Study On High Lung-Targeting Delivery System Of New Liposome Carrier

BackgroundThe high incidence of lung diseases such as lung cancer and tuberculosis is increasing in china. Our country has launched a lot of manpower and material resource into treatment of these intractable diseases. Though there are a series of important progression in the field of medicine therapy, the medicine results in high dosage, long period, serious side effects and drug tolerance because of the lack of target distribution in vivo. Lung-targeting delivery system is the ideal treatment strategies for lung diseases. Among the lung-targeting drug carrier, Liposomes, as a little toxic and well biocompatible carrier, more and more attention is being paid to them because they have many advantages such as sustained release drug, increasing tumor targeting, increasing pharmaceutical stability and improving Pharmacokinetics. But its research level, manufacture technique and industrialization need further improvement. Therefore, the key technology of liposome carrier delivery system research and industrialization are broken through, lung-targeting delivery liposome carrier system of lung cancer and tuberculosis drug and so on is developed, which are very necessary.ObjectiveThis study takes lung-targeting delivery system for treatment of non-small cell lung cancer and actual problem solved urgently in production as cut in point. Docetaxel (DTX) was chosen as model drug to prepare DTX-LP, and carried to lung through lung-targeting liposome carrier delivery system to improve therapy index and reduce side effects. Furthermore, the two key science problems of the bottleneck problem of production of liposome preparation and lung-targeting effect are effectively solved.Method1. A combination of solid dispersion and effervescent technology was used to prepare DTX-LP for the first time. Concretely speaking, first, the solid dispersion technology was used to prepare DTX proliposomes. Second, DTX-LP were obtained from DTX proliposomes by effervescent technology2. The morphology of liposome was observed by the light microscopy and transmission microscopy.3. The particle size and zeta-potential were determined by Malvern ZEN3600.4. HPLC analysis method was established and validated for analysis of DTX-LP preparation.5. Entrapment efficiency and the vitro release property of DTX-LP were studied by dialysis method.6. The significant influence factors on DTX-LP were studied using single factor method with particle size and morphology of liposome as index.7. The formulation and preparation of DTX-LP was optimized and initial determined using orthogonal design with particle size distribution, entrapment efficiency and stability as marker.8. HPLC analysis method was established and validated for determination of DTX in biosamples.9. On the basis of the initial optimized formulation and preparation, DTX-LP with various surface charges and particle size were prepared, and then the distribution of DTX was determined after intravenous administration of these liposomal DTX at 1.5h in rabbits to study lung-targeting effect and determined the best formulation and preparation.10. The vivo distribution and Pharmacokinetics of liposomal DTX were studied using pharmacokinetic method.11. Lung-targeting effect of liposomal DTX was evaluated with five targeting parameters as index.12. The toxity after intravenous administration of single-dose and series multiple dose of liposomal and injectable DTX was studied in rabbits.13. The transplantation tumor model of nude mice bearing A549 lung cancer was established to study inhibition effect of liposomal DTX on tumorResult一,Pharmacy study1. Formulation and preparation study: According to single factor, citric acid and sodium bicarbonate, surface temperature of solution on particle distribution, stability and entrapment efficiency was much significant. The initial optimized formulation and preparation was obtained using orthogonal design. The resulted DTX proliposomes were powder and (or) granules. The resulted DTX-LP were round in shape and dispersed well. Zeta-potential, particle size range and entrapment efficiency were about -23.4 mv, 0.90μm and 90.5%, respectively.2. Determination of the best formulation and preparation: On the basis of initial study, the best formulation and preparation was further determined with lung-targeting effect as marker by determination of vivo distribution of DTX-LP with various particle size and surface charges. The certain optimized formulation and preparation as follows: DTX, HSPC, cholesterol, tween-80 and citric acid were dissolved in ethanol with ultrasound instrument at 45℃. Then the solution was filtered through a 0.22μm hydrophobic membrane and placed in a round bottom flask. Under a constant stirring at 55℃, Sodium bicarbonate was added to evaporate organic to obtain solid granules (DTX proliposomes). Appropriate DTX proliposomes (1g) were hydrated under the effervescence produced by introduction of 5% sodium bicarbonate solution to obtain DTX-LP.3. DTX-LP quality study: The study of quality of samples prepared by the best formulation and preparation showed that the DTX proliposomes had good flow property and were completely hydrated with 5% sodium bicarbonate solution for 15min. The pH value of the resulted DTX-LP ranged from 5.2 to 5.8. The liposomes were round and dispersed well. The particle size, zeta-potential and entrapment efficiency were 0.95±0.12μm, -23±0.21mv and 90.57±0.32%. The in vitro release study showed sustained release behavior. The sample was stable for at least one year at 2-8℃in sealed containers in a dark place.二,Pharmacology and toxicity studies1. Analysis of vivo biosamples: The selectivity of the HPLC conditions is good, sample preparation method is appropriate, no significant interference peaks was observed. The recoveries were 80.9-113.4% and the precision values (expressed as RSD) of intra-and inter were below 10% for these biosamples. The extraction recoveries were more than 70%. The HPLC method could meet biosamples analysis.2. Lung-targeting effect study: The effect of liposomal DTX with various mean diameter and zeta-potential on biodistribution in vivo showed that drug concentration in lung for negatively charged DTX-LP of about same particle size was much higher than that of possively charged DTX-LP. Furthermore, DTX-LP of about 1μm diameter with negative surface charge of greater than 20mv showed the highest tissue distribution of DTX in lung.3. Tissue distribution and lung-targeting evaluation: The result of distribution in rabbits showed that drug concentration of DTX-LP in lung was markedly higher than that of DTX injection at different time points.As far as targeting parameters are concerned, the Re value of lung in the case of the liposomal DTX was 28.91, which was much higher than that of other tissues and blood. It showed that the liposome carrier greatly increase lung-targeting effect. When the Te and the ratio of Te_(liposomes) to Te_(injection) of lung were 1.00, compared with liver, spleen, kidney, the Te value of lung increased by a factor of 4.96, 8.51, 18.37, respectively, and the ratio of Te increased by a factor of 3.16, 23.0, 27.83, respectively. It showed that the liposome carrier markedly enhance selectivity to lung. The value of Ce in lung increased by a factor of 74.28 compared with DTX injection in the case of DTX-LP, however, the Ce value in liver, spleen and kidney only increased by the factors of 2.93, 1.09 and 1.41, respectively. Therefore, it was known that the liposome carrier changed completely DTX biodistribution in vivo in rabbits; furthermore, the drug was greatly concentrated in lung. The TCe value in lung was 63.41 in the case of liposomal DTX, which was higher than that of other tissues. The distribution ratio of DTX in lung was 6-folds greater than that of injectable DTX in rabbits. It can be seen from TQe value that only 10.445% of total DTX content was distributed in lung for DIX injection, however, 52.746% of total DTX content was concentrated in lung for liposomal DTX. It was helpful to treat lung cancer and had clinical application.4. Pharmacokinetic study: In blood, after intravenous administration of liposomal and injectable DTX in rabbit, three-compartment model gave the best fit to the plasma drug concentration time curves. AUC_(0-t) was 0.893and 2.130mg/L^*h, respectively. The t_1/2α and t_1/2β value of injectable DTX were 0.122 and 0.207h, respectively. However, the parameter of t_1/2α (0.023h) of liposomal DTX was shortened, which showed that the DTX-LP could rapidly distribute to targeting organ-lung, and the parameter of t_1/2β (2.186h) was longer, which showed that DTX-LP could prolong drug action. In lung, the kinetic parameter of liposomal and injectable DTX showed that drug concentration-time curve fitted to a two-compartment model and three-compartment model,respectively. AUC_(0-t) was 2428.65 and 84.00mg/L^*h,respectively.The t_1/2α value were 1.414 and 0.081h, and t_1/2β value were 8.808 and 3.481h, respectively.It can be seen from these results that significant difference was observed in kinetic parameter of liposomal DTX in lungs and in plasma. For lung-targeting DTX-LP, the pharmacokinetic parameter in blood could not reflect pharmacokinetic behavior in target organ-lung.5. Toxicity study: survive time of DTX-LP group was about one time higher than that of DTX injection group. On the other hand, only DTX injection group, significant change in pathological biopsy of various tissues was observed. The result showed that DTX-LP could reduce toxicity of chemotherapeutic drug.6. Pharmacodynamic study: The result of pharmacodynamics showed that the inhibition rate to A549 lung cancer of injectable DTX (12 mg/kg) and liposomal DTX (3, 6, 12 mg/kg) were 75.72%, 61.48%, 94.40% and 95.40%, respectively.Conclusion1. In this study, a combination of solid dispersion and effervescent technology was used to prepare DTX-LP successfully for the first time. This technology overcame bottleneck problems of liposome such as small product batch and quality instability and so on, which showed good foreground of industrialization.2. The new viewpoint of negatively charged liposome of about 1μm diameter showed the greatest lung-targeting effect was reported for the first time3. DTX-LP prepared in this study was efficient lung-targeting delivery carrier and could completely changed pharmacokinetic behavior of DTX to decrease dosage and improve therapeutic index, and reduce side effects, which are important for clinical application.4. The study is totally new without any report at home and abroad up to now and has our own independent intellectual property. The research will be helpful to promote development of proliposome preparation products and provide a more efficient drug delivery system for treatment of lung cancer, tuberculosis and other lung disease.