Preparation Of TPNP_DOX&PPNP_LOS Binary Co-delivery System And Its Biological Effects

Background Cancer is a malignant tumor that results from abnormal and uncontrolled cell division,invasion and destruction of surrounding tissues.Tumor heterogeneity is one of the characteristics of malignant tumors.Tumor heterogeneity is closely related to the microenvironment around tumor cells.Tumor structures include tumor cells and tumor microenvironment?TME?,which in turn consists of stromal cells and extracellular matrix?ECM?.Stromal cells,such as angiogenic vascular cells?AVCs?,infiltrating immune cells?IICs?,and cancer-associated fibroblastic cells?CAFs?.They can promote tumor invasion,proliferation,and metastasis by producing growth factors,chemokines,and matrix degrading enzymes.The tumor microenvironment interacts with tumor cells and plays a vital role in tumorigenesis,growth and metastasis.It also provides obstacles for drug delivery in nano-delivery systems,which greatly affects the sensitivity of tumor cells to drugs,which is one of the reasons for the resistance of anticancer drugs.The drug delivery system targeting the tumor microenvironment plays an important role in improving drug delivery and remodeling the tumor microenvironment.Only targeting tumor cells for targeted therapy may be ineffective for malignant tumors.Therefore,targeting cancer cells and tumor microenvironment is currently a new strategy for treating tumors.Cancer-associated fibroblasts?CAFs?are the major cell types in the tumor stroma and secrete a variety of extracellular matrix?ECM?components such as collagen,fibronectin and extracellular matrix-degrading proteases.Through the complex cell-cell interaction with tumor cells,it provides a growth environment for tumor cells and forms a barrier to the nano drug delivery system.In addition,CAFs also secretes a mass of growth factors such as vascular endothelial growth factor?VEGF?,specifically expresses various proteins such as alpha smooth muscle actin??-SMA?,fibroblast activation protein?FAP?and fibroblast-specific protein 1?FSP-1 keratin?.These matrix components can induce tumor fibrosis,angiogenesis and metastasis.In rerecently research,more and more studies have targeted CAFs to cleave its interaction with tumor cells.Therefore,the combination of nanotechnology-based treatment of tumor cells and cancer-associated fibroblasts may be a new treatment for anti-tumor therapy.The combination of anticancer drugs and anti-cancer associated fibroblast drugs in a binary blended nano-codelivery system,combined treatment of cancer cells and cancer-associated tumor cells,can reshape the tumor microenvironment,improve the bioavailability and anti-cancer efficacy of drugs.Aim We designed and prepared a binary blended co-delivery system named TPNP_DOX & PPNPLOS.The co-delivery system has two different nanoparticles?TPNP and PPNP?are loaded with two drugs for different cellular targets,one is the anti-fibrotic drug LOS,and the other is the anti-cancer drug DOX.It will then be physically mixed at different mass ratios for co-delivery of two small molecule drugs.In this study,two nanoparticles were targeted to cancer cells and CAFs of TME,respectively.We demonstrated that TPNP_DOX&PPNPLOS released LOS in the tumor microenvironment and release DOX in tumor cells.This step-by-step release drugs could kill CAFs and regulate the tumor microenvironment,thereby kill tumors more effectively,and achieve synergistic anticancer effects.Methods 1.Preparation and characterization: TPNP and TPNP_DOX were prepared by emulsion solvent evaporation method,and PPNP and PPNPLOS were prepared by solvent casting method.The particle size and surface potential of TPNP,TPNP_DOX,PPNP and PPNPLOS were detected.Using transmission electron microscopy to observe the surface morphology and dispersion of several nanoparticles and micelles,as well as the p H-sensitive morphological changes of TPNP nanoparticles and PPNP micelles.The p H-sensitive particle size changes of TPNP nanoparticles and PPNP micelles were detected.Preparation of doxorubicin?DOX?labeled TPNP and coumarin-6?C6?labeled PPNP,and after phically mixing that formatting of TPNP_DOX & PPNPC6.We observed they dispersion characteristics of the binary mixed co-delivery system by the confocal laser scanning electron microscope.Fourier transform infrared spectroscopy?FTIR?and X-ray diffraction?X-RD?to detecting the physical and chemical structure of nanoparticles and micelles;The UV-Vis spectrophotometer was used to measure the encapsulation efficiency and drug loading efficiency of the nanoparticles and micelles,as well as p H-sensitive release characteristics in vitro.2.The activate of CAFs: The transforming growth factor ?1?TGF-??was used to activate of NIH3T3 cells turn into the CAFs.Observation of ?-SMA protein expression by confocal laser microscopy using immunofluorescence technique.The expression of ?-SMA and FAP protein in the NIH3T3 cells were determined by Western Blot after treatment with different concentrations groups of TGF-?,and the appropriate concentration of TGF-? was selected.3.Biological effects: The cytotoxicity of TPNP and PPNP on Hep G-2 and NIH3T3 cells was measured by CCK-8 method to evaluate the safety of the material.The cytotoxicity of DOX,LOS,DOX+LOS,TPNP_DOX,PPNPLOS and TPNP_DOX&PPNPLOS on Hep G-2 and CAF cells were determined by CCK-8 kit,and IC50 values were calculated to evaluate the improvement of anticancer effect of binary mixed co-delivery system.4.Cell Uptake and Migration: Prepare TPNP_DOX,then confocal laser microscopy was used to observe the uptake and efflux of free DOX in Hep G-2 cells,and the uptake and migration of TPNP_DOX in Hep G-2 cells.Results 1.The test results show that several kinds of nanoparticles are spherical,uniform in particle size and good in dispersion.TPNP nanoparticles are about 170 nm,and PPNP micelles are about 130 nm.And there are different degrees of cleavage at different acidic p H values.Both the nanoparticles and the micelles are negatively charged,facilitating the dispersion of physical mixing and their transport in the blood,and can be internalized by endocytosis.Fluorescence tracer technology proves that the two nanoparticles and micelles are mixed and dispersed well after mixing.2.The results of FTIR and X-RD showed that the successful synthesis of TPNP,PPNP and the successful encapsulation of both drugs.Both nanoparticles and micelles have high encapsulation and drug loading efficiency.The drug release characteristics in vitro revealed that PPNPLOS micelles had weakly acidic microenvironmental response and could release drugs at p H=6.5.TPNP_DOX nanoparticles also displayed p H sensitive and could release drugs at p H=5.8.3.Cellular activation immunofluorescence results showed that the expression of ?-SMA protein with obvious red fluorescent label was observed by laser confocal microscopy after the activation of NIH3T3 by TGF-?.And the results proved that NIH3T3 were successfully activated into CAFs.Western Blot results showed that the TGF-? concentration of 5 ng/ml had the best expression of ?-SMA and FAP protein compared with other concentrations groups.4.The results of in vitro cytotoxicity experiments showed that the two drug delivery materials had low cytotoxicity on Hep G-2 and NIH3T3 cells,which demonstrated that the materials were basically safe.The cytotoxicity results of the drug formulations against Hep G-2 and CAFs indicated that the combined delivery of the anticancer drug DOX and the anti-fibrous drug LOS could improve the anti-cancer effect.And the cytotoxicity of binary blended co-delivery system were significantly higher than the mixing of two free drugs.It could reduce the dose of drugs and reduce side effects.5.The results of cellular uptake and migration pathway experiments indicated that free DOX could enter the nucleus of Hep G-2 cells.And TPNP_DOX nanoparticles could be ingested by Hep G-2 cells in 2 h-6 h and accumulated in the cytoplasm,and more and more DOX migrated and accumulated in the nucleus from 6 h to 24 h.It showed that both DOX were delivered to cells in 24 h and the red fluorescence appears in the nucleus than free DOX.This result revealed that TPNP_DOX nanoparticles could effeciently deliver the drug to the nucleus and accumulate in the nucleus to exert faultlessly its pharmacological effect.Conclusion In this study,we designed and prepared a dual p H-sensitive binary blended codelivery system,which that TPNP_DOX loaded with anticancer drug DOX and PPNPLOS loaded antifibrotic drug LOS.PPNPLOS could release LOS in the tumor microenvironment at p H=6.5 to effectively kill cancer-associated fibroblasts,eliminate the barrier of extracellular matrix,improve the tumor microenvironment,and improve the sensitivity of tumor cells to anticancer drugs.TPNP_DOX could effectively deliver cytotoxic drugs into tumor cells,and release the drug DOX at the tumor cell cytoplasm p H=5.8 to exert its pharmacological effects.The combination of two drug delivery systems could effectively kill tumor cells and tumor-associated fibroblasts,reduce the dose of anticancer drugs,reduce its toxic and side effects,enhance its anticancer effect,and achieve a good synergistic anticancer therapeutic effect.