The Design And Evaluation Of Stimuli-responsive Silica Nanoparticle Delivery System Loading Doxorubicin For Cancer Therapy

Mesoporous silica nanoparticles(MSN)with good biocompatibility serving as a typical kind of inorganic drug carrier possess special advantages:(1)adjustable pore size,and the pores designed according to the size of drug molecules can limit the drug aggregation by forming steric hindrance,making the drug highly stably dispersed in the pores in molecular state,amorphous state or microcrystalline state;(2)large surface area which could achieve high drug loading capacity,making MSN especially suitable for drugs with high dose demand in clinical use;(3)the preparation of MSN is simple and low cost,and the stable chemical properties combining with the rich silanol groups on the surface of MSN provide good structural foundations for further functional modification.This research used amphipathic polymer Pluronic and D-alpha-tocopheryl polyethylene glycol succinate(TPGS)to coat MSN,aiming to prevent premature drug release from the carrier before reaching the target site or target tissue,reduce side effects of drags,improve the dispersibility of the drug carrier,extend the circulation time of the carrier and improve drug efficacy.According to the unique physiological environment of tumor tissue,this research fabricated a kind of GSH redox-responsive drug delivery system based on MSN.First of all,by silane hydrolytic coupling reaction,3-mercaptopropyltrimethoxysilane was connected with the silanol groups of MSN through silicon ether bond,affording MSN-SH,then by mercaptosubstitution reaction,2,2’-disulphanediyldipyridine was connected with the thiol of MSN-SH,affording MSN-SS-Py,again by mercaptosubstitution reaction,the pyridine group of MSN-SS-Py was replaced by octadecanethio,obtaining hydrophobic MSN-SS-C,8,afterwards DOX was loaded into the carrier in methanol through physical adsorption method,then by centrifugation to obtain DOX/MSN-SS-C18,finanlly DOX/MSN-SS-C18 and Pluronic were mixed in chloroform,and Pluronic was coated onto DOX/MSN-SS-C18 through thin film hydration method,constructing DOX/PSMSN.Pluronics with different hydrophilic and hydrophobic ratios,including:P123,P105,F127 and L61 were used to coat MSN-SS-C18,and through investigating their dispersible stability,P123 was finally chosen to co-construct the system.Characterization results showed:the as-prepared MSN were spherical-like particles with a diameter about 150 nm possessing a well-ordered two dimensional structure;the specific surface area of MSN-SS-C18 was 877 m2/g,the pore diameter was 2.4 nm,indicating the modified group did not block the channel,the carrier was still suitable for loading drugs;while the specific surface area of PSMSN was sharply decreased to 36 m2/g,and the pores could not be detected,which suggested that P123 could be tightly binded with MSN-SS-C18,and efficiently cover the pores of MSN-SS-Cis by its own macromolecular structure,the proportion of P123 was 27%in PSMSN;the drug loading efficiency in DOX/PSMSN was 10.5%.In vitro drug release result showed that in pH 5.0 PBS without GSH,only 20%of the DOX in DOX/PSMSN was released within 24 h,while in the presence of 10 mM GSH,the cumulative release amount could be increased by 2.2 times,which demonstrated that the combination of octadecyl carbon chain and P123 in DOX/PSMSN could form a diffusion barrier helping to reduce the drug release from MSN before reaching the tumor tissure,and DOX/PSMSN had GSH redox-responsive drug release property.Cell experiment result showed:PSMSN could be endocytosed efficiently by 4T1 cells,and DOX/PSMSN achieved rapid intracellular drug release.Meanwhile,after 125 and 250,μg/mL of PSMSN co-cultured with 4T1 cells,the wound healing rates were 61%and 35%,respectively,while the control group reached up to 90%;the cell migration rates in transwell were 50%and 15%of the control group,demonstrating that PI 23 could endow PSMSN with the ability of anti-breast cancer cell metastasis.To further enhance the anti-cancer efficacy of the redox-responsive DOX/MSN delivery system,photothermal therapy(PTT)was introduced.First of all,MSN-SH was synthesized according to the method aforementioned.Then by mercaptosubstitution reaction,S,-(2-aminoethylthio)-2-thiopyridine hydrochloride was connected with the thiol of MSN-SH,obtaining MSN-SS-NH2,then by amidation reaction,the lipophilic near-infrared(NIR)light photothermal converting agent Cypate was linked with the amino group of MSN-SS-NH2,affording hydrophobic MSN-SS-Cy,finally DOX was loaded in MSN-SS-Cy and TPGS was coated on DOX/MSN-SS-Cy using the method aforementioned,constructing DOX/TCMSN for chemo-photothermal therapy.Characterization results showed:the specific surface area of MSN-SS-Cy was 786 m2/g,the pore diameter was 2.5 nm,indicating the modified group did not block the channel,the carrier was still suitable for loading drugs;while the specific surface area of TCMSN was sharply decreased to 42 m2/g,and the pores could not be detected,which suggested that TPGS could be tightly binded with MSN-SS-Cy,and efficiently cover the pores of MSN-SS-Cy by its own macromolecular structure,the proportion of TPGS was 25%in TCMSN;the drug loading efficiency in DOX/TCMSN was 12.2%.In vitro drug release result showed that in pH 5.0 PBS without GSH,only 27%of the DOX in DOX/TCMSN was released within 24 h,while in the presence of 10 mM GSH,the cumulative release amount could be increased by 1.8 times,and three times of NIR light irradiation all made the drug release in pulses,which demonstrated that the combination of Cypate and TPGS in DOX/TCMSN could form a diffusion barrier helping to reduce the drug release from MSN before reaching the tumor tissure,and TCMSN possessed an obvious GSH redox-and NIR light to heat-dual responsive drug release property.Taking 4T1 cells as the model cell,experiment result showed TCMSN triggered hyperthermia under NIR light irradiation could not only kill cancer cells directly,but also promote particle endo/lysosomal escape,improve intracellular DOX release from DOX/TCMSN;30%of TCMSN could be exocytosed from 4T1 cells through lysosome mediated pathway within 4 h,while after particle escape from endo/lysosomal rapidly under NIR light irradiation,the particle exocytosis was decreased by 18%,which indirectly increased drug retention time in cells;due to the existence of TPGS in TCMSN,ATP content in 4T1 cells was reduced by 30%in 2 h,which weakened the efflux of P-glycoprotein to the drugs and further extended the drug action time in cells.The in vivo biodistribution result showed that TCMSN(100 mg/kg)accumulated to a peak concentration at 2 h in tumor region,and 2 W/cm2 of NIR light irradiation could induce a temperature elevation of 11 ℃ at the tumor tissue,exceeding the temperature threshold required for PTT;observed from frozen sections of tumor tissue,NIR light greatly promoted DOX release from DOX/TCMSN in tumor region.The pharmacological experiment result showed NIR light increased the tumor inhibition rate of DOX/TCMSN by 1.3 times,the tumor inhibiton rate of DOX/TCMSN plus NIR light was 1.1 times than DOX solution.Compared with DOX solution,TCMSN group did not induce significant weight loss in mice during the treatment.The hematoxylin-eosin staining result demonstrated that no pathological changes were observed in the slices of the major organs for the DOX/TCMSN group,while DOX solution led to a partial myocardial fiber fracture which proved that TCMSN drug delivery system had a low toxicity and reduced the side effects brought on by the free DOX,and DOX/TCMSN combing with PTT would further enhance the anti-cancer effect.