| Background:Cervical cancer is the fourth most common cancer in women worldwide,and represents a major global health problem affecting middle-aged women.China is the country with serve prevention and treatment situation of cervical cancer,including the gradually increasing incidence rate and obvious trend of younger age.Chemotherapy is one of the main treatments for cervical cancer.Paclitaxel（PTX）,a broad-spectrum anticancer drug,is currently the standard frontline treatment of cervical cancer with low water solubility.To address the shortcoming,Cremophor EL and ethanol were utilized to dissolve PTX in the formulation of Taxol?to realize the solubilization of PTX,and as a result,Taxol?became the first clinically approved PTX formulation.Sadly,cosolvent-Cremophor EL induced toxicity and side effects bring huge suffering to the cancer patients,which greatly limited the clinical application of Taxol?.Nanomedicines offer a promising approach to address these tough issues,which can improve drug solubility,prolong circulation time and simultaneously enhance drug accumulation at tumor sites.Many PTX nanoformulations have been developed to reduce side effects and increase solubility of PTX,including Abraxane?,Apealea?,Genexol-PM?and Lipusu?.In fact,the biggest difference between them was the carrier.All of these nanomedicines have shown attenuated toxicity and increased solubilization of PTX,but they failed to show significantly prolonged overall survival and had shortcomings of low drug loading,drug leakage,and carrier-associated toxicity.Therefore,the development of new PTX nanoformulations is still undergoing.The new generation of stimuli-responsive smart nanomedicines have been developed and achieved controllable drug release and reduced side effects.A variety of tumor microenvironment-responsive nanomedicines have been developed in response to characteristicsof tumor microenvironment,such as weak acidity,high glutathione or reactive oxygen species（ROS）,hypoxia,and specific enzymes.Besides,some exogenous stimuli like light,ultrasound,magnetic fields and electric field,also can be employed to activate drugs.The structure or physicochemical properties of nanomedicines could be changed by these stimulis,achieving controllable drug release,reduced systemic toxicity and enhanced antitumor effects.In addition,the dimeric prodrugs tend to form stable nanoassemblies in aqueous solutions.Therefore,the formation of nanoassemblies needs greatly reduced carriers,which improve the drug content and reduce carrier-associated toxicity.Besides,the dimeric prodrug tactics in combination with stimuli-responsive nanomedicines by utilizing stimuli-responsive linkages could realize high drug loading capacity and specific drug release.As an emergingly non-invasive therapeutic modality,photodynamic therapy has attracted huge attention in cancer treatment due to its high selectivity,reduced side effects and repeatable treatment.Besides,photodynamic therapy shows great potency in the clinical treatment of cervical cancer due to the special cavity anatomy of the cervix.Light,photosensitizer and oxygen are necessary components involved in photodynamic therapy.Individually each component is non-toxic,but when photosensitizer is irradiated by light of the appropriate wavelength,the photochemical reactions result in the formation of cytotoxic reactive oxygen species that are responsible for tumor cell necrosis or apoptosis.The combination of chemotherapy and photodynamic therapy via nanotechnology can combat cancer through different mechanisms,thus maximizing therapeutic efficacy.This strategy has been widely investigated in recent cancer treatment researches for the superior anticancer effects.The condensed and heterogeneous extracellular matrix network in tumor represents a formidable and physical barrier that hinders intratumor drug delivery,limits the therapeutic efficacy of many anticancer therapies and induces drug resistance.Degradation of extracellular matrix components could lead to the reduced interstitial fluid pressure,the improved tumor blood flow and relieved tumor hypoxia,finally resulting in the improved distribution of nanomedicines throughout tumor.Recently,it has been reported that metformin,a traditional diabetes drug,could potentiate the anticancer activities of PTX against several cancer cells and fight fibrosis.As a result,metformin has emerged as a promising method to regulate tumor extracellular matrix and remodel tumor microenvironment.Purposes:（1）Design and construct light-activated binary PTX/porphyrin prodrug nanoparticles with self-boosted drug release.Then,explore its controllable drug release behavior,and investigate synergistic chemo-photodynamic therapy against He La cells.（2）Combine metformin with PTX dimeric prodrug nanoparticles（D-PTX@F127NPs）and investigated whether metformin could enhance the anticancer efficacy of D-PTX@F127 NPs.Methods:（1）The ROS-responsive paclitaxel/porphyrin prodrug with thioketal linker was synthesized via esterification reaction,and its structure and purity were characterized by proton nuclear magnetic resonance and electrospray ionization mass spectrometer.After that,the prodrug nanoparticles（Co-PT NPs）were prepared by nanoprecipitation without any adjuvants or carriers.The physicochemical properties,stability and co-assembly mechanism of Co-PT NPs were investigated by transmission electron microscopy,dynamic light scattering,UV-Vis spectroscopy and fluorescence spectroscopy.The confocal laser scanning microscopy was employed to investigate the cellular uptake of Co-PT NPs.Subsequently,the ROS production was detected using indocyanine green and the cellular fluorescent probe DCFH-DA.Then,high performance liquid chromatography was employed to verrify whether ROS could break thioketal linker and trigger PTX.Finally,the cytotoxic MTT assay,Annexin-V FITC/PI apoptosis detection,Calcein-AM/PI staining and tubulin immunostaining were used to explore the He La cell killing efficiency of Co-PT NPs with light irradiation.（2）PTX dimeric prodrug nanoparticles（D-PTX@F127 NPs）were prepared via nanoprecipitation.Transmission electron microscopy and dynamic light scattering were used to characterize the physicochemical properties and stability of D-PTX@F127 NPs.The antitumor effect of metformin and D-PTX@F127 NPs aganist He La and 4T1 cells was investigated by MTT assay,Calcein-AM/PI staining and crystal violet staining experiments.The in vivo anticancer efficiency,mechanisms and biological safety were studied by in vivo tumor inhibition experiment,Mason staining,Western blot experiment and HE staining experiment.Results:（1）First of all,the ROS sensitive thioketal linker was employed to connect PTX/porphyrin and successfully prepare homodimer prodrug,then construct carrier-free prodrug nanoparticles.Co-PT NPs exhibited uniform spherical morphologies with small sizes,narrow distributions and good stability,which can be effectively taken upby cancer cells in an energy-dependent and time-dependent manner.Under laser irradiation,the photosensitizer porphyrin prodrugs could generate ROS,inducing apoptosis and necrosis through oxidative stress.The generated ROS also can cleave the thioketal linkage,releasing the active PTX and inhibit the depolymerization of microtubules,thereby exerting cytotoxic effects.The MTT assay,Annexin-V FITC/PI apoptosis assay and Calcein-AM/PI staining assay demonstrated that Co-PT NPs synergized chemotherapy and photodynamic therapy to inhibit tumor cell growth.（2）The prepared PTX dimeric prodrug nanoparticles have good stability,small sizes,narrow distributions and uniform spherical morphologies.The combination with metformin significantly enhanced the toxicities of D-PTX@F127 NPs against He La and 4T1 cells.In vivo,pre-administered metformin can inhibit the secretion of TGFβ1and reduce the activation of CAF with high expression ofα-SMA,thereby reducing the content of collagen in tumor tissue,and enhancing anticancer efficiency of D-PTX@F127 NPs.In addition,no obvious systemic toxicities and organ damage were observed.Conclusions:（1）The prepared carrier-free prodrug nanoparticles can realize controllable drug release,and the chemo-photodynamic therapy could synergistically enhance antitumor efficiency.（2）The prepared PTX dimer prodrug nanoparticles show uniform morphologies and good stability.Metformin can enhance D-PTX@F127 NPs cytotoxicities against He La and 4T1 cells.In vivo experiments imply that merformin reduce the secretion of TGFβ,inhibit the activation of CAF with high expression ofα-SMA,thereby reducing tumor extracellular matrix and enhancing the anticancer efficacy of D-PTX@F127 NPs. |
