| Objective This study selected 7-ethyl-10-hydroxycamptothecin(SN-38)as the model of insoluble drug.Nanocrystal technology was used to increase the solubility of insoluble drugs.SN-38 nanocrystals were prepared and studied in vivo and in vitro,and the influence of particle size on behavior of nanocrystals was investigated.Thus,we evaluated the superiority and feasibility of nanocrystal technology applied to improve the efficacy of tumor chemotherapy.Methods The particle size and the physical stability at room temperature were used as evaluation indexs to determine the dosage of stabilizer.The influence of homogeneous pressure and cycles on the particle size of nanocrystals were investigated.Then,two SN-38 nanocrystals with different particle size were prepared by high pressure homogenization.The particle size,polydispersity index(PDI),Zeta potential,morphology and crystalline state of two SN-38 nanocrystals were determined via dynamic light scattering(DLS),transmission electron microscopy(TEM)and X-ray powder diffraction(XRPD),respectively.The in vitro release behavior of two SN-38 nanocrystals,SN-38 solution and physical mixture were evaluated by dialysis bag.Cellular uptake of two SN-38 nanocrystals of human fibrosarcoma cells HT1080 was evaluated by confocal laser scanning microscope(CLSM)when SN-38 solution was regarded as control.Cytotoxicity of two SN-38 nanocrystals and solution on MCF-7,HT1080 and Hep G-2 human cancer cells were evaluated by MTT assay.Pharmacokinetics of two SN-38 nanocrystals and solution in SD rats were evaluated.MCF-7 bearing mice were used to study antitumor efficacy and tissue distribution of two SN-38 nanocrystals and solution.Results Two SN-38 nanocrystals(SN-38/NCs-A and SN-38/NCs-B)were prepared via high pressure homogenization using different procedures.The mean size of SN-38/NCs-A and SN-38/NCs-B were(229.5±1.99)nm and(799.2±14.44)nm respectively.PDI values were 0.141±0.015 for SN-38/NCs-A and 0.202±0.067 for SN-38/NCs-B.Zeta potential of SN-38/NCs-A and SN-38/NCs-B were(-27.1±0.36)m V and(-27.6±0.21)m V respectively.Under TEM,SN-38/NCs-A and SN-38/NCs-B were both cone-shaped.The results of XRPD demonstrated that before and after the nanosizing process,the crystalline state of SN-38/NCsA and SN-38/NCs-B were not changed.The dissolution test showed that the dissolution velocity of SN-38 in vitro was improved significantly compared to the physical mixture,the dissolution rate of SN-38/NCs-A was significantly faster than that of SN-38/NCs-B;SN-38 nanocrystals had a lower dissolution rate when compared with solution.Cellular uptake studies showed that after 12 h,the amount of SN-38 in cells of SN-38/NCs-A group was much more than that of SN-38/NCs-B and solution group.Moreover,in SN-38/NCs-A group the drug was mainly distributed in cell nucleus.The amount of SN-38 in cells of SN-38/NCs-B and solution group was quite low and the difference was not significant,and the drug of the two groups were not observed in the cell nucleus.MTT assay showed that SN-38/NCs-A exhibited significant inhibition on MCF-7,HT1080 and Hep G-2 cells when compared with SN-38/NCs-B and SN-38 solution;SN-38/NCs-B was more potent against MCF-7 and Hep G-2 cells than solution,but the cytotoxicity of SN-38/NCs-B and SN-38 solution on HT1080 cells was not statistically different.Pharmacokinetic study in SD rats showed that SN-38/NCs-A could significantly increase the areas under the curve(AUC)of SN-38,decrease the clearance(CL),improve the mean residence time(MRT),and increase the bioavailability markedly;SN-38/NCs-B with larger particle size showed a different pharmacokinetic character with the largest CL and the smallest AUC.Antitumor efficacy study in MCF-7 bearing mice showed that the antitumor effect of SN-38/NCs-A was the strongest when compared with SN-38/NCs-B and SN-38 solution,and SN-38/NCs-B was more effective in inhibiting the tumor growth thanSN-38 solution.Besides,two SN-38 nanocrystals both did not exhibit obvious side effect.The tissue distribution study showed that although the amount of SN-38 accumulated in liver and spleen of the mice treated by SN-38/NCs-A and SN-38/NCs-B was significantly higher than that treated by solution,the two nanocrystals could markedly improve the drug accumulation in tumor tissue compared with SN-38 solution,and the amount of SN-38 in tumors of SN-38/NCs-A group was much more than that of SN-38/NCs-B group.Conclusions The average size of SN-38/NCs-A and SN-38/NCs-B were 230 nm and 800 nm respectively,and the crystalline state of SN-38 was preserved in the preparation process.Compared with SN-38 solution,nanocrystals could significantly improve the release rate,enhance the cellular uptake and cytotoxicity,increase the bioavailability and drug accumulation in tumor tissue,and had markedly antitumor efficacy with negligible side effect.It was obvious that particle size showed marked influence on the behavior of SN-38 nanocrystals.Compared to the 800 nm nanocrystals,the 230 nm nanocrystals exhibited higher cellular uptake and cytotoxicity,more favourable pharmacokinetics,more drug accumulation in tumor tissue and better tumor inhibition efficiency. |
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