Preparation And Physiochemical Characterization Of Sodium Bicarbonate Sustained-release Embolism Microspheres

Hepatocellular carcinoma(HCC)is a very common malignancy in the world with high mortality.Embolotherapy of hepatocellular carcinoma is safe and reliable because its operation is simple and less traumatic.A large number of lactic acid in tumor can make a slightly acidic microenvironment which influences embolization efficacy,tumor metastasis and regeneration.Some studies have shown that in the process of embolization,simultaneous administration of sodium bicarbonate injection,could effectively improve the tumor inhibition rate.In this study,ethyl cellulose was used as the microsphere material,and the antitumor drug doxorubicin and sodium bicarbonate were loaded into the ethyl cellulose microspheres by the solid-in-oil-in-oil(S/O/O)emulsification-solvent evaporation method.Combination of embolization,chemotherapy and tumor microenvironment improvement can significantly improve the efficacy of embolotherapy and effectively inhibit tumor growth,metastasis and regeneration.Ethyl cellulose was used as the microsphere material,and sodium bicarbonate microspheres(NaHCO3-MS)were prepared by the solid-in-oil-in-oil emulsification-solvent evaporation method.First,a sodium bicarbonate aqueous solution was injected into an ethyl cellulose solution in acetonitrile by a solvent diffusion method to prepare sodium bicarbonate nanosuspensions.Sodium bicarbonate nanosuspensions and soybean oil were used as internal oil phase and external oil phase for the preparation of sodium bicarbonate microspheres through S/O/O emulsification-solvent evaporation method.The effects of inner oil phase surfactants,internal and external oil phase volume ratio and emulsification temperature on the morphology,average particle size and distribution,drug content and in vitro release behavior of sodium bicarbonate microspheres were investigated.The results showed that the sodium bicarbonate microspheres prepared by using Tween 80 as internal oil phase surfactant,internal and external oil phase volume ratio of 1/9 and emulsification temperature of 25℃ had higher drug content.The average particle size was about 340μm and sodium bicarbonate could be released for more than 48 hours.Based on this preparation process,doxorubicin/sodium bicarbonate microspheres(DOX/NaHCO3-MS)were prepared.DOX/NaHCO3-MS had a number average particle size of 252 μm,a sodium bicarbonate content of 4.3%and a doxorubicin content of 0.6%.Sodium bicarbonate could be released for more than 72 hours,and doxorubicin could be released for more than 28 days.The same method was used to prepare doxorubicin microspheres(DOX-MS).The number average particle size of DOX-MS was about 230μm and the doxorubicin content was 0.6%.Doxorubicin could be released for more than 28 days.Rabbit hepatoma cell line VX2 and human hepatoma cell line Bel-7402 were used as model cells,and the cytotoxicity of the microsphere material was examined by tetrazolium blue colorimetry.Using NaHCO3-MS and DOX-MS as a contrast,tetrazolium blue colorimetric assay and viable cell staining were used to determine the cytotoxicity of DOX/NaHCO3-MS.Effect of DOX/NaHCO3-MS on cell culture pH was investigated simultaneously.The results showed that the viability of cells incubated with microsphere material at a concentration of 50 mg/mL was above 90%,indicating that the microsphere material was almost non-toxic to VX2 cells and Bel-7402 cells.NaHCO3-MS and DOX/NaHCO3-MS co-incubated with tumor cells,the pH of the culture medium increased to about 8.3.The effect of DOX-MS on cell culture pH was not obvious.In addition,NaHCO3-MS,DOX-MS and DOX/NaHCO3-MS had inhibition on the proliferation of tumor cells,in which DOX/NaHCO3-MS showed the strongest inhibitory effect.Rabbit hepatoma cell line VX2 was used as a model cell.The effect of lactic acid on cell viability in low glucose environment was investigated by tetrazolium blue colorimetric assay.The effect of lactic acid on the apoptosis of tumor cells in low glucose environment was examined by Annexin V-PI double staining.The results showed that:when the concentration of glucose in culture medium was 3mM,the survival rate of tumor cells after 48 hours was 55%;after adding 20mM lactic acid,the survival rate of tumor cells increased to 80%.In addition,lactic acid can also inhibit tumor cells apoptosis in a low glucose environment,suggesting that lactic acid can counteract the tumor cells from the death caused by glucose deficiency.Rabbit hepatoma cell line VX2 was used as a model cell.Using NaHCO3-MS and DOX-MS as a contrast,the effects of DOX/NaHCO3-MS on tumor cell survival rate,apoptosis and pH of culture medium were investigated in an environment containing 3mM glucose and 20mM lactic acid.The results showed that DOX/NaHCO3-MS could release sodium bicarbonate to neutralize lactic acid,and the pH of the cell culture increased to about 7.5,which showed the strongest inhibitory effect on the proliferation of tumor cells.In addition,Annexin V-PI double staining and JC-1 test results showed that compared with NaHCO3-MS and DOX-MS,DOX/NaHCO3-MS showed the strongest promotion effect to apoptosis and necrosis of tumor cells after co-incubation with tumor cells.In summary,the doxorubicin/sodium bicarbonate microspheres(DOX/NaHCO3-MS)prepared by the solid-in-oil-in-oil(S/O/O)emulsification-solvent evaporation method had an average particle size of about 250μm and could effectively encapsulate doxorubicin and sodium bicarbonate.DOX/NaHCO3-MS could achieve sustained release of doxorubicin and sodium bicarbonate,effectively improve the slightly acidic microenvironment in tumor caused by high expression of lactic acid,promote tumor cell apoptosis and effectively inhibit tumor cell proliferation.DOX/NaHCO3-MS is expected to be applied to safe and efficient embolotherapy of hepatocellular carcinoma.