Research On Co-delivery Nanoparticles Targeting Cancer Cells And Cancer-related Fibroblasts

BackgroundAt present,cancer is still a difficult medical problem to overcome,which seriously endangers human health.Clinically,the main treatments for cancer include surgery,chemotherapy and radiation therapy.Among them,the chemotherapy is still the preferred method,and it is also a synergistic method of surgery and radiation therapy.Classical chemotherapy often uses small molecule chemicals to interfere with cell division to promote tumor cell apoptosis or necrosis.Although such cytotoxic drugs have achieved good treatment effect as clinical first-line anti-cancer drugs,they also have some deficiencies,such as poor targeting,low bioavailability,large side effects and multidrug resistance.With the improvement of the treatment prescription,Li MC et al.developed the first effective metastatic combination chemotherapy programs for metastatic testicular cancer in 1969s.Combination chemotherapy is a method for treating cancer by two or more anticancer drugs with different pharmacological effects or different side effects,aiming to improve anticancer effect,reduce side effects and reduce the dosage of single chemotherapy drugs.Currently,the concept of combined chemotherapy has been widely accepted,and combined chemotherapy has become the standard method for many cancer treatments.With the development of nanotechnology in the pharmaceutical field,especially the development of nanoparticles?NPs?,based on the concept of combined chemotherapy,at least two anticancer drugs with different physicochemical properties and pharmacological mechanisms are loaded into the same nano drug delivery system?NDDS?,which is known as the nano drug co-delivery system?NDCDS?.Compared with the combination chemotherapy of using two free drugs,NDCDS has many significant advantages in breaking through the biological barrier,high targeting,and it can enhance penetration of cancer tissue,control of drug release,can better synergize chemotherapy drugs.However,in recent years,most NDCDS only loaded two or more drugs into the same type of nanoparticles,which named single nanoparticles for drug co-delivery?SNDCD?.The advantage of SNDCD is that it can significantly increase the accumulation of model drugs in the tumor area and produce a synergistic effect to enhance the anti-tumor effect.However,there are also some disadvantages.For example,due to its different physical and chemical properties of the two loaded drugs,the drug loading rate and the encapsulation rate are different,so the dose of each drug cannot be quantified accurately when the drugs was delivered.In addition,when the drugs were loaded in the same physical space of nanoparticles,it may cause physical or chemical interactions between the drugs during the drug-loaded nanoparticles was stored or delivered,which may ultimately affect the therapeutic effects.In recent years,the tumor microenvironment?TME?has been widely concerned,a growing number of studies have shown that TME plays an important role in occurrence and migration of tumors.Tumors are complex tissues that include not only malignant cancer cells,but also genetically stable stromal cells such as endothelial cells,fibroblasts,and many other immune cells,as well as the extracellular matrix?ECM?they produce.This combination of tumor cells,stromal cells and extracellular matrix is named the tumor microenvironment?TEM?.The key cells in TME are used as drug targets for antitumor therapy.AimBased on the theory of TME and combination chemotherapy,we propose a scientific hypothesis that the anti-cancer drug?VCR?and anti-fibrotic drug?PFD?are separately loaded into NPs(CPNP_VCR and PPNP_PFD)to improve the anti-tumor effects.Binary blended nanoparticles for drug co-delivery?BBN-DCD?were innovatively designed to deliver small molecule drugs to two key target cells?A549 and CAFs?.And the synergistic effects of the nano drug co-delivery system?NDCDS?in anti-tumor therapy were evaluated.Methods1.Preparation and characterization of NPsWe have successfully prepared empty nanoparticles CPNP and PPNP,as well as drug-loaded nanoparticles CPNP_VCR and PPNP_PFD by emulsion-solvent evaporation method and solvent casting method,respectively.And the physical and chemical characterization of prepared nanoparticles were characterized.The surface morphology and dispersion of nanoparticles were observed by transmission electron microscopy.A suspension of the traceable nanoparticles CPNP_Rh123 and PPNP_C6carrying the fluorescence was observed by laser confocal microscopy,in order to evaluate the mixed aggregation of the two nanoparticles.The particle size and surface charge of the nanoparticles were analyzed.Fourier transform infrared spectroscopy?FTIR?and X-ray diffraction?X-RD?were successfully used to detect whether the free drug was successfully loaded into the nanoparticles.The UV-Vis spectrophotometer was used to examine the encapsulation efficiency and drug loading efficiency of the nanoparticles,as well as the release in vitro.2.Cell culture and induced activation of NIH/3T3 in vitroThe model cells we selected were human lung adenocarcinoma cell A549 and mouse embryonic fibroblast NIH/3T3.In order to obtain cancer-associated fibroblasts?CAFs?,we induced the activation of NIH/3T3 cells by transforming growth factor TGF-?1 in vitro.And the expression of the marker protein of CAFs,?-smooth muscle actin protein??-SMA?and fibroblast activation protein?FAP?,were detected by Western blot to evaluated whether NIH/3T3 cells was successfully activated into CAFs cells.3.Cytotoxicity experiment in vitroAll cytotoxicity assays were measured by using the WST-1 kit.First,we evaluated the non-toxic or low toxicity of nanomaterials.Secondly,we examined the cytotoxic effects of free drugs and drug-loaded nanoparticles on A549 cells or CAFs cells,and calculated the half maximal inhibitory concentration(IC₅₀)and the synergistic index(CI₅₀)to evaluate the improvement of the anticancer effect of our nano drug co-delivery system?NDCDS?.4.Biological effects and mechanism studiesIn the study of biological effects,we established a non-direct contact co-culture model of A549 cells and CAFs cells in vitro.The co-culture model was treated with different administration methods for 48 h,and the proliferation of A549 cells was observed.The apoptosis of A549 cells was detected by flow cytometry to evaluate the proximity effect of CAFs cells on proliferation or apoptosis of A549 cells.In addition,we also evaluated the reversal effect of PFD on CAFs cells by Western blot.The apoptosis of A549 cells was detected by flow cytometry to evaluate the proximity effect of CAFs on A549 cells.Result1.Preparation and Characterization of NanoparticlesThe particle size of several nanoparticles that we prepared is 100-200 nm,and it had a uniform particle size and good dispersion.Since the surface of the nanoparticles is negatively charged,even if the aqueous solution of CPNP_VCR and PPNP_PFD was mixed,there is no aggregation of the two nanoparticles.The results of infrared and X-ray diffraction showed that the free drug has been successfully loaded into the nanoparticles.And nanoparticles had a high encapsulation and drug loading efficiency for both drugs.In vitro drug release experiments showed that both of CPNP_VCR and PPNP_PFD were pH-sensitive.2.Activation of NIH/3T3 cellsThe activation effect of NIH/3T3 cells showed TGF-?1 concentration-dependent and time-dependent.Western blot bands were analyzed quantitatively by using ImageJ software,we found that TGF-?1 treated NIH/3T3 cells at a concentration of 10 ng/ml for 48 h with the best activation.3.Cytotoxicity experiments in vitroThe nanomaterials were less cytotoxic to A549 cells and CAFs cells,indicating that they can be used in drug delivery vehicles.The cytotoxicity of the drugs showed that the cytotoxicity of CPNP_VCR was significantly enhanced,and when CPNP_VCR and PPNP_PFD were co-delivered,there was a certain synergistic effect.4.Biological effects and mechanism studiesThe results of biological effects and mechanism studies showed that the cell density of A549 cells was significantly higher than that of A549 cells alone when A549 cells and CAFs cells were co-cultured,indicating that CAFs cells can promote the growth and proliferation of A549 cells to a certain extent.When the co-culture model was co-delivered,the results showed that the growth of CAFs cells was inhibited,and the therapeutic effect of the anti-cancer drug VCR could be significantly improved.At the same time,we also found that the anti-fibrotic drug PFD?also an inhibitor of TGF-?1?not only induced the transition of CAFs cells from activated to a resting state,but also inhibit the TGF-?1 induced activation of CAFs cells.ConclusionIn this study,we have innovatively designed and prepared binary blended nanoparticles for drug co-delivery?BBN-DCD?:CPNP_VCR and PPNP_PFD.The nano drug co-delivery system?NDCDS?can significantly enhance the toxic effect of the anti-cancer drug VCR on tumor cell A549 by inhibiting the proliferation of cancer-associated fibroblast?CAFs?.