Construction Of Polysaccharide-Based Prodrugs Of Hydrophobic Molecules And The Application In Colon Cancer Therapy

Colon cancer poses a serious threat to human health due to high incidence and mortality.Prodrug micelles which demonstrate superiorities in increasing solubility,tumor-targeting ability and tumor-microenvironment responsive drug release have been applied to the delivery of hydrophobic chemotherapy drugs.Unfortunately,a long circulation process is ineluctable for traditional administration routes,resulting in the limited concentration of drug accumulation at tumor tissue.In recent years,oral colon-specific drug delivery system(OCDDS)has brought about widespread attention due to organ-targeting drug delivery to colon lesions,showing good application prospect in colon cancer therapy.Among them,polyelectrolyte complexes(PEC)based on polysaccharide derivatives have unique characteristics.Nevertheless,researches on the combination of PEC with prodrug micelle systems were still unavailable.In this study,hydrophobic anticancer drugs were grafted to hydrophilic polysaccharide derivatives to construct amphiphilic prodrug micelles.Then,prodrug micelles were applied to encapsulate hydrophobic chemotherapeutic drugs for synergistic drug delivery and PEC composed with polysaccharide derivatives were modified on the surface of micelles to achieve oral colon-specific drug delivery.Effects of responsive linkers,ratio of hydrophobic sides to hydrophilic backbones,and different chemotherapeutic drugs loaded in the hydrophobic cores on self-assembly ability,particle size distributions,tumor microenvironment response and cytotoxicity of prodrug micelles were investigated in detail.Besides,endocytosis efficiency,endocytosis pathway,subcellular distribution,tumor penetration,bio-safety and in vivo antitumor activity of the prodrug micelles were comprehensively evaluated.This study could provide a reference for the construction of oral colon-specific prodrug micelle systems in the application of colon cancer treatment.First,curcumin(CUR)was grafted to quaternary ammonium group substituted hydroxyethyl starch(QHES)by a two-step esterification to form amphiphilic prodrug which could self-assemble into micelles.Effects of different linkers construcing prodrugs,i.e.thioether bond(QHES-2S-CUR),disulfide bond(QHES-SS-CUR)and carbon-carbon bond(QHES-CCCUR)on morphologies,size distributions,tumor microenvironment response,endocytosis efficiency and cytotoxicity of prodrug micelles were investigated.Results showed that QHES2S-CUR could self-assemble into spherical micelles with an average particle size of 217.28±12.62 nm and a PDI of 0.129 ± 0.033 in water.The average particle size of the micelles formed by QHES-SS-CUR was larger than that of QHES-2S-CUR,while QHES-CC-CUR was lack of conformational stability and easy to form large aggregates.Meanwhile,the types of linkers also exerted an impact on tumor microenvironment response of prodrugs.QHES-2S-CUR demonstated rpaid tumor microenvironment-sensitive drug release,with 73.57 ± 4.22%of curcumin released under the stimulation of H2O2 over 24 h.Drug release rate of QHES-SS-CUR was relatively slower,while QHES-CC-CUR was insensitive to ROS.Among the three prodrug micelles,QHES-2S-CUR linked by thioether bond showed the fastest endocytosis efficiency and the strongest cytotoxicity due to small diameters and rapid drug release under the trigger of tumor microenvironment.Then,curcumin prodrug micelles(QHES-S-CUR)with different drug loading capacity(DLC)were prepared to know the effect of hydrophobic payload/hydrophilic backbone ratios on self-assembly abilities and hydrophilicites of prodrug micelles.Correlations between prodrug micelle structures and microenvironment response as well as cytotoxicity were investigated to establish the structure-function relationship.Besides,endocytosis pathway and subcellular distribution were explored,and tumor penetration abilitiy and in vivo antitumor activity were evaluated.Results showed that the self-assembly behavior of QHES-S-CUR micelles was significantly influenced by DLC and an increase of DLC prompted the self-assembly of QHESS-CUR into micelles with smaller particle diameters and uniform size distributions.QHES with a DLC of 9.79 ± 0.78%demonstrated strongest hydrophilicity,tumormicroenvironment response and cytotoxicity.In addition,caveolae-mediated uptake and mitochondrion-targeting delivery of QHES-S-CUR was validated in CT-26 cells.Accroding to the caveolae-mediated endocytosis,an enhanced penetration to distal region of tumor was performed in three-dimensional multicellular spheroid models.Moreover,QHES-S-CUR prodrug manifested valid inhibition to tumor growth and good safety in the in vivo antitumor assays,which indicated its potential application in colon cancer treatment.Furthermore,QHES-S-CUR prodrug micelles were applied to the construction of synergistic drug delivery systems.The stability of curcumin/chemotherapy drug mixtures was assessed by molecular simulation,by means of which mean energy and centroid distance were calculated,and main conformation was displayed.Based on the results of molecular simulation,chemotherapy drugs were loaded in the hydrophobic core of QHES-S-CUR to prepare synergistic prodrug micelles.Variations of morphologies and size distributions of synergistic prodrug micelles with the raito of chemotherapy drug/QHES-S-CUR ratios were carried out.Meanwhile,drug release behavior,cytotoxicity,subcellular distribution and penetration ability of synergistic prodrug micelles were investigated.After that,CT-26 bearing mouse models were established to evaluate the safety and antitumor activity.Molecular simulation results revealed that the mixture of curcumin and 7-ethyl-10-hydroxycamptothecin(SN38)had the lowest energy and the shortest centroid distance,implying the strongest interactions between curcumin and SN38.Therefore,SN38 was enpasulated in the QHES-S-CUR to construct synergistic drug delivery system(SN38@QHES-S-CUR)with good stability.The mass ratio of SN38 to QHES-S-CUR had a significant effect on morphologies and size distributions of SN38@QHES-S-CUR.With the ratio increasing from 1:10 to 1:20,SN38@QHES-S-CUR changed from rod-like morphology to nanomicelle structure and SN38 encapsulated in SN38@QHES-S-CUR was amorphous.At a mass ratio of 1:20,SN38@QHES-S-CUR showed a quick response to tumor microenvironment,mitochondrial targeting and deep penetration in tumor.In the in vivo antitumor assays,SN38@QHES-S-CUR exhibited more efficient antitumor effect than irinotecan hydrochloride(CPT-11)in CT-26 bearing mouse models,which verified the synergistic action of curcumin combined with SN38.In order to modify SN38@QHES-S-CUR with PEC shell,sodium cellulose sulfate(NaCS)was adsorbed on the surface of SN38@QHES-S-CUR by electrostatic interaction(i.e.NaCS/SN38@QHES-S-CUR).Weight ratios of NaCS/QHES-S-CUR were adjusted to explore its effect on morphologies and surface potentials of micelles.Stability of NaCS/SN38@QHESS-CUR in gastrointestinal environment and effects of cellulase hydrolysis on endocytosis efficiency,subcellular distribution and tumor penetration were conducted to forecast the prospect in OCDDS for colon cancer therapy.Additionally,antitumor efficiency was assayed in CT-26 bearing mouse models.The results showed that NaCS/SN38@QHES-S-CUR presented negative surface potential and PEC layers were thickening with the adsorption of more NaCS on the surface.NaCS/SN38@QHES-S-CUR showed good stability in simulated gastrointestinal environments while curcumin and SN38 were responsively released under the trigger of reactive oxygen species(ROS)in simulated tumor microenvironment.Besides,endocytosis efficiency,mitochondrion-targeting and tumor penetration of NaCS/SN38@QHES-S-CUR were improved in response to cellulase hydrolysis.Overall,in vivo safety and stronger antitumor effect than CPT-11 were proved in CT-26 bearing mouse models.Finally,in order to improve the stability of hydrophobic drug loaded drug delivery systems,supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer(SAAHCM)was applied to the micronization of curcumin-loaded emulsion with polysaccharide as matrix.Mechanism of particle formation and effects of operation parameters including mass flow ratio of CO2 to liquid,pressure and temperature in the mixer on size distributions were investigated in detail.The chemical stability,crystalline state and thermal behavior of particles were analyzed,and stability of drug loaded particles in simulated gastrointestinal tract and enzyme response in colon were evaluated by in vitro drug release experiments.Micronization of the emulsion by SAA-HCM could be considered as two continuous processes.In the mixer,polymer solved in oil phase was solidified and nucleated with the extraction of organic solvent by supercritical carbon dioxide(SC-CO2).In the second step,water of droplets was evaporated in the precipitator and particles were gradually formed.Operating parameters of SAA-HCM had a significant effect on particle size distributions.High gas/liquid raio,low mixer pressure and elevated mixer temperatue prompted narrow size distributions.Solid state characterization showed that curcumin loaded in partilces was in an amorphous state.In addition,particles showed great stability in simulated gastrointestinal environment while an enzyme responsive drug release in simulated colon environment.The nano-in-microparticles prepared by SAAHCM are superior in avoiding aggregation and improving the redispersibility of nanoparticles.In conclusion,prodrug micelles based on polysaccharide derivatives for hydrophobic anticancer molecules were sucssessfully prepared,which were further applied to OCDDS for synergistic chemotherapy.This study highlights the construction of novel prodrug system with organ-targeting ability,deep tumor penetration and accurate intracellular distribution,with the expectation to be a promising drug devliery strategy in targeting therapy for colon cancer.