| In the study, Triptolide (TP) was chosen as a model, and 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) and PEG-5000 modified to prepare invisible cation liposome (PEG-TRX-TP-LP). The delivery system used different size of glomerular filtration system and highly affinity of TRX-20, with positively charged in physiological pH, TRX-20 specifically binded to chondroitin sulfate proteoglycan which had high expression on the cell surface,it maked the drug delivery have characteristics of kidney targeting. With the help of TRX-20, liposome and cell membrane fusion, which maked the triptolide release into the lesion site and developed its anti-inflammatory effects. This invisible cationic liposome had advantage of easily fused with cells,while, modified with PEG can increase stability in blood circulation.The first part narrated the synthesis and identification of cationic ligand(TRX-20). Using the preparation of Pinner salt to prepare TRX-20, using 3,5-dihydroxybenzonitril with 1-bromopentadecane as material yielded 3,5-dipentadecyloxybenzonitrile. The treatment of 3,5-dipentadecyloxybenzonitrile with Hcl in methanol and chloroform at room temperature followed by ammonia in methanol and chloroform by refluxing gave rise to trx-20 as colorless crystals (yield 86%). During the whole action, using thin layer chromatography track process, the structure identified by MRI.The second part described the characterization and preparation of cationic lipid(PEG-TRX-TP-LP), the preparation used film-ultrasonic using the entrapment efficiency, size, PDI as factors investigated the effects of ratio of phospholipid (HSPC) and cholesterol (Ch), the ratio of drug and total lipids, solubilizing content and loading temperature. Ultimately, the prescription was Ch:HSPC:TP=2:8:1, with 0.2% Pluronic F-68 isotonic buffer solution of PBS, at a temperature of 50 to hydrate and ultrasonic, pressed film to obtain the PEG-TRX-TP-LP.Because of the unstable of the liquid liposome system, the study using appearance, rehydration, particle size, entrapment efficiency as factors of freeze-dried powder injection to invest the different content of freeze-drying protective to protect liposome, finally, we determined in 1% lactose+1% sucrose+1% mannitol as the freeze-drying protective agent for preparing frozen powder injection.In order to evaluate the characteristics of kidney delivery of PEG-TRX-TP-LP in vitro, reference the preparation of PEG-TRX-TP-LP we prepared fluorescence probe liposomes loaded coumarin-6(TRX-C-6-LP)with different content of TRX-20, the entrapment rate was 99%. Evaluating the drug delivery through the primary cultured of MCs. The scanning of microscopy confocal showed that TRX-C-6-LP with 10% TRX-20 had relatively stronger fluorescence and low toxicit, so using 10% TRX-20 to prepare TRX-C-6-LP and evaluating the uptake of TRX-C-6-LP combined with BCA quantitative. The result showed that with the increase of total lipid, the uptake increased, comparing with liposomes control (C-6-LP) the intake of TRX-C-6-LP had statistically significant difference (P< 0.05).The fifth part evaluated the treatment of invisible cation of liposome loaded triptolide (PEG-TRX-TP-LP) in vivo. Applying C-BSA to build glomerulonephritis. Using SPSS combined with pathological examination compared 24 h urinary protein and BUN, Scr, UA, Cys-c in blood. Urinary protein results showed that four weeks later the model group, urine protein reached 17.42±2.43 mg, blank group reached 8.30±1.11 mg (P< 0.05); after the treatment of PEG-TRX-TP-LP urinary protein decreased to 11.18±1.89 mg/24h, while the model control group was 23.98±2.97 mg/24h and the normal group was 8.94±2.19 mg/24h, the TP group still exacerbate the proteinuria and even caused death, each group had significant difference (P< 0.05), The statistic data showed the PEG-TRX-TP-LP could reduce kidney injury, the pathological sections also confirmed this result.All the result clearly indicated the PEG-TRX-TP-LP could accumulate in kidney more and cure glomerulonephritis more efficient through in vitro and in vivo experiment. The PEG-TRX-TP-LP might serve as promising kidney delivery system of anti-inflammatory drug. |
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