Study On Bone Marrow Targeting Polybutylcyanoacrylate Nanoparticles Of All-Trans Retinoic Acid

Objective: To develop a new idea, new method and new technique for bone marrow targeted drug delivery system (BMTDDS). Leucocythemia was a kind of serious malignant tumors of hemopoietic system, commonly called "blood cancer", the features of which were a significant reduction of normal hematopoietic cell, comprehensive of proliferation of leukemia cell in bone marrow and other hematopoiesis tissue, and infiltrating other tissues, leading to exhaustion of normal function of hematopoiesis. Utilized extensively to cure of apoptosis of APL now, ATRA (All-trans-retinoic acid), which is mainly used to cure kinds of dermatosis formerly, has the virtues as high abs remission rate (above 90 percent), low incidence rate of dispersed intravascular coagulation and having no side effect of bone marrow depression and so on. Bone marrow targeting ATRA-PBCA-NP was prepared by modern target-oriented drug delivery technique, in which the biodegradable polybutyl cyanoacrylate (PBCA) was used as carrier and ATRA was selected as a model drug. The surface morphology, diameter and size distribution of nanoparticle were studied. To establish the quality control standard for ATRA-PBCA-NP, the encapsulation efficiency, drug loading capacity, in vitro release and stability were also examined. At the same time, pharmacokinetics and tissue distribution in rabbits and mice were investigated to evaluate its virtues of bone marrow targeting. The mechanisms of BMTDDS were discussed to break through the limitation of treatments for bone marrow diseases and provide scientific theoretical basis for BMTDDS.Methods: The emulsification evaporation method was used to prepare ATRA-PBCA-NP. According to the results of the effects of different variables,the uniform experimental design was applied to optimize the technology of preparation. The properties of nanoparticle as diameter and size distribution were observed by transmission electric microscope (TEM) and laser dispersive analyzing apparatus for granularity. Micro-electrophoresis apparatus was used to measure Zeta electric potential. The encapsulation efficiency, drug loading capacity, drug concentration in blood and tissues were assayed by ultra centrifugation at low temperature. In vitro release of ATRA from nanoparticles was performed by dialysis film method. The pharmacokinetics in rabbits and tissue distribution in mice of the ATRA-PBCA-NP and ATRA injection were investigated.Results: The diameter of the nanoparticles was remarkably determined by the quantity of BCA, PluronicF-127, Dextran-70, pH and stirring duration ,et al; The optimization is as follows: BCA was 0.06ml; PluronicF-127 was 75 mg; Dextran-70 was lOOmg; pH was 1.4; stirring duration was 5h. Three batches of ATRA-PBCA-NP were prepared using the optimized formulation. From the transmission electron microscope observation, the nanoparticles were spherically shaped and ranged in size between 15 and lOOnm. The particle size distribution was 47.7nm. UV spectrophotometry method was established as the quality controlled standard, there was a good linearity of the drug concentration within the range of 0.7-6.2 u g/ml. The linear equation was y=0.1450x+0.0037 (r=0.9999) . The average recovery was 99.3% and the RSD (intra- and inter-day) were less than 2%. The drug loading and incorporationefficiency were 16.12% and 83.67% respectively.The in vitro release behavior of ATRA from nanoparticles could be described by double phase dynamic model and could be expressed by the following equation: 100-R = 42.34-e^*9'+ 58.69?e"011'(ro =0.9961, r, =0.9998). The release mechanism was diffusion. The in vitro release profiles of ATRA stuff could be described by first order dynamic model and could be expressed by the following equation: ln(l00-R) = -2.3532/ + 4.8444 (r= 0.9946) .The freeze-dry injection of ATRA-PBCA-NP was prepared. During storedat 40°C for 3 months, the properties and drug content did not alter. The stability was distinctly affected by light irradiation in 10 days. 60Co radiation had no effect on surface morphology and content of ATRA.HPLC method was established to investigate the drug content and drug distribution in rabbits and mice. The recovery of drug in blood of rabbits was more than 95% and the RSD (intra- and inter-day) were less than 2%. The recovery of drug in tissues of mice was more than 90% and the RSD (intra-and inter-day) were less than 6%. There was a good linearity within the rage of 2~1000ng/ml.The concentration-time courses of ATRA conformed to a single compartment model. The half-life was 0.916h.The concentration-time courses of ATRA-PBCA-NP conformed to a double compartment model. The half-life was 6.636h, which was prolonged remarkably compared with ATRA injection The result indicated that the ATRA-PBCA-NP had a good sustained release efficacy. Compared with ATRA injection, the relative distribution percentage of ATRA-PBCA-NP in bone marrow enhanced from 2.27% to 8.14%.Conclusion: ATRA has remarkable effects in curing of APL, but it has some disadvantages such as first pass effect, short half-life (ti/2=l.l~l .8h) , side effect and drug tolerance. Its physic-chemical characteristic was not stable in the condition of heat or light. The ATRA-PBCA-NP may increase the drug concentration in bone marrow. It also can decrease the dosage, the side effects and improve the therapeutic index (TI). The results of in vitro release and pharmacokinetics in vivo showed that the ATRA-PBCA-NP had a good sustained release efficacy and the half-life of ATRA was prolonged from 0.916h to 6.636h.The tissue distribution results of ATRA-PBCA-NP in rats indicated that the ATRA-PBCA-NP had a remarkable bone marrow targeting efficacy.