Development Of Silk Fibroin Nanoparticles Loaded With ICG Based On Supercritical Fluid Technology For Tumer Photo-thermal Therapy

Photo-thermal therapy(PTT)has garnered enormous attention clinically over conventional cancer therapeutic strategies because of its advantages such as minimally invasiveness and targeted therapy.PTT agents transform the absorbed light energy to heat energy,which kills the targeted cells.Indocyanine green(ICG)is one of the biocompatible near-infrared(NIR)PTT agents with photoactive features,however,limited in its clinical application due to lack of targeting,prone to biodegradation,and photo-instability.To overcome this,we firstly developed silk nanoparticles(SF NPs)using supercritical antisolvent(SAS)process and investigated their physicochemical properties and bio-safety.Then,ICG-SF NPs were prepared.The physicochemical properties of the ICG-SF NPs were also well-characterized using various techniques.Meanwhile,the in vitro release characteristics at different pH values,photothermal effect,stability,and bio-safety of ICG-SF NPs were investigated.The intracellularly delivered ICG-SF NPs exhibited significant activity in MCF-7 against ICG.Herewith the respective tasks are discussed in brief.Initially,we have synthesized the SF NPs by SAS process under the conditions provided(CO2 venting rate-20 g/min,flow rate-0.5 mL/min,temperature 30oC,and pressure-10 MPa).Further,the physical characterization showing the morphology and particle size distribution has been demonstrated.The results indicate that SF NPs have possessed good sphericity and uniform particle size with an average size of 100nm;FITR spectra and XRD recordings suggest that the secondary structure of silk fibroin has changed after SAS treatment;the residual amount of hexafluoroisopropanol was only 0.051%;hemolysis rate is less than 5%.Further,ICG is impregnated into SF carriers following the above mentioned conditions and confirmed by various methods.The ICG loading percentage was calculated as 4.5%.The in vitro investigations including photothermal properties and stability of ICG-SF NPs were investigated.The results demonstrate that the aqueoussolutions of ICG and its equivalent amount in ICG-SF NPs has shown similar temperature rising curve under NIR irradiation.In addition,photo-thermal efficiency increased significantly with time(8 days)demonstrating the stability of ICG impregnation in SF.The residual amount of hexafluoroisopropanol is only 0.051%and hemolysis rate is less than 5%.Eventually,the in vitro antitumor efficacy of ICG-SF NPs was investigated by recording cell proliferation rate by CCK-8 kit and further chronicled the apoptotic morphology evidence by Calcein/PI fluorescent staining assay.ICG-SF NPs solution(50 ?g/mL equivalent amount of ICG)in the dark has no toxic effect on cells.However,it has shown the promising effect of assassinating MCF-7 and Hela cells under NIR laser irradiation.Confocal laser scanning microscope(CLSM)results disclosed that ICG-SF NPs are well-internalized into MCF-7 cells.In summary,we have developed the ICG-SF NPs by SAS process,making it highly stable,biocompatible and multifunctional.These particles enhanced the photo-thermal performance of ICG than the ICG alone.Due to the deep tissue penetration of NIR light,excellent biocompatibility and antitumor efficacy of ICG,ICG-SF NPs has a potential to be used for further research,such as in combinatorial chemotherapy and PTT for cancer treatment.