| BackgroundCondyloma acuminatum（CA）is a sexually transmitted disease caused by human papilloma virus（HPV）which could infect epithelium cells of damaged mucocutaneous by unclean sexual contact.The histology is characterized by papilloma hyperplasia and clinical neoplasm or tumor.The incidence rates of CA is increasing annually,which is one of the most common sexually transmitted diseases（STDs）in our country now.HPV infection is very common not only in China,but in the world.It is reported that the percentages of HPV infection in genitourinary tract for female are 5%-49%.Continuous HPV infection are at increased risk of cervical cancer.With the increasing number of female CA patients,the rate of female cervical HPV infection and the incidence of cervical cancer are increasing year by year.According to the research report,there are approximate half a million new cases of cervical cancer annually in the world and 250,000 people died as a result,while there are approximate 13500 new cases of cervical cancer annually in China and 80000 people died as a result,whose incidence and mortality is in the third of the world,which meets the trend of making the patients more younger.It has been evidenced that persistent infection of cervical mucous membrane with high-risk HPV are closely related to high-grade cervical intraepithelial neoplasia（CIN）and cervical cancer by a great deal clinical epidemiology research and clinical and experimental study.HPV is also responsible for all most squamous cell carcinomas and more than 70%of adenocarcinoma.The problem of HPV infection of the female genital tract has been a hotspot in medical science.There are plenty of options of treating HPV infection.The most ideal treatment for HPV infection is to target the virus or infected cells directly and eradicate the virus from the tissue,but due to lack of effective anti-virus methods and difficulty in complete removal of virus-infected cells,all of the existing treatments have high recurrence rate.Podophyllotoxin（POD）is a natural substance isolated from podophyllotoxin lignans.It has significant cytotoxic activity and is one of the most effective first-line drugs recommended by the WHO for the treatment of CA.The most commonly prescribed forms of POD is tincture clinically.But it also has low permeability on vaginal and cervical mucosa,poor targeting,low adhesion,high toxicity by system absorbing and low effect on latent infection and subclinical infection of HPV.Moreover,it could cause large mucosa irritation,subsequently ulceration and secondary infection after the application of that in local mucosal,therefore its application of HPV infection of cervix and vagina was limited.In order to overcome the limitation that POD cannot applicatted in the genital mucosa,we should develop a new POD formulation.Solid lipid nanoparticles（SLN）is the controlled release system which had lower toxicity and higher compatibility.Potential problems also exist in SLN,such as limited drug-loaded ability and the leaking of drug during storage and high level of water in the aqueous dispersions.Nano lipid carriers（NLC）is the second-generation lipid nanoparticles developed from solid lipid nanoparticles（SLN）in the early 21st century.Unlike SLN,it uses low toxic,good biocompatible,biodegradable lipid blends as the carrier and encapsulates the drug in lipid membrane.So it can control drug release,avoid drug degradation and have good targeting like SLN,but also overcome the problems of SLN such as low drug loading,easy to leak during storage.Therefore it is a very promising new drug carrier.Our preliminary study found that POD on nano-drug carriers had good skin targeting and were able to eliminate HPV infection.Although to the best of our knowledge no research has been conducted on nano POD carrier that used to anti-HPV in the cervical mucos,some people has reported the successful preparation of nano anti-HIV mucosal drug.Combined with above mentioned excellent characteristics of NLC,there is a good opportunity to develop NLC-based drugs for podophyllotoxin mucosal agents in the treatment of female vaginal,cervical HPV-related genital infection and potential applications on tumor.Therefore,with the financial support from National Natural Science Foundation,we used modified low temperature curing-emulsion evaporation to preopare podophyllotoxin nano lipid carriers（POD-NLC）and applied it to the genital mucosa of Tibet Mini-Pig then observe the quantizing analysis on dynamic concentration keeping and absorption of POD-NLC by the confocal laser scanning microscope and High Performance Liquid Chromatographic Analysis.Our research could be an experiment support for the development of new mucosal-targeting drug in the field of medical preparations.This research has very high technological content and great future in application.Object1.Determined the envelopment efficiency（EE）by the method of high velocity centrifuge-High Performance Liquid Chromatography.2.To study the distribution regular pattern of POD in animal mucosa aftert applying POD-NLC suspension and POD tincture on Tibet Mini-Pig cervical mucosa.3.Investigate the change of serum concentrations of POD-NLC aftert applying POD-NLC suspension on Tibet Mini-Pig cervical mucosa.Methods1 Preparation and characterization of Podophyllotoxin nanostructured lipid carriers（POD-NLC）Based on single factor and orthogonal experimental design optimized prescription,the preparation of POD-NLC was carried out via modified low temperature curingemulsion evaporation method using glycerin monostearate as solid lipid material,caprylic/capric triglyceride as liquid lipid material and PF188 as mixed surfactant.The sample was characterized by appearance,particle size,encapsulation efficiency and stability:the particle size was measured by SEM and Zeta-sizer;the encapsulation efficiency was detected by high speed centrifugal-high performance liquid chromatography（HPLC）.2.POD-NLC was experimental group and control group was taken with POD tincture.Two drugs were applied on pig genital mucosa under the principle of random and comparison.Aftetr a regulated time the mucosa samples were taken and made into optical sections.Confocal laser scanning microscopy was used to detected the fluorescence intensity,comparing their different and distribution regular pattern in mucosa.3.Two kind of drugs,POD-NLC and POD tincture,were applied on pig genital mucosa under the principle of random and comparison.The serum concentration of POD was detected by High Performance Liquid Chromatographic.The results were statistically analyzed.4.Statistical software SPSS 13.0 was used to conduct statistical analysis for all the experimental data.The data was interpreted by X±S and numbers between the two groups were compared using two independent samples t test（Independent-Samples T Test）.Factorial design variance analysis was used to analysis the Fluorescence intensity of mucosa.Repetitive measure analysis of variance was used in analyzing serum concentration at different groups.Comparison among multiple groups was using single factor analysis of variance（One-Way ANOVA）.Multiple comparisons were addressed by Bonferroni test and heterogeneity of variance was addressed by Welch and Tamhane’s T2.When P was<0.05,the difference was statistically significant.Results:1.The encapsulation efficiency of POD are analysied by high velocity centrifuge-high performance liquid chromatographic（HPLC）method,and the EE are 84.9%、80.9%and 83.0%repectively.2.Confocal laser scanning microscopy shows that unit area fluorescent amount of epidermal and dermal was highest after 8h and 4h in control group（1465.568/μm2 and 1109.3733/μm2）respectively,and then quick reduced;But unit area fluorescent amount of epidermal and dermal was rather low before 4h in experimental group,and gradually increased after 6h,and was highest after16h and 12h（1001.990μm2 and 587.9933/μm2）.The unit area fluorescent amount of epidermal and dermal were significantly different in all time of the experiment group and control group（P<0.05）,except that in dermal at 8h.Variance analysis shows that main effect of group of drug has statistical significance（F=66.726,P<0.001）,main effect of time has statistical significance（F=885.837,P<0.001）,main effect of position has statistical significance（F=1018.538,P<0.001）.Interactive effect also has significant difference.3.POD serum concentration of test group is always lower than that of experimental groups.The highest POD concentration value of t experimental group is（14.2787 ± 0.3346）μg/ml,and that of control group is（42.4557 ± 0.3202）μg/ml（P<0.001）.The AUC of tincture is 1.3-fold higher than that of experimental group.Conclusion:1.The method was quick,accurate,feasible and applicable.It was suitable to assay the entrapment efficiency of the durg.2.POD-NLC was slowly releasing and can last longer time in mucosa.3.With topical application of POD-NLC suspension,the systemic absorption was significantly lower than that of 0.5%POD tincture,suggesting that the POD-NLC is far safer than the POD tincture. |
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