| Doxorubicin hydrochloride (DOX) is valid for various cancer types, while its clinical application is seriously restrained by its severe cardiotoxicity and spinal toxicity, and this is one of the reasons why DOX is a second-line theraputic drug for cancer despite its strong potency. The aim of this thesis was to increase the therapeutic effect and decrease the toxic and side-effect of DOX by using phamaceutical techniques, and to prepare bevacizumab-modified doxorubicin hydrochloride-loaded albumin nanoparticles which has the function of active tumor-targeting and long circulation. Vascular endothelial growth factor (VEGF) are overexpressed on tumor cells, accordingly bevacizumab which has special affinity to VEGF was employed as an active-targeting preparation’s target head. Specified crosslinger, NHS-(PEG) n-MAL (which has a molecular weight of about 3500), was adopted to link bevacizumab to drug-loaded albumin naoparticles using the method of chemical modifying.The main methods, content and conclusions are as followed.UV-vis spectrophotography was applied to analyze the in vitro content of doxorubicin. The linear regression equation showed good in the range of 0 to 40μg/mL. The precision and accuracy were consistent with the requisition of methodology. DOX was soluble in water.The method of desolvation, stablizing-crosslinking was adopted to prepare DOX-loaded albumin nanoparticles. With partially measuring the particle size, monofactorial experiments were performed, on the basis of which the central composite design-response surface methodology (CCD-RSM) was then employed in order to optimize the preparation of DOX-loaded albumin nanoparticles. The optimized formula and techniques were as followed, albumin concentration of 17mg/ml, amount of DOX of 2mg/ml, pH value is 9 and percentage of albumin theoretic crosslinking of 125%. Under this condition, it gave rise to the actual average value of mean particle size (151±0.43 nm), zeta potential (-18.8±0.21 mV), drug loading efficiency (21.4±0.70%) and drug entrapment efficiency (76.9±0.21%).0.2% phosphotungstic acid dyeing showed the appearence of Dox-A-Nps was uniform, sphericity or sphericity-like and was stable in the condition of 4℃. The behavior of drug release of prepared Dox-A-Nps was also studied, which showed a burst in the initial stage, then a stable and delayed release.Chemical modification was used to prepare bevacizumab-modified Dox-A-Nps, and CCD-RSM was also applied to optimize the corresponding mono-antibody mediated albumin nanoparticles. The method of determination of drug loading and entrapment efficiency was established. The amount of 2-Iminothiolane hydrochloride, weight ratio of Dox-A-Nps and bevacizumab, and the amount of heterobifunctional crosslinkers, NHS-(PEG) n-MAL were studied, with the particle size, drug loading and entrapment efficiency as evaluation index. The optimal formula were 50μg of 2-Iminothiolane hydrochloride,27.5 mg/mg of weight ratio of Dox-A-Nps and bevacizumab, and 8.8 mg of NHS-(PEG) n-MAL. The prepared immuno-nanoparticles had particle size of (216.1±2.31) nm, drug loading of (28.93±0.94) % and entrapment of (80.39±2.83)%. ELISA kit was used to detect the activity of bevacizumab, before and after the chemical crosslinking,which showed a good preservation. Bevacizumab\modified Dox-A-Nps were stable in the circumstance of 4℃, and had a delayed and stale release in the study of in-vitro drug-release behavior.Fluorescence spectrophotometry was applied to determine the amount of DOX in rats’ plasma. The pharmacokinetics of DOX solution, Dox-A-Nps and Bevacizumab-modified Dox-A-Nps in rats was investigated in the aid of WinNonlin software. Compared with that of DOX solution, the tl/2β, AUC of Dox-A-Nps and Bevacizumab-modified Dox-A-Nps were all prolonged or increased.The model of compartment was a two-compartment one.Fluorescence spectrophotometry was also employed in the study of distribution of DOX in mice. The results showed that the formulation group could decrease the distribution of DOX in mice’s heart, which reveald it could indirectly decrease the toxic and side effects, and increase the therapeutic effect of DOX.MTT method was used to study the inhibitory rate (IR) of different preparations on MCF-7 cell line. The IR of Bevacizumab-modified Dox-A-Nps was higher than the other group when the amount of DOX was the same. Different weight ratio of Bevacizumab and Nps had different IR, in which the ratio of 1:3 gave rise to the highest IR. Flow cytometry was chosen to evaluate apoptosis made by different preparations. The results showed that Bevacizumab-modified Dox-A-Nps could induce more late-apoptosis. Preliminary research of pharmacodynamics of drug-loaded albumin immuno-nanoparticles has been done. Model of BALB/C-nu nude mice bearing MCF-7 cell was established. Tumor volume growth curve, tumor weight percent and tumor growth inhibition rate were studied in the existence of saline (as control), Bevacizumab alone, Dox-A-Nps and Bevacizumab-modified Dox-A-Nps. Except saline, the rest groups all showed valid to the nude mice bearing tumor, in which Bevacizumab-modified Dox-A-Nps had a best therapeutic effect. |
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