| Cancer is a major disease that seriously threatens human health.At present,surgery,radiotherapy,chemotherapy and immunotherapy are the four main methods of treating cancer,of which chemotherapy dominates.However,the poor water solubility,low bioavailability,narrow therapeutic window,and serious toxic and side effects of chemotherapy drugs have seriously hindered their clinical application.Nano preparations have significantly improved the problems of solubility,biodistribution and bioavailability of chemotherapy drugs,but their relatively low drug load and the use of a large number of inactive excipients bring potential safety risks and high costs.Therefore,in this project,the active polyphenol silybin(Sil)and metal ion Fe(Ⅲ)were used to form a metal-polyphenol network coated with dihydrotanshinone I nanocrystals(DHT NC),and a multidrug carrier-free nanocomposite(DHT NC@SF MPN)with high drug load and high safety was constructed for the treatment of gastric cancer and its mechanism of action was elucidated.The principle of its action is that the metal-polyphenol network coated on the surface of DHT NC@SF MPN was decompositions in the tumor acidic microenvironment,releasing DHT NC,Sil and Fe(Ⅲ).DHT NC promotes apoptosis of tumor cells,Sil inhibits proliferation and metastasis of tumor cells,and Fe(Ⅲ)triggers ferroptosis,and the three synergistic effects to achieve chemotherapy/chemokinetic therapy of tumors.1.Preparation and characterization of DHT NC@SF MPN nanocomplexesFirstly,DHT NC was prepared by precipitation,and the structure of DHT NC prepared by different dispersants was investigated by X-ray diffraction(XRD),and povidone K30 was selected as the dispersant.The optimal preparation process of DHT NC was further determined by orthogonal tests using particle size and polydisperse coefficient(PDI),and the final DHT NC particle size was 224.0 ± 0.7 nm,and the PDI was 0.49 ± 0.05.Secondly,The metal-polyphenol network formed by the coordination of Sil and Fe(Ⅲ)coats DHT NC.The composition of the nanocomplexes was characterized by infrared method,and the results showed that the nanocomplexes were successfully loaded into DHT and SF MPN,which proved that the nanocomplexes were successfully constructed.The morphology of the nanocomplexes were characterized using SEM and TEM,and the results showed that the nanocomplexes were approximately spherical.In order to optimize the physicochemical properties of DHT NC@SF MPN,the dosage ratio of Sil to Fe(Ⅲ)was investigated by particle size,PDI,drug load and encapsulation rate and pH sensitivity,and the results showed that the DHT NC@SF MPN nanocomplexes obtained by coating DHT NC with a ratio of Sil to Fe(Ⅲ)molar ratio of 3:1 were well dispersed,with particle sizes of 262.12±5.61 nm,PDI of 0.269 ±0.013,and high stability.DHT and Sil have higher loads,56.08%and 41.88%,respectively,and the DHT encapsulation rate is 55.28%.At the same time,the rapid release of nanocomposites within 1 h at pH 6.0 was 25.35%and 41.00%for DHT and Sil,and 63.2%and 94.6%for DHT and Sil at 72 h,respectively.2.Evaluate DHT NC@SF MPN nanocomplexes at the cellular levelThe in vitro antitumor effect of DHT NC@SF MPN carrier-free nanocomplexes was evaluated by human cancer cell HGC 27.The cytotoxicity results showed that DHT NC@SF MPN nanocomplexes had obvious cytotoxicity,and the IC50 value was 51.70%and 71.03%of DHT and the mixture of DHT+Sil+Fe(Ⅲ),respectively.The results of apoptosis experiments showed that nanocomplexes could promote apoptosis,and the apoptosis rate increased by 46.98%and 6.07%compared with DHT and the drugs mixture,respectively.The results of cell migration experiments showed that the effect of nanocomplexes in inhibiting cell migration was significantly higher than that of DHT and the drugs mixture.The transport process of nanocomplexes within the package of HGC 27 cells was investigated.The results of uptake behavior experiments showed that the uptake of nanocomplexes by cells was time/concentration-dependent.Endocytic protein inhibition experiments show that nanocomplexes enter cells mainly through Lipid raft/fossa proteinmediated pathways.Organelle colocalization results showed that nanocomplexes were mainly distributed in mitochondria,lysosomes and endoplasmic reticulum.3.Investigate and evaluate DHT NC@SF MPN nanocomplexes at the animal levelThe pharmacokinetics and tissue distribution of DHT NC@SF MPN nanocomplexes were investigated by SD rats,and the results showed that the bioavailability of the nanocomposite group was 1.86 times and 1.59 times that of the physical mixture of DHT and the drugs mixture,respectively,while the mean retention time and half-life were extended,and the nanocomposites were mainly distributed in liver tissues.The HGC27-CDX nude mouse tumor model was established to evaluate the efficacy and safety of DHT NC@SF MPN nanocomplexes.In vitro pharmacodynamic studies have shown that DHT NC@SF MPN nanocomplexes can inhibit tumor growth,and its tumor growth inhibition rate is 39.74%,which is significantly enhanced compared with the inhibition rate of DHT and the drugs mixture,but has no significant difference with the clinical use of paclitaxel liposome.Biodistribution studies have shown that nanocomplexes can accumulate at the tumor site for a long time after intra-tumoral injection,effectively avoiding the harmful effect of drugs on normal tissues.H&E-stained sections and TUNEL-stained sections showed that nanocomplexes promoted tumor tissue necrosis and tumor cell apoptosis.The results of western blot experiments showed that the nanocomplex upregulated the expression of caspase3 and caspase8,downregulated the expression of Bcl-2 to promote apoptosis of tumor cells,while downregulated the expression of MMP2 and MMP9 inhibited tumor metastasis,increased the expression of ferroptosis-related protein TFRC,and decreased the expression of GPX4,indicating that ferroptosis was activated.The safety evaluation results showed that the nanocomposites had low toxicity to normal tissues and high safety.In summary,DHT NC@SF MPN carrier-free nanocomplexes were constructed,the nanocomplexes have the characteristics of high drug load and pH-responsive drug release,and the efficacy in vivo and in vitro is better than the physical mixing of DHT,Sil and Fe(Ⅲ)drugs,and its anti-tumor mechanism is regulate to the expression of apoptosis,metastasis and ferroptosis-related proteins,and inhibit tumor growth and metastasis through multi-target mode of action,which provides new ideas for the development of nanodrugs. |
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