Novel Formulations Of Camptothecins:Study On The Relationships Among Preparation Methods,Particle Microstructures And Properties

Camptothecin(CPT) and 10-hydroxycamptothecin(HCPT) are natural pentacyclic alkaloids isolated from extracts of the Chinese tree Camptotheca acuminate. Although numerous studies show that CPT and its analogues(CPTs) are the most potential agents against a wide spectrum of human cancer, therapeutic applications of unmodified CPTs are hindered by their poor solubility, instability and unexpected toxicity. To overcome the limitations of CPTs in clinical therapy, the development of various formulations is gaining increasing attention. In this work, novel formulations of CPT and HCPT were prepared by appropriate preparation methods and using suitable carrier materials, where great efforts were focused on establishing the relationships among preparation methods, particle microstructures and properties. And the influence mechanism of operating parameters, particle formation mechanism, modification mechanism of carrier materials, drug release and targeting mechanism were investigated in detail. The major contents are as follows:(1) Micronized formulation. Recrystallization and micronization of CPT was conducted using supercritical anti-solvent(SAS) technology, where the influence of solvents on particle microparticles was discussed in detail by combining the solvent properties with the physical phenomena involved in the SAS process. The processed CPT microparticles were flake-like, and much thinner and more uniform than the lamelliform CPT raw material. Solvents with higher the ratio of density and viscosity, lower surface tension and lower solvation power are prior to form smaller CPT microparticles with lower crystallinity, which help to improve the solubility of CPT in biological liquids. Furthermore, the addition of ethanol into dimethyl sulfoxide(DMSO) improves the properties of DMSO and decreases the CPT solubility, which is benefit to obtain smaller particles, and the particle size decreased significantly with the increase of ethanol volume ratio(R). The chemical structure and crystalline structure of CPT had no change after the SAS process. However, the crystallinity of obtained CPT microparticles was less than that of raw CPT, and varied for different solvents. Results of the solubility study in vitro proved that the solubility of CPT could be effectively increased by the SAS micronization, especially CPT particles obtained by using chloroform and ethanol/DMSO(R ? 0.5). But the results of solvent residue analysis indicated that chloroform was less suitable than ethanol/DMSO mixtures for micronizing CPT.(2) Liposomal formulation. SAS technology was used to produce HCPT proliposomes(HCPT/PL), where the effects of different variables on HCPT/PL formation were investigated using an OA25(55) orthogonal experimental design. The formulations were evaluated quantitatively using an overall desirability function. SEM and TEM images showed that spherical or clavate HCPT/PL were obtained under different processing conditions, and the drug loading(DL) has a significant effect on the morphologies. Under the optimized conditions, clavate HCPT/PL with mass median diameter(Dp50) = 209.8 ± 38.4 nm, DL = 5.33% and encapsulation efficiency(EE) = 85.28% were obtained. For the optimized HCPT/PL, the residual DCM meets the ICH requirement, and part of the encapsulated HCPT still maintains its crystalline state. It also showed that HCPT/PL sustained the HCPT release rate successfully, where the drug release of the optimized HCPT/PL followed the first order kinetics, and the drug diffusion mainly corresponded to a Fickian diffusion mechanism during the first 10 hour.(3) HCPT nanocrystals loaded zein(HCPT NC/Zein) microspheres. Zein, a group of alcohol-soluble proteins extracted from corn gluten meal, was selected as the carrier material for CPTs delivery. A novel built-in ultrasonic dialysis process(BUDP) was established to overcome the difficulties associated with existing techniques for self-assembly of zein microspheres and fulfilling the encapsulation of CPTs. Results indicated that zein microspheres with controllable particle size and narrow size distribution were self-assembled using BUDP. The formation mechanisms of zein microspheres and the influence mechanisms of the operating parameters were discussed in detail, where a semi-empirical model was developed for a system capable of precise microsphere formation and scaling up BUDP. After that, HCPT NC/Zein microspheres were obtained by combining the SAS process with BUDP, where the effects of the process parameters and the formation mechanism of HCPT NC/Zein microspheres were discussed in detail. Under the optimized conditions, HCPT NC/Zein microspheres with Dp50 = 1.10 ± 0.12 μm, DL = 5.98% and EE = 95.68% were obtained. The further characterizations demonstrated that HCPT NC was successfully incorporated into the interior of zein microspheres. Furthermore, it is presented that HCPT NC/Zein microspheres sustained HCPT release rate successfully, where HCPT fast released in the first 20 h, then the release trend followed zero order kinetics and reached 70% in 82 h.(4) Folate receptor-mediated targeting formulation. Zein was conjugated with folic acid(FA) to prepare nanoparticles for targeted delivery of CPTs. The reaction process was monitoring by the on-line ATR-UV spectrophotometer, where the absorption peak of FA at 367 nm was disappeared and a new peak around 342 nm was generated gradually during the activation reaction. Then the activated FA was conjugated with the amino groups of zein by amidation reaction, where the ATR-UV characteristic peak at 342 nm almost did not change. The conjugation degree was quantified by UV-VIS spectrophotometer. Successful conjugation was further evidenced by FI-TR and 1H-NMR. FA-Zein nanoparticles were prepared by BUDP, results indicated that FA conjugation facilitated the formation of small nanoparticles with good dispersibility and stability, where particle size varied from 820.2±195.0 nm to 356.8±68.7 nm, and zeta potential ranged from-24.8 ± 4.22 mV to-40.5 ± 6.00 mV, which depended on the conjugation degree. Cell uptake studies were carried out in folate receptor positive Hela cells and negative A549 cells, the results showed that FA conjugation resulted in little increase in cellular uptake by A549 cells but large increase in Hela cells. HCPT NC loaded FA-Zein nanoparticles were prepared by combining the SAS process with BUDP, the results showed that smaller particle size, better stability and drug release behaviors than those with nonconjugated zein nanoparticles. In vitro cytotoxicity test indicated that HCPT NC loaded FA-Zein nanoparticles had obvious cytotoxicity for both Hela cells and A549 cells after 48 h, which had greater potency than free HCPT. These results suggest that FA-Zein is a potential carrier material for encapsulation and enhanced delivery of anticancer drugs.