A Preliminary Study On The Synergistic Phototherapy And Immunomodulatory Effects Of Targeted Nanoparticles Based On Iodinated-cyanine Dyes

Photodynamic therapy?photodynamic therapy,PDT?has the advantages of high selectivity,minimally invasiveness,and low toxicity.It is one of the efficient phototherapy strategies for cancer treatment,which can effectively destroy local tumor cells and induce systemic anti-tumor immunity.However,current nanotechnology-assisted photosensitizers have disadvantages such as poor permeability to deep tumors or low singlet oxygen production,which hinders them to be effective immunomodulators.In this paper,a new type of targeted nanocarrier?HA-PEG-CyI,HPC?was constructed by inducing the self-assembly of PEGylated CyI?iodinated-cyanine?with attaching ligand HA on the surface.It can be used as an immunomodulator for synergistic phototherapy,which can exert PDT/photothermal therapy?PTT?effects to kill tumors,and induce the occurrence of anti-tumor immune responses,thus reducing tumor recurrence.CyI is an iodinated-cyanine dye with enhanced singlet oxygen generation ability as well as excellent photo-to-photothermal and near-infrared fluorescence imaging properties.Hydrophobic CyI and PEG were covalently bound through an amide reaction,and then the targeted ligand HA molecule was linked to synthesize a therapeutic nanocarrier HPC.¹O₂fluorescent probe SOSG was used to investigate the photodynamic effect of HPC in vitro.The photothermal effect of HPC in vitro was measured by thermocouple thermometer.The cytotoxicity and hemolytic toxicity of HPC were tested to evaluate its in vitro safety.To explore the metabolism of nanocarriers,the in vivo pharmacokinetic and biodistribution ofHPC was studied.The cellular uptake of HPC in 4T1 cells stained with LysoTracker Green DND-26 was observed by a confocal laser scanning microscope.The uptake mechanism of HPC by 4T1 cells was investigated using a confocal microscope,stained with Hoechst33342.The photodynamic effect of HPC at the cellular level was evaluated using 2',7'-dichlorodihydrofluorescein diacetate?DCFH-DA?as a fluorescent probe to detect reactive oxygen species?ROS?.The photothermal effect of HPC at the cell level was detected by a thermocouple thermometer and infrared camera.The in vitro synergistic effect of HPC was investigated by MTT method,confocal microscope and flow cytometry.A subcutaneous ectopic breast cancer model of BALB/c mice was established,and the in vivo distribution of HPC,the PTT/PDT properties and the synergistic anti-tumor efficacy were investigated via tail vein injection.ELISA kits and flow cytometry were used to quantify the relevant immune factors and immune cells in the tumor in order to investigate the effect of HPC,as an immunomodulatory,to stimulate the anti-tumor immune response.The results showed that HPC has good water solubility and of targeting ability to cancer cells,while retains the characteristics of high singlet oxygen production of CyI,which has a positive correlation with concentration and laser power density.Moreover,HPC could produce enough singlet oxygen for PDT treatment in a short time?5 min?.Under the near-infrared light irradiation?808 nm,0.96 W/cm²?,the temperature of the HPC solution can rapidly rise to above 43°C within 5 minutes,which could be used for tumor ablation.Within the therapeutic concentration range,HPC has no obvious cytotoxicity and hemolytic toxicity,demonstrating good biocompatibility.In vivo pharmacokinetics and tissue distribution results showed that HPC is mainly metabolized by the liver and excreted through the kidneys,with no accumulation in the body to cause long-term toxicity issues.In vitro studies showed that HPC could be taken up by 4T1 cells through the CD44 receptor-mediated endocytosis pathway after 4 h incubation.Under near-infrared?NIR?light irradiation,HPC could produces effective intracellular ROS and heat to kill cancer cells.In vivo anti-tumor studies showed that HPC has strong tumor-targeting capabilities.With the help of NIR laser irradiation,it can achieve the synergistic anti-tumor effect of multiple phototherapy,and induce the generation of systemic anti-tumor immunity,thus inhibiting tumor recurrence.Above all,this new type of therapeutic nanocarrier HPC can be efficiently enriched into tumor sites and used for near-infrared imaging to diagnose tumors.Moreover,HPC can combine PDT with PTT for synergistic cancer treatment,and further induce anti-tumor immune response,leading to secondary death of tumor cells and reducing tumor recurrence.This multifunctional nanocarrier HPC can be used as a promising multifunctional cancer treatment strategy with great potentials for clinical applications.