Study On Polyrotaxane-Camptothecin Conjugate (Prodrug) Drug Delivery System

Cancer is one of the disease that greatly threatened human's health, people who pursuit in medicine is endeavoring to find a specific medicine to cure cancer. It is reported by the World Health Organization that patients who suffered from cancer is increasing greatly year by year. The number of cancer patients is about 1.2 million and more than 900 thousand died of cancer every year in China. Meanwhile, the pharmacotherapy of cancer is progressing rapidly and new anti-cancer drugs is emerging prominently. The anti-cancer effect of Camptothecin,Paclitaxel and Vitamin A were believed to be the most important discovery in the 90's. Camptothecin (CPT) is a natural anti-tumor alkaloid isolated from Chinese tree Camptotheca acuminate which is widly distributed in the yangtze river,southern sichuan,guizhou and yunnan area.The anti-cancer effect of natural Camptothecin is remarkable, and the clinical therapeutic efficacy of CPT on digestive tract tumor,leukemia,bladder carcinoma,ovarial cancer is very strong, but it can caused severe side effects such as bone marrow depression,vomiting and hemuresis. Camptothecin is hard to dissolve in water and ester therefore it's unsuitable to prepare eligible formulation and further restrain it's application in clinical. Considering the disadvantages of CPT and it's derivatives, new dosage forms such as prodrug,microsphere,liposomes is emerging to increase the solubility,stabillity of lactonic ring and to prolong the blood circulation.A supramolecule is a system of two or more molecular entities held together and organized by means of intermolecular non-covalent binding interactions. The studies on supramolecular structures involving macrocycles have been a fascinating research area because they not only serve as models for understanding natural supramolecular self-assembly and molecular recognition, but also provide precursors for designing novel nanomaterials for electronics, and biomedical and pharmaceutical applications.Recently, CD-based polyrotaxanes and polypseudorotaxanes, the inclusion complexes composed of multiple CD rings threaded on a polymer chain with or without bulky end caps, have led to interesting developments of supramolecular biomaterials such as hydrogels and biodegradable polyrotaxanes. These biomaterials could be used for controlled drug delivery based on mechanisms which are different from the conventional systems. Over the last decade, there have been a lot of developments of drug delivery systems based on CD-based polyrotaxanes, which were used in nasal drug delivery, in peptide and protein delivery, in ophthalmic drug delivery, and in many other areas.As new drug carriers, polyrotaxanes shows it's unique feature:1. Polyrotaxanes were composed by CD and linetype PEG, and they were both water soluble,safe and without side-effect。2. The abundant hydroxyl group in CD can be actived, and then linked to the drug to form the stable compound of polyplex.3. The terminal moieties of polyrotaxanes are first degraded to generate the release of the drug-immobilized a-CDs. These drug-immobilized a-CDs may act as a CD carrier. The covalent bonds between the drug and the a-CDs are then subjected to hydrolysis to release the active drugs to enter the nucleus.In a word, the supramolecular structure and good biocompatibility make it as a novel drug delivery system in the future.The clinical limitations of anticancer drugs are often reflected in their low specific choices of tumor cells, thus lead to severe damage to normal cells. Therefore, an ideal delivery system should target the tissue,organ or cells after administration, and maintain the blood concentration for a certain time to play a pharmacodynamics. Searching for highly effective,low toxicity formulation is becoming more and more important in clinical.The targeted delivery mediated by folic acid/folate receptor is getting more and more concern in recent years because it is founded that folate receptor express in most of the cell membrane of malignant tumor but little in normal cells. folic acid is useful in targeting tumor cells and posses of strong abilities to binding the folate receptor, so that it can be efficiently mediated to tumor cells.Therefore, we proposed the ideas of preparing polyrotaxane-camptothecin conjugate drug delivery system to improve the solubility and drug loading of CPT.The folice acid were further linked with PR carrier to increase the selectivity of tumor tissue and reduce the side effects in clinical.The main frame of polyrotaxane-camptothecin conjugate were composed of a-cyclodextrin and PEG-BA (4000). folic acid/Tyr were used as bulky end caps to blocked the two ends of PEG. Then actived the abundant hydroxyl group of CD with SA, and linked to CPT to form the polyrotaxane-camptothecin conjugate, the structure were confirmed by infrared spectrum,1H NMR.The release of CPT by hydrolysis from the PR-CPT conjugates were also examined in vitro in phosphate buffer (pH 7.4). As data shows, a constant release profile was observed without significant burst release of the drug in 3,6,12,24 hours. The cumulative percentage release rate were about 80% after120 hours. Dissolution data were then examed by SPSS 13.0 software to find the best dissolution curve model. The results showed that CUB is the best dissolution curve model.The content determinne were preform with HPLC method:Acetonitrile:water (30:70); flow rate lml/min; detection wavelength 254nm; injection volume 10μl. The retention time of CPT is 14.5 min. Solubility test showed that the polyrotaxane-camptothecin conjugate were increased by 7 times comparing with CPT.Content determination showed good linearty between 0.017～17μg, detect wavelength 254nm, and the samples were completely separated. Drug loading is about 8%, the same as reported in the literature.MTT methord were used to examine the cytotoxity effect of PR-CPT conjugates against ovarian cancer A2780 cells in vitro. The growth inhibition rate and IC50 showed dose-effect relationship between the inibition rate and concentration. The IC50 of CPT,FA-PR-CPT and Tyr-PR-CPT were 5.2,34.8 and 97.8mg/L seperately. The IC50 of FA-PR-CPT is about 2.8 times lower than Tyr-PR-CPT, suggesting that FA-PR-CPT might be specific targeting to tumor cells.The cells' proliferation affected by polyrotaxane-camptothecin conjugates was observed by microscope. The result showed that Camptothecin-polyrotaxane conjugates had significant effect on ovarian cancer cell line A2780 in a dose-dependent relationship; The cells' apoptosis after treatment with polyrotaxane-camptothecin conjugates was observed by FCM. The result showed that the anti-tumor effect of Camptothecin-polyrotaxane conjugates may be achieved by inducing cells'apoptosis.In vitro cytotoxicity against A549 human lung adenocarcinoma cell line were performed. The IC50 of CPT,FA-PR-CPT and Tyr-PR-CPT were 4.9,33.9 and 39.5mg/L, and showed no significant different between FA-PR-CPT and Tyr-PR-CPT. This result suggested folate conjugate may be targeting tumor tissue through folate receptor mediated.Acute toxicity test showed that after administration,100% mortality were founded in the maximum dose 5mg/10g, and zero death in minimum dose 5mgx0.74/10g. LD50 of PR-CPT conjugate were 2.30 mg/10g and 2.45 mg/10g.Anti-tumor effect of PR-CPT conjugate against S180 mice invivo showed significent effect when comparing with control group. Pathology results indicated it's anti-tumor mechanism may be through reducing the blood supply to tumor site to cause cell necrosis.HCPT were used as internal standard in pharmacokinetics test, Chromatographic conditions as the mobile phase:Acetonitrile:1% triethylamine (35:65), flow rate lml/min, inject volume:20μl. The blood concentration-time curve and Pharmacokinetic parameters showed significent increasing AUC, extending the half life and reducing the elimination rate of PR-CPT conjugate.