodynamic therapies were investigated in detail.The research contents and results are summarized as follows:1 Design of CSAD nanoparticles,investigating the effect of degree of substitution on nanoparticlesThrough two steps amidation reaction,CSAD conjugates were synthesized by grafting DOCA to CS via ADH as the linker.Three kinds of CSAD were synthesized with different DS of DOCA(DS ranged from 2.5%-7.0%).The critical aggregation concentration(CAC)ranged from 0.027 to 0.050 mg/ml.CSAD nanoparticles prepared by probe sonication method were confirmed with sphere morphology.The average particle size of CSAD was 160-190 nm,and zeta potential ranged from-18 mV to-27 mV.The hemolysis ratios of CSAD nanoparticles with different DS were below 5%,which demonstrated their excellent biocompatibility.DTX loaded CSAD nanoparticles(DTX-CSAD)were prepared by a sonication-dialysis method.When the drug-conjugate ratio was 3:10,the drug loading(DL)was 11.8%with size distribution of approximately 180 nm.Morphology of DTX-CSAD was spherical in shape with a uniform size distribution.Two steps release have been obvioused in CSAD drug release,initial burst release and followed countrolled release.CSAD with higher DS showed an increase trend in drug release,for example,the release percentage rose from 75%to 86%at 120 h when increasing the DS of DOCA.Cellular uptake result indicated that CSAD nanoparticles could be internalized with CD44 receptor-mediated endocytosis,and the uptake amount was affected by the time as well as the concentration of nanoparticles.In addition,DTX-CSAD could effectively kill tumor cells.For instance,when the concentration of DTX was 1?g/ml,the availability of MCF-7 cells was 49.2%,46.9%and 42.9%,respectively,when cells were incubated in DTX-CSAD with increased DS in 48 h.The results above implied that the DS of DOCA in CSAD played an important role in self-assembly,drug loading and release behaviors.Experimental data indicated that with the increase of DS,the average size,CAC and drug release rate reduced.This could be explained by the fact that increasing hydrophobic segments could improve hydrophobic interaction and made it easier for conjugates to self-assembly,thereby contributing to the tight hydrophobic core.After loading DTX,the hydrophobic interaction between DTX and DOCA were reinforced,which leaded to the inhibition of drug release.Moreover,the uptake and cell killing ability were also gradiently improved by DS increase.On investigating the DS,desired drug release and therapeutic effects can be achieved via the alteration of DS of hydrophobic segments,which contributes to guideline of the application of polymer nanocarrier.2 Design of redox/enzyme responsive CSCD nanoparticles and investigation on the anticancer effectCSCD conjugates were synthesized by grafting DOCA to CS via CYS as linkage.Redox/enzyme responsive CSCD nanoparticles self-assembled by conjugates in water phase,which were used for the delivery of DTX.The CAC value was low(0.027-0.050 mg/ml).CSCD nanoparticles showed sphere morphology with uniform distribution,the average particle size was 130-170 nm and zeta potential ranged from-18 mV to-22 mV.The hemolysis ratios of CSAD nanoparticles were below 5%.DTX loaded CSCD(DTX-CSCD)nanoparticles were prepared by sonication-dialysis method.When the drug-conjugate ratio was 3:10,the DL reached 15.6%,and the size distribution was approximately 175 nm with spherical in shape.CSCD exhibited great stimuli-responsive properties.When they were incubated in simulated TEM(GSH and Hyal-1),the size distribution changed from single peak between 100-200 nm to double peaks.It was mainly due to CS could be degraded by Hyal-1 while the disulfide bonds could be cleaved under GSH,which leads to the disruption of hydrophilic-hydrophobic balance as well as the disaggregation of the structure.In addition,the responsive drug release was assessed.The release rate increased under the presence of GSH or Hyal-1,further rise was observed with the synergetic effect of GSH and Hyal-1.These results demonstrated the redox/enzyme sensitive properties of CSCD nanoparticles.In cellular level,cellular uptake result indicated that CSCD nanoparticles could be internalized effectively,which was mainly depended on CD44 receptor-mediated endocytosis with consume of energy.It was also confirmed that small amount of CSCD nanoparticles could be internalized by clathrin and osmotic pressure mediated uptake.DTX-CSCD could effectively kill tumor cells.For instance,when the concentration of DTX was 50?ig/ml,the cell availability was below 20%when cells were incubated in DTX-CSCD in 72 h.Pharmacokinetics study and tissue distribution were evaluated in Wistar rats and C57 mice,respectively,and DTX-CSAD nanoparticles were also investigated as the non-redox sensitive control.Pharmacokinetics study showed that DTX-CSCD and DTX-CSAD could significantly prolong the residence time of DTX in blood circulation and lower the drug clearance rate.The half-time of DTX-CSCD and DTX-CSAD were 4.3 and 3.5-folds,respectively,higher than that of Taxotere(?).Tissue distribution indicated that DTX-CSCD and DTX-CSAD could increase the drug distribution in tumor,for example,the DTX concentration of DTX-CSCD and DTX-CSAD in tumor were 6.1 and 3.2-folds,respectively,higher than that of Taxotere(?).Moreover,the distribution of nanoparticles in heart was reduced,which decreased the non-specific toxicity.Compared to Taxotere(?),higher concentration of DTX-CSCD and DTX-CSAD was observed in lung,which may conducive to the antimetastasis role of these nanoparticles in lung.DTX-loaded nanoparticles showed significant anticancer and antimetastasis effects.Anticancer results demonstrated that two nanoparticles could dramatically inhibit the tumor volume,the volume of DTX-CSCD,DTX-CSAD and Taxotere(?)were 12.6%,25.6%and 38.4%,respecticely,of normal saline(NS)group.In addition,the H&E and TUNEL results further indicated that DTX-CSCD and DTX-CSAD nanoparticles contributed to tumor apoptosis and necrosis,achieving the goal of tumor growth inhibition.In addition,DTX-loaded nanoparticles could decrease the number of tumor nodules in lung as well as the pathological results compared to that of NS group.Meanwhile,tumor immunohistochemistry results showed that the metastasis related protein COX-2 was reduced in DTX-CSCD,DTX-CSAD groups.Compared with DTX-CSAD group,DTX-CSCD groups presented smaller tumor volume with higher percentage of apoptosis cells.These results exhibited the advantages of redox/enzyme responsive CSCD nanoparticles in drug release and anticancer ability.3 Design of redox/enzyme responsive crosslinked nanoparticles(X-NPs)and the chemo-sonodynamic therapeutics in anticancer effectBased on the design of CSCD nanoparticles,we wanted to increase the stability of nanoparticles and combine multiple anticancer methods.In this part,Ce6,an ultrasound sensitive agent,was chosen as the hydrophobic segment in order to achieve sonodynamic therapy(SDT).The combination strategy of crosslinking and redox responsiveness was used as well.We hoped this multifunctional nanocarrier could exhibit excellent anticancer effect.Amphiphilic CS-Ce6-LA conjugated was synthesized by grafting Ce6 and LA to CS via ADH.CS-Ce6-LA could self-assemble to non-crosslinked nanoparticles(NX-NPs).After crosslinking via intermolecular disulfide bonds,redox/enzyme responsive X-NPs could be prepared for the delivery of DTX,achieving synergetic chemo-sonodynamic therapeutics for cancer treatment.NX-NPs showed sphere morphology with uniform distribution,the average particle size was 133-233 nm and zeta potential ranged from-12 mV to-22 mV.After crosslinking,X-NPs exhibited lower size distribution(118-197 nm)compare to NX-NPs,and zeta potential ranged from-12 mV to-22 mV.The hemolysis ratio of X-NPs was below 5%.DTX loaded X-NPs(DTX/X-NPs)were successfully prepared.When the drug-conjugate ratio was 3:10,the DL reached 16.6%,and the size distribution was approximately 160.9 nm.X-NPs exhibited great colloidal stability with slightly increase in size when they under the incubation of solvents with high ionic strength,or organic solvent or under the high dilution.In comparison,it was difficult for NX-NPs to stay stable,and portion of them were easily disaggregated to unimers.Interestingly,X-NPs showed great redox/enzyme responsive properties with obvious disaggregation of structure.In addition,DTX/X-NPs could reduce the release rate compared to that of DTX/NX-NPs.For example,after 72 h,about 62.3%of DTX was release from DTX/NX-NPs but the percentage of DTX released from DTX/X-NPs was below half of that number.However,under the effect of GSH or GSH/Hyal-1,release percentage increased to 72.3%and 97.8%,respectively.In comparison,GSH did not impact the drug release of DTX/NX-NPs.These results demonstrated that X-NPs could dramatically increase the stability and slow down drug release rate in general environment,while they were able to de-crosslinking within tumor cells and subsequently lead to rapid drug release.The results further indicated the advantages of combination strategy of crosslinking and stimuli responsiveness,this strategy also perfectly solved drug release issue of chemical crosslinking nanoparticles.In cellular level,cellular uptake results indicated excellent uptake efficiency of X-NPs.In addition,DTX/X-NPs+SDT with chemo-sonodynamic therapeutics exhibited prominent tumor cell killing effects.In specific,the effect of DTX/X-NPs+SDT was greater than that of DTX/X-NPs(single chemotherapy).The cytotoxicity increased when the ultrasound duration increased from 1 min to 3 min.As for the cell apoptosis of SDT,the apoptosis percentage of X-NPs+SDT was about 2-fold higher than that of Ce6+SDT,which may be due to the higher internalization of X-NPs.The mechanism of SDT that induced the apoptosis of tumor cells was further investigated.These results showed that SDT contributed to the production of reactive oxygen species(ROS),and jc-1 probe results revealed the decrease of mitochondrial membrane potential(MMP)under the effect of SDT.PCR results confirmed change of apoptosis related mRNA expression in mitochondrion and Western Blot results indicated the increased expression of apoptosis related proteins.Based on these results,the process of SDT induced cell apoptosis could be described as follow:after the internalization of X-NPs,Ce6 contributed to SDT under the stimulation of ultrasound,leading to the production of ROS.ROS could induce cell apoptosis via activating mitochondrial-caspase pathway.In other words,mitochondrial was damaged by ROS which lead to MMP down-regulation.Expression of Bax increased and expression of Bal-1 decreased.Subsequently,downstream cytochrome C was activated with enhanced expression,which induced cleavage of caspase-9 followed by degradation of caspase-3.The cleavage of caspase-3 contributed to the cleavage of PARP,which may play a role in the post-process of apoptosis.Pharmacokinetics study in rats showed that DTX/X-NPs could change the existence modality of DTX,significantly increased the concentration and prolonged the residence time of DTX in blood circulation.The ex vivo imaging study of mice tissues was performed by recording the fluorescence intensity at designed time points post injection of Ce6 solution or X-NPs.X-NPs were found to effectively enhance the Ce6 signal intensity and increase the retention time of Ce6 in tumor.Compared to Ce6,more X-NPs were observed in lung,which may help nanoparticles play a role in antimetastasis in lung.Anticancer results demonstrated that chemo-sonodynamic therapies(combo)were superior to chemotherapy in tumor volume inhibition.After 18 days,the the volume of DTX/X-NPs+SDT(combo),X-NPs+SDT(SDT),DTX/X-NPs(chemotherapy)and Taxotere(?)were 0.6%,10.0%,14.9%and 61.8%,respectively,of normal saline(NS)group.In addition,DTX/X-NPs+SDT(combo),X-NPs+SDT(SDT),DTX/X-NPs could decrease the number of tumor nodules in lung compared to that of NS group.Meanwhile,tumor immunohistochemistry results showed that the metastasis related protein COX-2 and uPA were both reduced in DTX/X-NPs+SDT(combo),X-NPs+SDT(SDT),DTX/X-NPs groups.It was notable that effects of DTX/X-NPs+SDT(combo)group was optimal.These results exhibited the advantages of redox/enzyme responsive X-NPs in rapid drug release in TEM and SDT induced cell apoptosis with the synergetic chemo-sonodynamic therapies.Notably,DTX/X-NPs+SDT(combo)and X-NPs+SDT(SDT)groups improved the infiltration of CD8+ cytotoxicity T cells(CTLs),and the percentage of these two groups were 1.6%and 1.4%,respectively.In comparison,the figures for other three groups without SDT including NS,Taxotere(?),and DTX/X-NPs(chemotherapy)were blow 0.3%.The interferon-y(INF-y)infiltration was also increased due to SDT,leading to anticancer immune response.These results demonstrated the potential of SDT for application of tumor treatment and paved a way for tumor immune therapy. |
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