Multifunctional Nano Drug Delivery Systems Based On Phototherapy For Synergistic Effects Targeting Oral Squamous Cell Carcinoma

Background and Objective:Oral squamous cell carcinoma（OSCC）is a common malignant tumor of oral and maxillofacial region.The traditional treatment methods against OSCC mainly include surgery resection,radiotherapy and chemotherapy.However,the oral and maxillofacial region is not only closely related to beauty,but also bears the important functions such as chewing,pronunciation,swallowing and gland secretion.Hence,surgical resection often brings irreversible damage to patients from physiological and psychological aspects.Besides,radiotherapy,chemotherapy and other adjunctive cancer treatment methods can rarely achieve complete cure of OSCC.Therefore,more and more attentions have been paid to various new anti-tumor treatment methods,such as phototherapy,gene therapy,immunotherapy and molecule-targeting agents et al.Phototherapy includes photothermal therapy（PTT）and photodynamic therapy（PDT）.The principles of PTT and PDT are to use photothermal conversion agent and photosensitizers to trigger the heating effect and the production of reactive oxygen species（ROS）under the corresponding wavelength excitation of laser irradiation,so as to directly kill tumor cells,close tumor neovascularization and activate the anti-tumor immunity of the body,respectively.Compared to traditional OSCC therapies,PTT and PDT are more minimally invasive and more controllable treatments,as well as not easier to produce drug resistance.Nano drug delivery system can used to carry photosensitizers,photothermal converters,chemotherapy drugs,genes and other therapeutic factors,and further modify nanoparticles with tumor targeting material,not only can overcome the defects of single drug preparation,such as large side effects,poor tumor selectivity and stability,but also can achieve significant precise delivery capability of drugs to tumors and synergistic anti-tumor effect,which has a broad application prospect in OSCC treatment.In order to realize the combination of optical therapy and other treatments（chemotherapy and gene therapy）against OSCC,two kinds of nanoparticles with simple structure and diverse functions were designed:1.Regarded c RGD peptide and TL owning ROS responsive bond breaking ability as target ligand and chemical bridge,respectively,a ROS-responsive nanoparticle（c RGD-PEG-TL-DOX/HP,RPTD/HP）based on maleimide and amino polyglycol（PEG）which co-loaded photosensitizer hematoporphyrin（HP）and chemotherapy drug doxirubicin（DOX）was constructed.The precise tumor delivery and controlled release of two therapeutic factors were achieved,and the further synergistic effect of PDT in combination with chemotherapy was remarkably exhibited in killing CAL-27 cells.2.Regarded Poly（β-aminoester）s（PBAE）and Poly（lactic-co-glycolic acid）（PLGA）as nanocarrier materials,a cancer cell membrane（CCM）coated biomimetic nano drug delivery system（CCM@PBAE/PLGA/ICG-Nrf2-si RNA,M@PPI-si RNA）which co-loaded indocyanine green（ICG）and Nrf2-si RNA was constructed.The creation could amplify the photodynamic effect by regulating the antioxidant mechanism in SCC-25cells and the modification of homologous cancer cell membrane,thus realized the combined anti-tumor efficacy of PDT,PTT and gene therapy.In this paper,the preparation and characterization of nanoparticles were introduced,and the anti-tumor effect of nanoparticles in combination with laser irradiation in vitro and in vivo were evaluated.Methods:First part:Study on OSCC targeted and ROS responsive nano therapeutic system1.Preparation and characterization of nanoparticle:PTD was synthesized by coupling DOX and PEG via ROS responsive chemical bond TL,and then the sulfhydryl group in c RGD reacted with maleimide at the end of PTD to synthesize anti-tumor targeting prodrug RPTD.The chemical structures of PTD and RPTD were investigated using ¹H NMR and IR.The ROS responsive bond breaking property of TL was investigated in H₂O₂solution.Finally,the HP was loaded by RPTD nanocarrier to prepare RPTD/HP nanoparticles using nano precipitation method.The morphology,diameter and distribution of RPTD/HP nanoparticles were respectively measured using transmission electron microscope and dynamic laser scattering method.Theπ-πstacking of RPTD and HP was verified by fluorescence spectrometry.The ability of RPTD/HP nanoparticles to trigger ROS formation under laser irradiation was investigated by SOSG fluorescence probe,and the ROS responsive drug release capacity of RPTD/HP nanoparticles was investigated by in vitro dynamic dialysis method.2.Evaluation of the antitumor effect of RPTD/HP nanoparticles in vitro:The uptake of PTD/HP and RPTD/HP nanoparticles by OSCC cell line CAL-27 and human oral epithelial cell line HOEC and the subcellular distribution of DOX under laser irradiation were examined by confocal microscopy and flow cytometry,so as to evaluate the OSCC targeting of RPTD/HP nanoparticles in vitro.DCFH-DA was used to detect cellular ROS production triggered by RPTD/HP nanoparticles under laser irradiation,thus to evaluate the PDT effect of nanoparticles in vitro.The damage of mitochondrial membrane was investigated using fluorescence probe method,and the subcellular distribution of Cytochrome c（Cyt c）was observed using immunofluorescence technique,so as to preliminarily explore the mechanism of apoptosis induced by PDT.The inhibitory effect of RPTD/HP combined with laser irradiation on the growth of CAL-27 cells in vitro,the induction of apoptosis and the cell cycle arrest were investigated,and the synergistic antitumor effect of nanoparticles combined with PDT and chemotherapy was evaluated.3.Evaluation of the antitumor effect of RPTD/HP nanoparticles in vivo:The CAL-27 tumor bearing nude mice model was established by subcutaneous injection.PTD/HP and RPTD/HP nanoparticles were labeled with Cy5.5 fluorescein and the distributions of PTD/HP/Cy5.5 and RPTD/HP/Cy5.5 nanoparticles in vivo were detected after intravenous injection,so as to evaluation of OSCC targeting of RPTD/HP nanoparticles.The tumor bearing mice were intravenously injected with free drugs and nanoparticles and exposed to laser irradiation once every 5 days for consecutive 4 times.During the treatment,the weight and tumor volume of mice were monitored.The main organs and tumors were separated for further histopathological analysis,including hematoxylin/eosin staining（H&E）and CD31immunohistochemical staining after the treatment,so as to comprehensive evaluate the inhibitory effect of RPTD/HP nanoparticles combined with PDT and chemotherapy on tumor growth in CAL-27 tumor bearing mice.Second part:Study on homologous cancer cell membrane coated biomimetic nano therapeutic system1.Preparation and characteristics:Regarded PBAE and PLGA as nanocarriers,ICG loaded PPI nanoparticles were prepared using double emulsion method.PPI-si RNA nanoparticles were prepared by positive and negative charge adsorption between PPI nanoparticles and Nrf2-si RNA.The cancer cell membrane（CCM）of OSCC cell line SCC-25 was extracted and encapsulated on the surface of PPI-si RNA nanoparticles through extrusion method to prepare M@PPI-si RNA nanoparticles.The morphology,diameter and zeta potential of M@PPI-si RNA nanoparticles were measured using TEM and dynamic laser scattering method,respectively.Agarose gel electrophoresis was used to explore the best proportion of PPI nanoparticles and Nrf2-si RNA.SDS-PAGE gel electrophoresis was used to investigate the retention of CCM related proteins on M@PPI-si RNA nanoparticles.The drug-loading rate and PTT/PDT effect of ICG and M@PPI-si RNA were respectively measured using UV-photometer.2.Evaluation of the antitumor effect of M@PPI-si RNA nanoparticles in vitro:The uptake and subcellular distribution of PPI-si RNA and M@PPI-si RNA nanoparticles in human melanoma cell line B16,HOEC and OSCC cell line SCC-25were detected using laser confocal microscopy and flow cytometry,so as to evaluate the OSCC targeting and the ability to deliver Nrf2-si RNA for escaping from endosomes/lysosomes of M@PPI-si RNA nanoparticles.The expression of heat shock protein 60（Hsp60）were detected using immunofluorescence technique,so as to evaluate the PTT effect of M@PPI-si RNA nanoparticles.The production of cellular ROS was detected by ROS fluorescence probe DCFH-DA.Meanwhile,immunofluorescence technique was used to analyze the co localization of Cyt c and mitochondra to evaluate the PDT effect of M@PPI-si RNA nanoparticles and its apoptotic mechanism.Western blotting was used to detect the expression level of Nrf2 and its downstream regulated transcription genes GCLC and GCLM in SCC-25cells,so as to explore the mechanism of amplified PDT effect induced by Nrf2-si RNA.MTT assay,living and dead cell staining and apoptosis kit were used to investigate the inhibitory effect of M@PPI-si RNA nanoparticles on the growth of SCC-25 cells in vitro under laser irradiation.3.Evaluation of the antitumor effect of M@PPI-si RNA nanoparticles in vivo:The SCC-25 tumor bearing nude mice model was established by subcutaneous injection.M@PPI-si RNA nanoparticles were intravenously injected,and then the tissue distribution and tumor retention were investigated by in vivo imaging system to evaluate the in vivo OSCC targeting of nanoparticles.After intravenous injection,local laser irradiation was performed on mice tumors.During the treatment,infrared thermography was used to monitor the temperature change of tumor.SOSG fluorescence probe was used to detect the ROS production of tumor tissue,while immunohistochemistry was used to detect the expression of HSP60 and Nrf2 protein in tumor tissue.Meanwhile,the weights and tumor sizes of mice were monitored.After treatment,the main organs and tumors were separated for further histopathological analysis,including hematoxylin/eosin staining（H&E）and CD31immunohistochemical staining,so as to evaluate the therapeutic effect of M@PPI-si RNA nanoparticles combined with PTT/PDT and Nrf2-si RNA on OSCC.Results:First part:Study on OSCC targeted and ROS responsive nano therapeutic system1.The RPTD was successfully synthesized and its chemical structure was confirmed by IR and ¹H NMR.RPTD assembled with hydrophobic photosensitizer HP throughπ-πstacking and hydrophobic interaction in aqueous medium to form RPTD/HP nanoparticles,thus realizing the effective co loading of HP and DOX.The size and PDI of RPTD/HP were 186.2 nm and 0.156,respectively,and showed core-shell spherical structure with uniform distribution.RPTD/HP nanoparticles exhibited good dispersion and stability in vitro,and showed significant photodynamic effect.The TL could efficiently cleavage in the ROS environment,and laser irradiation can promote the rapid release of DOX from RPTD/HP nanoparticles,which proves that the nanoparticles has ROS responsive drug release performance.2.In CAL-27 cells,the cellular internalization of RPTD/HP nanoparticles was significantly higher than that of PTD/HP nanoparticles,but there was no significant difference between two kinds of nanoparticles in HOEC,which indicated that the surface modification of c RGD peptide mediated the effective targeting of RPTD/HP nanoparticles to CAL-27 cells in vitro.The RPTD/HP nanoparticles triggered the production of ROS in CAL-27 cells under laser irradiation,and activated mitochondrial apoptosis pathway,which include mitochondrial membrane damage and Cyt c release from mitochondria to cytoplasm.The generated ROS promoted the release of DOX,which showed a significant synergistic effect of PDT and chemotherapy in terms of cell killing,apoptosis and cell cycle arrest.3.RPTD/HP/Cy5.5 nanoparticles showed significantly better tumor targeting and retention ability than PTD/HP/Cy5.5 nanoparticles in CAL-27 tumor bearing mice after intravenous injection,which indicated that the modification of c RGD peptide endows RPTD/HP nanoparticles with significant in vivo OSCC targeted ability.RPTD/HP nanoparticles combined with laser irradiation inhibited the growth of the tumor to the greatest extent and effectively closed the neovascularization under the laser irradiation.At the same time,RPTD/HP nanoparticles showed good biosafety.There was no significant change in the weight of mice during the treatment,and no pathological damage in the main organs was appeared after treatment,indicating that the synergistic treatment strategy based on RPTD/HP nanoparticles has good in vivo biosafety.Second part:Study on homologous cancer cell membrane coated biomimetic nano therapeutic system1.The PPI and PPI-si RNA nanoparticles have spherical morphology and dense internal structure,with an average particle size of 159.4 nm and 168.3 nm,respectively,and zeta potential of+43.6 m V and+26 m V.The drug loading rate and encapsulation rate of ICG in PPI nanoparticles were 1.9%and 91.8%,respectively.CCM was extracted from SCC-25 cells and successfully coated on the surface of PPI-si RNA nanoparticles by extrusion.The particle size and zeta potential of M@PPI-si RNA nanoparticles were respectively 190.5 nm and-38 m V.PPI nanoparticles have high adsorption capacity for Nrf2 si RNA,while M@PPI si RNA successfully retains the protein characteristics of CCM surface.M@PPI-si RNA nanoparticles showed significant thermal and ROS promoting effect under laser irradiation,indicating that the nanoparticles have good PTT and PDT properties in vitro.2.The PPI-si RNA and M@PPI-si RNA nanoparticles exhibited almost identical internalization in HOEC and B16 cells,but the uptake of M@PPI-si RNA nanoparticles by SCC-25 cells was significantly higher than that of PPI-si RNA nanoparticles,indicating that the modification of homologous CCM promoted the internalization of nanoparticles by OSCC cells.Through the delivery of M@PPI-si RNA nanoparticles,Nrf2-si RNA can escape the endosome/lysosome and enter the cytoplasm to exert RNA interference effect.M@PPI-si RNA nanoparticles combined with laser irradiation significantly upregulated HSP60 protein expression,and triggered amount of intracellular ROS production,which confirmed its good PTT/PDT effect.Meanwhile,compared with PPI nanoparticles,PPI-si RNA and M@PPI-si RNA nanoparticles significantly reduced the protein expression of Nrf2,GCLM and GCLC,thus showing more significant cytotoxic and apoptosis inducing effects,promoting more Cyt c release from mitochondria to cytoplasm.The results indicated that the targeting effect of homologous CCM and the antioxidant inhibited effect of Nrf2-si RNA amplify the PTT/PDT efficiency of ICG.3.After administration via tail vein,M@PPI-si RNA nanoparticles showed significantly better tumor targeting and retention ability than PPI-si RNA nanoparticles in SCC-25 tumor bearing mice,further indicating that the surface modification of homologous CCM endow M@PPI-si RNA nanoparticles with active targeting ability to OSCC.Combined with laser irradiation,M@PPI-si RNA nanoparticles induced local temperature rise and significantly increased the expression level of HSP60,which confirmed its good PTT effect in vivo.Meanwhile,it also triggered the generation of ROS in tumor tissue and down regulated the expression level of Nrf2,which confirmed its significant PDT effect in vivo.The results of treatment in tumor bearing mice showed that M@PPI-si RNA nanoparticles combined with laser irradiation could significantly inhibit the growth of tumor,induce apoptosis or necrosis and inhibit tumor angiogenesis in mice,indicating that the homologous targeted strategy and anti-oxidation inhibited effect mediated by Nrf2-si RNA amplified the anti-tumor effect of PTT/PDT.Conclusion:In this study,we designed and constructed two kinds of multi-functional nano therapy systems with simple structure,which realized the efficient co-loading,targeted delivery,controlled release of photosensitizers.The multi-functional nano therapy systems could also exert the synergistic inhibition function on OSCC through the combination of phototherapy including PTT and PDT with other treatment methods such as chemotherapy and gene therapy.The first multi-functional nano therapy system is named RPTD/HP nanoparticles,which is based on c RGD as the targeted ligand,TL with ROS responsive bond breaking ability as the connecting bridge.This nanoparticles,which have both OSCC targeting and ROS responsive drug release performance,have obtained the synergistic inhibited effect of PDT and chemotherapy against OSCC.The second multi-functional nano therapy system is named M@PPI-si RNA nanoparticles,which co loaded with ICG and Nrf2-si RNA and coated with homologous CCM.The PTT/PDT effect of ICG is amplified by homologous tumor targeted delivery and regulation of antioxidant resistance mechanism,so as to obtain the synergistic inhibited effect on OSCC.Accordingly,the study provide promising strategies for the clinical treatment of OSCC.