All-Trans Retinoic Acid/MWCNTs-OH Composite Nanofibers Based On Electrospinning For Combined Differentiation And Photothermal Therapy Of Cancer Stem Cells

ObjectiveCancer cells in tumors usually exist in different phenotypic states that have different functional properties.Stem cell theory believes that tumors are derived from cancer stem cells(CSCs).which are characterized by self-renewal,unlimited growth potential,and clonal tumor initiation.CSCs have been identified in many solid tumors including breast cancer,brain tumors,lung cancer and melanoma.The CSCs may become resistant to radiation therapy and chemotherapy,evade the immune system,and spread metastatically.Therefore,the treatment of CSCs is crucial in the entire tumor treatment process.In this study,polycaprolactone(PCL)nanofibers containing hydroxylated multi-walled carbon nanotubes(MWCNTs-OH)and all-trans retinoic acid(ATRA)was fabricated via electrospinning method,and combined with photothermal therapy and differentiation therapy was developed for treatment of CSCs.Methods1.Electrospinning technology was used to prepare electrospun fiber membranes with different concentrations of MWCNTs-OH,ATRA and PCL,and the surface morphology and diameter of the fibers were observed by scanning electron microscope(SEM).Raman and X-ray diffraction(XRD)were used to analyze the state of ATRA,MWCNTs-OH and PCL in the fibers.The fiber membranes were tested for its hydrophilicity,hydrophobicity and mechanical properties.2.The electrospun fiber membranes containing different components were respectively placed in phosphate buffered saline(PBS)and the back of Kunming mice,and the changes of temperature in the medium under near-infrared irradiation(NIR,808nm,1.5 W/cm2)were detected in different time periods to observe the photothermal performance of the electrospun fiber membranes.3.High-performance liquid chromatography(HPLC)was used to detect the drug loading and encapsulation efficiency of fiber membranes loaded with ATRA.Furthermore,the ATRA released from the fiber membranes into PBS with or without NIR irradiation was detected at specified time points.4.Immunofluorescence,western blot(WB),and flow cytometry were performed to detect the expression of CD 133,a marker of glioma stem cells(GSCs),after co-culture with themembranes for a period of time to assess of the effect of membranes on differentiation ofGSCs.5.CCK-8 was used to detect the toxicity of different component fiber membranes toGSCs with or without NIR irradiation,and clarify the effect of different treatment modes onGSCs.6.The MWCNTs-OH-containing membrane was placed on the back of Kunming mice.After 15 days,hematoxylin-eosin staining(H&E)was used to observe biocompatibility which was evaluated by damage degree of the liver,lung and kidney tissues of Kunming mice after transplanting membranes loaded with MWCNTs-OH.In addition,GSCs were injected at the dorsal right side of Balb/c nude mice to make tumor models,and the membranes of different components were placed in the site of tumors,and the tumor size in the presence or absence of NIR irradiation was measrued to evaluate the antitumor effect of the membranes in vivo.Results1.The fiber with 0.2%w/v MWCNTs-OH had a smooth diameter and a uniform shape.In addition,the fiber diameter decreased with the addition of MWCNTs-OH;Raman spectroscopy shows that the MWCNTs-OH and ATRA chemical functional groups in the fiber remain unchanged;XRD shows that when loading with 0.2%w/v MWCNTs-OH,the peak width and intensity of PCL did not change significantly.However,after loading different concentrations of ATRA,the characteristic peak intensity of PCL became lower and wider;the water contact angle showed that the hydrophilicity of the membrane did not change significantly after loading MWCNTs-OH and ATRA;the mechanical properties of the membrane after loading ATRA was significantly reduced.However,loading MWCNTs-OH could ameliorate the marked decrease in the tensile strength of the scaffolds after the addition of ATRA.2.Under NIR irradiation,the membrane containing 0.2w/v%MWCNTs-OH raised the temperature of the medium to 48.92℃ for 30s,and reached 90℃ for 90 s.Therefore,the addition of MWCNTs-OH could rapidly increase the temperature of the stent under NIR irradiation to reach the "treatment temperature range"(42-47℃),and the membrane possessed high quality of the photothermal performance in vivo and in vitro.3.The drug loading and encapsulation rate of the fiber membrane were high,The burst release phenomenon was slight,and the release of ATRA could be accelerated under near-infrared irradiation.4.The results of immunofluorescence,western blot(WB),and flow cytometry showed that after a period of co-culture with ATRA fiber membranes,the expression of CD 133 decreased.5.The activity of GSCs was maintained after co-cultivation with the highest concentration ATRA fiber membrane for 3 days.Compared with the group of MWCNTs-OH membrane,the GSCs co-cultivated with MWCNTs-OH and ARTA membrane had lower viability under NIR irradiation for 2min,and prolonging the NIR irradiation time to 3min could further reduce the viability of GSCs.6.Liver,lung and kidney of mice with and without MWCNTs-OH membrane were not significantly changed;under NIR irradiation,the tumor size of mice with MWCNTs-OH and ARTA membrane were statistically different from that of the control group,and the effect of treatment was obvious.ConclusionsThe PCL/ATRA/MWCNTs-OH biodegradable sustained-release membranes were constructed by electrospinning technology.With smooth morphology,uniform diameter,good photothermal performance,differentiation therapy and good biocompatibility the membranes possessed good antitumor effect.Therefore,this system may be a potential treatment to reduce the recurrence of cancer for localized therapy after surgery.