Construction And Anti-tumor Effect Of PLGA Nanoparticles Loaded With CTD Which Are Modified By Hyaluronic Acid

Fighting poison with poison is an important idea for Chinese medicine to treat cancer.Cantharidin（CTD）is an effective component of the traditional Chinese venom of the traditional Chinese medicine,and has been shown to inhibit various malignant tumors.However,CTD has poor water solubility and a narrow safety range（10-80 mg for adult mortality）,which limits its clinical application.The construction of nanoparticle-based drug delivery systems has the advantages of improving the water solubility of the drug,reducing the toxicity of the drug,and improving the bioavailability.Modification of active targeting molecular ligands on the surface of nanocarriers can improve targeting,enhance efficacy of drug,and reduce toxicity to non-targeting sites.Hyaluronic acid（HA）is a non-toxic targeting ligand that is overexpressed on the surface of cells such as liver cancer,breast cancer,and gastric cancer.Combining CTD with nanoparticle drug delivery system and modifying the active targeting ligand HA on the surface is of great significance for enhancing the targeting of CTD and reducing toxic side effects.Construction of the polylactic acid-glycolic acid copolymer（PLGA）nanoparticles（HA-PLGA-CTD）drug delivery system with CTD which are modified by hyaluronic acid（HA）:PLGA nanoparticles were prepared by emulsification solvent evaporation method,and the principle of electrostatic coupling was utilized.,HA was adsorbed on the surface of PLGA nanoparticles.Single factor study the impact of cetyltrimethylammonium bromide（CTAB）concentration in aqueous phase,PLA（polylactic acid）/PGA（glycolic acid）ratio,PLGA molecular weight,HA molecular weight,HA solution concentration,organic solvent and feed ratio to nanoparticles.The preparation process was optimized by orthogonal design of particle size,potential and encapsulation efficiency.The optimized formulation was:CTAB solution with a concentration of 1.5%,PLA/PGA ratio of 75/25,PLGA molecular weight of7000,HA molecular weight of 6700,HA concentration of 2%,organic solvent of acetone,and feed ratio of 20:1.Quality evaluation of HA-PLGA-CTD nanoparticles:（1）The particle size,PDI and potential of the nanoparticles were determined by a laser particle size analyzer.The average particle size of the nanoparticles was 157.3±7.13 nm,the PDI was 0.194±0.042,and the average potential was-12.7±1.94 mV.（2）The morphology of the nanoparticles was observed by electron transmission electron microscopy（TEM）.The nanoparticles were spherical or spheroidal in uniform size.（3）To investigate the stability of the nanoparticles,the nanoparticles were good stable after 30 days of storage,diluted 100 times,and added with bovine serum albumin（BSA）of 0.9%NaCl and heparin sodium of 200 U/mL.（4）In vitro hemolysis experiments investigated the hemolytic properties of nanoparticles.When the concentration of nanoparticles reached 2 mg/mL,the hemolysis rate was less than 5%,indicated good biocompatibility.（5）The release characteristics of nanoparticles were investigated by in vitro dialysis method.After 1 h,the cumulative release of nanoparticles reached 69.23%.After 24 h,it reached 90.47%,and the release was more complete.In vitro and in vivo anti-tumor studies of HA-PLGA-CTD nanoparticles:（1）Establish a BALB/c mouse liver cancer H22 subcutaneous transplantation tumor model,and divided the mice into model group（normal saline,0.1 mL/10 g）,positive group（cyclophosphamide,20 mg/kg）,CTD prodrug group（400μg/kg）,PLGA-CTD nanoparticle group（non-targeted preparation,400μg/kg）and HA-PLGA-CTD nanoparticle group（targeted preparation,400μg/kg）,tail vein injection,administered every other day.Tumor volume and tumor inhibition rate were calculated,and tumor tissue sections were observed by HE staining.The results showed that the inhibition rates of the positive group,the CTD prodrug group,the PLGA-CTD nanoparticle group and the HA-PLGA-CTD nanoparticle group were 49.83%,47.95%,52.69%,and 64.89%,respectively.The area of tumor necrosis in the tumor tissue of the HA-PLGA-CTD group was significantly larger than that of the CTD original drug group and the PLGA-CTD group.（2）MTT assay was used to investigate the inhibitory effects of CTD,PLGA-CTD nanoparticles and HA-PLGA-CTD nanoparticles on the proliferation of HepG2,MCF-7 and A549.At the 24h,the results showed that the IC50 of CTD drug against HepG2,MCF-7 and A549 were 4.04±0.17μg/mL、3.54±0.08μg/mL、3.28±0.08μg/mL;the IC₅₀ of PLGA-CTD drug against HepG2,MCF-7 and A549 was were2.71±0.03μg/mL、3.22±0.18μg/mL、2.15±0.43μg/mL,respectively.The IC50 of HA-PLGA-CTD nanoparticles to HepG2,MCF-7 and A549 were respectively 2.26±0.03μg/mL、2.43±0.13μg/mL、1.83±0.03μg/mL;the apoptosis of MCF-7 cells was detected by DAPI staining.The results showed that the same concentration of HA-PLGA-CTD nanoparticles was more easy to cause apoptosis of tumor cell.The results showed that the targeted preparation HA-PLGA-CTD nanoparticles could significantly enhance the anti-tumor effect.Preliminary targeting studies of HA-PLGA-CTD nanoparticles:（1）coumarin-6（C-6）PLGA nanoparticles（PLGA-C-6）and HA-PLGA-C-6 nanoparticles were constructed,the results showed that PLGA-C-6 had a particle size of 178.87±8.26 nm and a concentration of8.06±0.03μg/mL.HA-PLGA-C-6 has a particle size of 185.39±3.43 nm and a concentration of 7.37±0.01μg/mL.（2）Using C-6 as a model drug,fluorescence microscopy and fluorescence microplate reader were used to investigate the uptake of HepG2 and MCF-7.At 4 h,the uptake rates of HepG2 to C-6,PLGA-C-6 and HA-PLGA-C-6 were 34.55±1.18%、35.93±1.34%、and 42.70±0.23%respectively;the uptake rates of MCF-7 to C-6,PLGA-C-6 and HA-PLGA-C-6 were 33.51±2.14%、35.15±0.99%、and42.24±5.43%,respectively.The results showed that the uptake rate of tumor cells in each group was time-dependent,and the uptake rate of HA-PLGA-C-6 was higher.（3）HA pretreatment of tumor cells to investigate the mechanism of cell entry.At 2 h,the uptake rates of HepG2 in the HA-PLGA-C-6 group and the hyaluronic acid pretreatment group were46.43±0.08 and31.85±0.98%,respectively;MCF-7 was transparent to the HA-PLGA-C-6 group.The uptake rates of HA-pretreatment group were 37.74±6.27 and 30.38±4.60%,respectively.The uptake rate of MCF-7 to HA-PLGA-CTD nanoparticles was significantly decreased after pretreatment,indicating that the nanoparticles may be affected by the receptor-mediated endocytosis enters.（4）To compare the poison of the original drug and nanoparticles of CTD to the non-targeting organ of the mouse,we had determined the histopathological sections of each organ by calculating the organ index of the mouse,measured the biochemical indexes of the mouse serum,and HE staining.The results showed that the spleen organ index of the HA-PLGA-CTD group was significantly reduced compared with the original drug group and the PLGA-CTD group.Compared with the PLGA-CTD group,the renal index of the HA-PLGA-CTD group was significantly reduced;the pathological section showed that the heart,liver,spleen and kidney tissues of the HA-PLGA-CTD group were alleviated;HA-PLGA-CTD nanoparticles reduced the levels of UA and AST in mouse serum.The results showed that HA-PLGA-CTD nanoparticles reduced the toxic side effects on other organs,indicating that HA modified nanoparticles were targeted.The HA-PLGA-CTD nanoparticles prepared by the emulsification solvent volatilization method have uniform particle size,a negative charge on the surface,and a uniform spherical shape.The stability was good under the conditions of 30 days of storage,50 times dilution,and various factors in the blood.No hemolysis,good biocompatibility.The release is more complete within 24 h.The prepared HA-PLGA-CTD nanoparticles can significantly enhance the anti-tumor effect in vivo and in vitro,reduce damage to the spleen and kidney,and improve the targeting.