| Objective: This study selected paclitaxel as the model drug of insolubility. Anew nanoparticle drug delivery system—paclitaxel nanocrystal was prepared;paclitaxel commercial injection formulation was regarded as a control drug andantitumor effect of paclitaxel nanocrystals were studied in vivo and in vitro; thus, weevaluated the superiority and feasibility of nanocrystal technology applied to improvethe efficacy of tumor chemotherapy.Contents: The formulation and preparation of paclitaxel nanocrystals werestudied and mainly evaluated through index as appearance, particle size and potentialto determine the optimal formulation composition and optimum preparationconditions; paclitaxel nanocrystals were physically characterized through particle sizeanalyzer, transmission electron microscopy, X-ray diffractometer and DSC. The size,morphology and crystalline characteristics of paclitaxel nanocrystals were measuredthrough characterization; the study established a method for the determination ofpaclitaxel content. The solubility and in vitro release characteristics of paclitaxelnanocrystals and paclitaxel material were proceeded as a comparative study toevaluate the nano effect of paclitaxel nanocrystals in such aspects as to improve drugsolubility and release rate; the cytotoxicity experiment preliminarily evaluated theeffect of paclitaxel nanocrystals on killing cancer cells; pharmacokinetics study ofpaclitaxel nanocrystals and commercial injection formulation were proceeded as acomparative study to evaluate the characteristic of two formulations inpharmacokinetics; then, a nude mice tumor model was established to proceed thepharmacodynamics of paclitaxel nanocrystals, using commercial injection formulationas a reference preparation; finally, the tumor targeting of paclitaxel nanocrystals wereevaluated by in vivo imaging technology.Methods: Paclitaxel nanocrystals were prepared by using anti-solventprecipitation. Prescriptions, preparation and freeze-drying process were selected bysingle factor test; particle size, Zeta potential and particle morphology of nanocrystals were determined by dynamic light scattering and the transmission electronmicroscopy; the crystalline characteristics and phase transition temperature ofpaclitaxel nanocrystals was identified through X-ray diffraction and DSC; the contentof paclitaxel was measured by HPLC method; the solubility and in vitro release testsof paclitaxel nanocrystals and paclitaxel material were proceeded by shaker methodand dissolution method; cytotoxicity of paclitaxel nanocrystals and commercialinjection formulation on MCF-7human breast cancer cells were evaluated by MTTassay and compared by drawing cell survival rate curves, then statistical analysis wascarried on; pharmacokinetics characteristics of paclitaxel nanocrystals andcommercial injection formulation were studied by LC-MS method; thetumor-burdened nude mice model was established to evaluate the pharmacodynamicsof paclitaxel nanocrystals and commercial injection formulation by index as tumorinhibition rate, while the tissue distribution of two formulations in tumor-burdenednude mice was evaluated; the paclitaxel nanocrystals and commercial injectionformulation were fluorescence labeled and the targeting efficacy in tumor site wereevaluated by in vivo imaging system.Results: The initial formulation composition of poloxamer407and DOSS weredetermined through single factor test, and the optimized process to remove organicsolvent and most surfactants was vacuum-filtration; paclitaxel nanocrystals thatprepared had a small size, a stable potential and a uniform distribution. The averageparticle size was194.9nm, the PDI was0.138and the Zeta potential value was-29.6mV; the average particle size of paclitaxel nanocrystals lyophilized powder afterredissolved was217.5nm, PDI was0.149and Zeta potential value was-27.3mV,without a significant change in parameters; TEM images showed that paclitaxelnanocrystals had a rod-like morphology with a smooth surface and a uniformdistribution; X-ray diffraction measurement showed that the majority of paclitaxelnanocrystals existed as amorphous structure, only with several kind of shapelycrystalline structure; DSC measurement showed the phase transition temperature ofpaclitaxel nanocrystals decreasing; paclitaxel nanocrystals can effectively improve thedrug solubility and in vitro release rate; the cytotoxicity effect of paclitaxelnanocrystals and commercial injection formulation on MCF-7cells were associatedwith drug concentrations and effect time. Two formulations were significantly inhibited the proliferation of cancer cells.The plasma concentration-time profiles of paclitaxel nanocrystals andcommercial injection formulation in mice were described by a two-compartmentmodel. The t1/2,αwere (2.909±0.067) and (3.696±0.063) min，t1/2,βwere (69.41±0.73)and (53.94±0.62) min，AUC(0-∞)were (276700±960) and (464160±710) μg min/L，CL were (0.036±0.011) and (0.022±0.010) L/(min kg), respectively; the drugconcentration in liver and spleen was significantly increased for paclitaxelnanocrystals comparing with commercial injection formulation (P<0.01), and lower inheart and kidney (P<0.001), but had no significant difference in lung;pharmacodynamic evaluation showed that the drug concentration in tumor wassignificantly increased for paclitaxel nanocrystals comparing with commercialinjection formulation (P<0.01), with more accumulation and a longer time. Antitumorevaluation showed the significant antitumor effect of paclitaxel nanocrystals andcommercial injection formulation at a high dosage. Experiment showed that PTXnano’s tumor weight inhibition rate was85.51%while PTX injection was91.06%;PTX nano’s tumor volume inhibition rate was81.83%while PTX injection was86.55%. Nonetheless, PTX injection had a serious toxicity with a mice mortality rateof83.3%(five deaths at six), while PTX nanocrystals did not cause animal death.Paclitaxel nanocrystals effectively improved the survival condition and raised thesurvival rate in mice; fluorescence imaging showed that the retention time ofcommercial injection formulation in nude mice was short, and the main accumulationsites were heart, lung, kidney, and bladder, without obvious fluorescence at tumor.However, the retention time of paclitaxel nanocrystals in nude mice was longer,especially at and near the tumor site had a large amount of accumulation, and couldmaintained a high concentration for a long time.Conclusions：The average particle size of paclitaxel nanocrystal prepared byanti-solvent precipitation was~200nm, which had a rod-like morphology and a stablepotential. Various parameters of paclitaxel nanocrystals lyophilized powder afterredissolved did not have a significant change; after prepared into nanocrystals, drugmolecule was transformed from shapely crystalline structure into amorphous structure,with a decreased phase transition temperature; paclitaxel nanocrystals had greatlyincreased the solubility and in vitro release rate; paclitaxel nanocrystals had strong cytotoxicity effect, which could significantly inhibited the cancer cell growth;paclitaxel nanocrystals could rapidly distributed into the surrounding tissue andmainly absorbed by the liver and spleen, and effectively reduced the toxicity in heartand kidney, and increased the accumulation in tumor tissue; simultaneously, paclitaxelnanocrystals could effectively restrained the growth of the tumor with a significanteffect, which had clinical significance to decrease the side effects.In summary, paclitaxel nanocrystals delivery system constructed by nanotechnology had good tumor targeting, which had a large amount of accumulation atthe tumor site and had a significant antitumor effect, while reducing the side effects ofcommercial injection formulation. Overall, paclitaxel nanocrystals are feasible newformulations used in clinical tumor treatment, which has research significance inclinic. |
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