| BackgroundCondyloma acuminatum(CA) belongs to one of the sexually transmitted diseases(STDs), which is frequent in clinic, rating to the secondary position, and human papilloma virus(HPV) is the pathogen of CA, having the characteristic of high relapse rate. Occationally, some of CA might convert into squamous cell caicinoma or the cancer of the cervix, therefore CA is the dangerous disease to human's health, which can't been neglected by us. Podophyllotoxin (PPT) which comes from podophylltom resin is a natural and living material with significant cell toxicity. A count 0.5%PPT tincture is a very effective of medicine which has been recommended by WHO as the first line medicine to controlling CA, however some side effects such as strong skin irritation, the curing effect not lasting long, hard to wipe out dormant infection of HPV, poor skin targeting, easily observed absorbing toxicity and so on are present. Such side effects limit its further application clinically. Solid lipid nanoparticles(SLN) has becoming more and more hot as drug carrier to skin in the recent years, which is thought as the next generation preparation of liposome to carry medicine. Most studies showing that SLN has good physiology compatibility, has good skin targeting, can regulate drug to slow-release or controlled release, can avoid chemical degradation of unsteady medicine, and can reduce side effects such as skin irritation etc. Therefore. SLN has the good development foreground as drug carrier in local application on skin.we used the solid fat material- stearic acid and stearamide with low toxicity and good biocompatibility as drug carrier to adsorb or wrap up PPT, taking Brij78 and soybean lecithinsoybean as composite surfactant, by the method of modified emulsion evaporation in high temperature and solidification in low temperature to prepare SLN suspension containing PPT. But PPT-SLN suspension can't last long in room temperature, some of which developed hydrolytic decomposition, crystallization, floccule deposition. We prepared PPT-SLN freeze drying powder and gel, study its physicochemical characteristics, and study the feature of dermal targeting and slow-release of PPT-SLN (Podophyllotoxin-loaded solid lipid nanoparticles) gel.AIM1. To investigate technological process of PPT-SLN powder and PPT-SLN gel.2. Study the physicochemical characteristics of PPT-SLN powder.3. Study the feature of dermal targeting and slow-release of PPT-SLN gel.METHODS1. using the method of modified emulsion evaporation at a high temperature and solidification at a low temperature to prepare PPT-SLN suspension, then put different cryoprotectors such as 15% trehalose, 15% mannitol and combination of the two ones (each 5%) into the same kind of suspension to prepare PPT-SLN powder, putting carbomer into PPT-SLN suspension to prepare PPT-SLN gel.2. Transmission electron microscope, Image-Pro Plus 6.0 were used to study what PPT-SLN looks like and calculate the particle diameter (trehalose and mannitol have two kinds of condition, that is 2×2 factorial design). High-performance of liquid chromatography was used to calculate the entrapment efficiency. Powder appearance and dissolution, as well as the stability when stored at 4℃were all studied by these techniques to evaluate the effect of different adjuvant on the powder.3. 28 rats of wistar were randomized distribute to 14 groups (that is 2×7 factorial design), and shave two place of abdomen skin of each rat by electric razor, then apply PPT-SLN gel and PPT tincture to Skin of abdomen of wistar rats in the same time. After0.5,1,2,4,8,16,32 h, put rats to death by dislocating certebrae colli, and separate the skin of abdomen contained drug from not, and make rapid frozen section of these skin specimens, without fixing by formalin, then rapidly make tissue pathology frozen section, thickness of which is about 4-6um. By using confocal scanning laser microscopy, we can obtain confocal images and quantify them by assigning every pixel gray scale intensity value which ranges from 0 (black) to 255 (white). By this way we acquired 50 piece of fluorescence intensity values every section, and 200 piece of fluorescence intensity values every group.4. The Acquired data was described by (X|-)±S, making use of SPSS 13.0 software, by the method of factorial design numerical data two-way analysis of variance, and if numerical data between groups have multiple level, use LSD analysis, if there are two kinds of level, use independent sampler t analysis, to dispose the acquired data, comparing whether there are significant difference in these data (size of test is P<0.05).RESULTS1. The prepared suspension of PPT-SLN is translucent and thin milk in color, exhibiting uniform texture and light blue milk-like appearance. The surface of PPT-SLN powder contained 15% trehalose or combination of 5% trehalose and 5% mannitol looked slake and porous, which of PPT-SLN powder contained 15% mannitol looks smooth, dense, cake-shaped. The prepared gel of PPT-SLN looks translucent, light milk color, exhibiting uniform and exquisite texture, and exhibiting slight viscous external appearance.2. PPT-SLN powder contained 15% Trehalose or combination of 5% Trehalose and 5% Mannitol dissolved in water rapidly, about 20 seconds. PPT-SLN powder contained 15% Mannitol was hard to dissolve. Ultrasonic oscillation could be conducted with assistance of external force. When stored at 4℃for 24 hours, 1 month, 3 months and 6 months, the powder samples showed no significant differences of appearance and dissolution. The particles of PPT-SLN powder was round or ellipse, with even distribution. There was no significant difference before and after cryopreservation. Particle diameters of PPT-SLN powder without cryoprotectors and with Trehalose, Mannitol or combination of the two was (82.65±18.43) nm, (94.78±21.94) nm, (114.63±21.42) nm, (109.26±16.15) nm separately. Variance analysis shows that main effect of group of trehalose hasn't statistical significance (F=1.802, P=0.181), and main effect of group of mannitol has statistical significance (F=158.579, P=0.000) , interactive effect has significant difference; t analysis shows that mannitol and the combination one have no statistical significance (t=1.442, P=0.152) , between trehalose and no cryoprotector, there is statistical significance (t=3.560, P=0.001) . The entrapment efficiency of PPT-SLN powder with cryoprotectors and with Trehalose, Mannitol or combination of the two were 87.4%, 86.2%, 79.6%, and 80.3% separately.3. After 0.5,1,2,4,8,16h, the fluorescence intensity of PPT-SLN gel were 10.77±1.71,27.27±2.52,29.26±3.74,40.80±5.88,110.53±18.69,8.50±1.20, comparing means of numerical data exhibit P<0.05, the fluorescence intensity of PPT tincture were 68.32±4.31,91.84±7.52,126.95±13.40,42.79±3.24,14.02±2.11. Variance analysis shows that main effect of group of drug has statistical significance (F=47.269, P=0.000) , main effect of time has statistical significance (F=l 146.565, P=0.000) , interactive effect has significant difference. Interactive effect contour map shows that in 0.5,1,2,4h, the fluorescence intensity of PPT is 6.34,3.36,4.34,1.05 times of that of PPT-SLN gel, PPT-SLN gel distribute slowly, but in 8 h, the fluorescence intensity is 7.88 times of that in tincture, and in 16 h which didn't subside totally. According to this, I prepared scatter plot, which apparently exhibit that in 2 hours PPT tincture concentrates to epidermis mostly, in 4 hours, the density decreases gradually, while in 8 hours PPT-SLN gel concentrates epidermis mostly, and decrease gradually.CONCLUSION1. We prepared PPT-SLN powder and gel successfully, and the appearance of PPT-SLN gel is satisfying.2. Trehalose is a kind of good cryoprotector. The small particle diameter, high entrapment efficiency and good stability of PPT-SLN powder containing 15% trehalose are satisfying, and the preparation process is practical.3. Distribution of PPT-SLN gel and PPT-SLN tincture in epidermis has significant difference, and PPT-SLN gel has the feature of epidermal targeting and can release slowly before 32 hours.
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