Intracellular Tracking Of Drug Release From PH-sensitive Polymeric Nanoparticles Via FRET For Synergistic Chemo-photodynamic Therapy

In recent years,the synergistic application of photodynamic therapy and chemotherapy has significantly promoted the treatment of tumors.A series of new nano drug delivery systems for synergistic chemo-photodynamic therapy have been well constructed.The delivery efficiency of bioactive molecules has been successfully improved and problems such as the difficulty of real-time monitoring the release process of anti-tumor drugs and the poor effect of single drug treatment have been solved.It has been reported that porphyrin,a photosensitizer,could induce the production of singlet oxygen under the stimulation of near-infrared laser and has a good therapeutic effect on tumor.The fluorescence emission wavelength range?500-600 nm?of epirubicin?EPI?coincides with the fluorescence excitation wavelength range?520-600 nm?of porphyrin?Por?.Therefore,when the space distance between epirubicin?EPI?and porphyrin?Por?is close enough,the condition of FRET effect could be emerged.Based on this study,we designed a pH sensitive drug delivery system with both photodynamic and chemotherapeutic effects:Por was introduced into the drug carrier as photosensitizer,EPI was loaded into the hydrophobic nucleus of nanoparticales,and the tumor treatment process was monitored in real time through the potential FRET effect between the two.By introducing the cyclic penetrating peptide cRGD,the role of cRGD could improve the efficiency of drug-loaded narnoparticals into the tumor;Polyhistidine segments was introduced to the carrier,for the low pH in lysosomes,leading to its protonation and triggering the rapid release of drugs.At the same time,we introduced photodynamic therapy photosensitizer and antitumor drugs which could produce FRET effect for monitoring the real-time release of drug process,and achieved synergistic treatment of PDT and chemotherapy.We synthesized Polymer materials Por-PCL-PH-PEG-cRGD by amidation and Esterification reaction.Their structures were identified by ¹H-NMR and GPC.Nanoparticales were prepared by dialysis method.Dynamic light scattering was used to measure the average diameter of the nanoparticales at various p H values,the morphology of the polymer was observed by transmission electron microscopy,the loading efficiency and encapsulation efficiency of EPI were determined by ultraviolet spectrophotometer,the cumulative release rate of EPI was measured by the dialysis bag method;CCK-8 method was used to investigate the cytotoxicity of nano delivery system with CT26 cells,a 3D tumor sphere model was established in vitro.The effect of drug loading system on the permeability of solid tumor tissue was investigated by scanning tumor spheres with confocal laser microscopy.The uptake efficiency of CT26 cells to drug loading system was investigated by flow cytometry,a CT26 tumor-bearing mouse model was established to investigate the effect of drug-loaded nanoparticales on the anti-tumor effect,and in vivo imaging technology was used to investigate the distribution of drug-loaded nanoparticales.The results of ¹H-NMR and GPC indicated that the target products were successfully synthesized.Spherical EPI-loaded nanoparticales with the average size of 234±6.1 nm was procured with negative charge,the loading efficiency and encapsulation efficiency of EPI were 14.9±1.5%and 74.6±2.3%respectively.The 72 h cumulative release ratio of EPI from nanoparticales was 30%under pH 7.4,whereas that under pH 5.0 was 82%which showing a certain pH dependence.The cellular uptake of EPI loaded cRGD-PEG-PH-PCL-Por was monitored in real time by the FRET efect between EPI and cRGD-PEG-PH-PCL-Por.The polymeric narnoparticals combined PDT and chemotherapy showed signifcant anticancer activity both in vitro(IC₅₀=0.47?g/m L)and better therapeutic efcacy than that of free EPI in vivo.The results of in vitro tumor penetration investigation experiments found that the fluorescence intensity of drug-loaded nanoparticles was significantly higher than that of traditional PEG-PCL drug-loaded narnoparticals,and multifunctional drug-loaded narnoparticals could better penetrate into solid tumors.Flow cytometry results show that drug-loaded nanoparticles are more easily taken up by CT26 cells whose receptors are not saturated with cRGD,indicating that cRGD has a certain active targeting effect.In vivo imaging results show that drug-loaded nanoparticales are more likely to accumulate in tumor sites than free drug sand less liver distribution.In the evaluation of the CT26 xenograft tumor model in vivo anti-tumor effect,drug-loaded nanoparticales play a better anti-tumor effect than free EPI effect.This study provided a nano assembly method for intracellular drug release tracing and cooperative photodynamic therapy,which could achieve effective tumor treatment through real-time drug release monitoring,dynamic cell imaging and anti-tumor activity.This multi-functional drug delivery system could not only overcome the obstacles in the process of delivery of antitumor drug in vivo,but also monitor the drug release process,while applying the use of PDT and chemotherapy synergistic treatment to efficiently improve the treatment of cancer.