Dermatoxicology Study On Solid Lipid Nanoparticles Containing Podophyllotoxin

Background:The condyloma acuminatum(CA) is from the human papilloma virus(HPV)infection and is clinical familiar sexually transmitted disease, withhigh relapse as the characteristic, which is the only next to gonorrheaoccurence and is second currently as the our country disseminationparoxysm, while parts of CA can convert into the scale cancer or the templeneck cancers, therefore the CA have endangered mankind's health and becomethe problem we can't neglect. Podophyllotoxin(PPT) from podophylltumresin is natural and live material with significant cell toxicity. 0.5%PPTtinctura is the most effectively a glimmer of medicine to cure CA as WHOto recommend, while the existence of some disadvantages such as big skinirritation, short function time, hard to wipe out potential infectionto HPV of skin, poor skin target, easily observed absorbing toxicity andso on has limitted its further application in the clinic. The solid lipidnanoparticles(SLN) has become hot point of study on the target medicinecarrier to skin in the recent years, which is thought as the nextgeneration preparation of liposome to carry the medicine. Studies have found SLN has good physiology compatibility, can control the medicineto release, has good target to skin, can avoid chemical degradation ofunsteady medicine of and can reduce skin irritation of medicineetc. Therefore, SLN has the good development foreground as the medicinecarrier to give by skin. Currently, there is still not a complete andsystemic study on dermatoxicology of the concerning PPT at home andabroad, the foundation research of the SLN containing PPT is just juststart, its partial applied safety also did not appear in newspaper athome and abroad. To increase the content of PPT in the skin, to strengthenits target function to pathological changes organize, to prolong itsfunction time to the pathological changes cell, to attain the treatmentresult of raise the CA and reduce the CA to relapse, and to attain thepurpose of its part and whole body poisonous side effect possibly lowerat the same time, we used the solid fat material-stearic acid andstearamide with low toxicity and good biocompatibility as the medicinecarrier to adsorb or wrap up PPT, taking Brij78 and soybeanlecithinsoybean as composite surfactant, using the method of modifiedemulsion evaporation at a high temperature and solidification at a lowtemperature to prepare SLN containing PPT, and carrying out the firststep experimental research on the technological process, the quality andthe safety of SLN containing PPT applied on skins.Purpose:1. To investigate technological process of SLN containing PPT.2. To estimate the quantity of SLN containing PPT.3. To estimate the safety of SLN containing PPT applied on skins.Method:1. According to the single factor and just hand over the experiment to design excellent the prescription, taking the solid fat material-stearic acid and stearamide as the medicine carrier, taking Brij78 andsoybean lecithinsoybean as composite surfactant, we used the method ofmodified emulsion evaporation at a high temperature and solidificationat a low temperature to prepare SLN containing PPT.2. SLN containing PPT's morphology was examined by transmissionmicroscop. Particle-diameter-analysator and High-performance of liquidchromatography were employed to determine the particle size, potentialand trapment efficiency of PPT in the nanoparticles respectively. ThepH value of nanoparticles was measured by PH-meter.3. Choose 220 rats of Wistar and 120 guinea pigs of Fmmu. SLNcontaining 0.5%,0.1%,0.05%PPT and without PPT were prepared using themethod of modified emulsion evaporation at a high temperature andsolidification at a low temperature, at the same time used 0.5%PPTtinctura as control.①To carry out acute cutaneous stimulation test,40 guinea pigs were randomly divided into 10 equal groups: intact skingroups and dermal injury groups with SLN containing 0.05%,0.1%,0.5%PPTand without PPT and 0.5%PPT tinctura. The experimental method:24 hoursbefore the beginning of the experiment, guinea pigs of 10 groups weresubjected to localized depilation of the skin of the back to the leftand right of the spine. Dermal injury was produced by scraping the skin#with the surgical knife blade until oozing of blood began to appear. Anauto-contrast method was adopted in which changes in the skin on bothsides of the spine of the same animal were compared. In guinea pigs of10 groups, the depilated area to the left of the spine was smeared bythe single application with 0.1mL of SLN containing 0.05,0.1%,0.5%PPT and without PPT and 0.5%PPT tinctura respectively while the right partwas left for blank control, subsequently the depilated area was sealedby self-making the single layer plastics thin film and double layer gauzes.Respectively 1, 24 and 48 hours after doing away with the dressing andcleaning medicine, to observe if there were erythema, oedema and so onat local part and instauration circumstance and times of theabove-mentioned variety. To carry out many times cutaneous stimulationtest, another 40 guinea pigs were divided, treated and observed asdescribed above except that medicine and control substances were appliedonce daily for 14 consecutive days.②To carry out acute cutaneoustoxicity test, 110 rats were randomly divided into 11 equal groups: intactskin groups and dermal injury groups with SLN containing 0.5%,0.1%,O.05%PPT and without PPT and 0.5%PPT tinetura and normal controlgroups. The experimental method: After rats of 11 groups were givedlocalized depilation of the skin of the back to the left and right ofthe spine, the depilated area to the spine was smeared by the singleapplication with 1mL of SLN containing 0.05,0.1%,0.5%PPT and withoutPPT and 0.5%PPT tinctura respectively, subsequently observed some indexesof all rats for 14 consecutive days such as body weight of rats, quantityof a meal, breathe daily, physique, eye, central nervous system, armsand legs activity, excrement, death and so on. To carry out long-termcutaneous toxicity test, another 110 rats were divided, treated andobserved as described above except that medicine and control substanceswere applied for 0.1mL once daily for 30 days consecutive days. 24 hoursafter giving the medicine to animals at the end time, all rats wereanesthetized, subsequently taking the blood 3-4mLs of animals to carry on the hematology and blood bio-chemical test, simultaneity to carry onhistopathology examine of skin and confocal laser scanning microscopeto scan with skin slice from the center district of the smeared skin.③To carry out dermal allergy test, 40 guinea pigs were randomly dividedinto 4 equal groups: SLN containing 0.5%PPT group, 0.5%PPT tinctura group,negative control group and positive control group. The experimentalmethod: After guinea pigs of 4 groups were gived localized depilationof the skin of the back to the left and right of the spine, the depilatedarea to the left of the spine was smeared by the single application with0.1mL of SLN containing 0.5%PPT,0.5%PPT tinctura,SLN without PPT,1%2, 4, 2 nitric-chlorine benzene, subsequently the depilated area wassealed by self-making the single layer plastics thin film and double layergauzes. 6 hours after doing away with the dressing and cleaning medicine.7 days and 14 days after this, it was repeated once in same wayrespectively, total for 3 times. 14 days after in the end time takingsensitized medicine, the right part of the depilated area was given with0.1mL of sensitized medicine, 6 hours after cleaning sensitized medicine,instantly observing the dermal allergy reaction, 24, 48 and 72 hours afterobserving the skin reaction again.4. Statistics and analysis: Statistic software SPSS13.0 was used forstatistic analysis. Measurement data were expressed with (?)±S. T testwas used for significant test with two groups. One-way Annova analysiswas used for significant test with 3 groups or more than 3 groups.Differences were significant if P＜0.05.Result:1. The prepared suspension of SLN containing PPT exhibited translucent, thin milk color, even quality and the light blue milk lightexternal appearance.2. PPT-SLN appeared as round shape or ellipse. The mean diameter ofparticles, potential and the entrapment efficiency of SLN containing PPTwere 75.3nm±26.2nm, 23.2mV±3.1mV and 86.4% respectively. PH of theprepared was 4.66±0.18.3. Studied 220 rats and 120 guinea pigs, all enter the result analysis.The result of study on dermatoxicology of SLN containing PPT exhibited:①No irritative dermal reaction was induced by single or repeatedapplications of SLN containing PPT and its matrixs in guinea pigs. Noirritative dermal reaction was induced by single applications of 0.5%PPTtincture in intact skin in guinea pigs. There was obvious irritativederma] reaction in injured skin by single applications of 0.5%PPTtinctura in guinea pigs. There was obviously irritative dermal reactionin intact and injured skin by repeated applications of 0.5%PPT tincturain guinea pigs, Among them stimulating emergence of a guinea pig of damagedskin groups was of the earliest stage and serious.②No acute toxicreaction was showed by single large dose applications of SLN containing0.1%,0.05%PPT and without PPT in rats. Short date of systemic absorbingtoxicity was easily showed by single large dose applications of SLNcontaining 0.5%PPT and 0.5% PPT tincture in rats, which were even obviousin injured skin groups.③No long-term toxic reaction was obviouslyshowed by low dose everday applications of SLN containing PPT and withoutPPT and PPT tincture in rats. A slightly acute inflammatory reaction couldby observed primarily in epidermis histopathology by applications of SLNcontaining 0.5%,0.1%PPT. Among them, inflammatory reaction of 0.5% density group appeared relative earlier: The hair growth obstacle wasshowed by applications of SLN containing 0.5%PPT: A severe whole layerskin inflammatory reaction and obvious hair growth obstacle was showedby applications of 0.5%PPT tinctura.④Medicine was mainly enriched togather in the epidermis layer, hair follicle and its surroundings in ratsof SLN containing PPT groups but medicine content was very few in dermisand subcutaneous tissue. Compared with PPT tinctura, it had better skintarget; The quantity of enriched medicine in each layer of skin in ratswas increased along with the increment of the medicine density.⑤Noallergic dermal reactions was induced by PPT tincture, SLN containingPPT and its matrix in guinea pigs.Conclusion:1. The SLN containing PPT has the craft in brief, the quantity is moreideal.2. The experimental study shows that within the scope of validobservation time and certain density, the SLN containing PPT and PPTtincture all has no serious systemic absorbing toxicity to experimentalanimal, but compared SLN containing PPT with PPT tincture, the formerhas some advantages such as the little partial reaction, light reactiondegree, late adverse reaction, good skin target etc.