| BackgroundCondyloma acuminatum（CA）is caused by human papillomavirus（HPV）and are usually transmitted sexually and it often occurs in the anus and external genitalia.According to reports,in 17 to 25 age group of sexually active person in France rates of detection and high risk HPV infection are the highest,22.1%and 14.7%respectively and the prevalence of CA decreases with age.In our country,in the year of 2003 the incidence rate of CA is 56.66/100000,male cases 429,970 accounting for 58.86%,female cases 300,480 accounting for 41.14%,and the ratio of male and female is 1.43:1.The CA reported accounts for 21.21%of the totale reported sexually transmitted diseases in 2003.The pathogen of warts is human papillomavirus,a small DNA virus.Human is the only host of HPVs in nature.HP Vs infection can cause the proliferation of squamous epithelium in human skin and mucous membranes.At present,HPVs are divided into more than 100 kinds of subtypes by molecular biology techniques,and different types of HPVs infection can cause different clinical manifestations.CA is mainly caused by HPV-6,11,16,18,31,32,33,35,39,42,43,44,51,52,53,54.According to the relationship between HPV and cancer,HP Vs can be further divided into low-risk and high-risk type.Low-risk types mainly include 6,11,42,43,44 and high-risk types mainly include 16,18,31,33,35,39,51,51.A lot of research in recent years has demonstrated that HPV is pathogenic for genital cancers,Especially,the occurrence of cervical intraepithelial lesions（CIN Ⅱ/Ⅲ）and cervical cancer of women associate with HPV infection.There is no monotherapy in genital warts treatment.The major clinical methods for genital warts include local medication,physical methods,surgery,systemic therapy,photodynamic therapy and vaccine therapy emerging in recent years.Those methods are ineffectively and can’t completely clear the HPV infection and prevent the recurrence of genital warts,which cause a heavy financial and psychological burden to patients and the patients’ families,and resulting in negative social impact.The exact treatment of genital warts how to reduce relapse and the prevention of transfer to cancer is the major problem to be solved for the medical community.Podophyllotoxin（POD）is a cytotoxic compound isolated from podophyllotoxin lignans,mainly used for anti-tumor and anti-virus.0.5%POD tincture is one of the most effective first-line drugs recommended by the WHO for the treatment of CA.There is large mucosa irritation when 0.5%POD tincture is used on glans penis,anus,vagina,cervix and other mucosa.The short duration of 0.5%POD tincture action in the vagina make it inefficient for subclinical infection and latent HPV infection therapy.Absorption too much POD of the body may cause serious adverse side effects,including nausea,dizziness,vomiting,diarrhea,abdominal pain,thrombocytopenia reducing,leukopenia,abnormal liver function,ataxia and peripheral nerve palsy that limits its further application in clinical.In order to overcome the limitations of POD application on mucosa,we need to develop novel dosage forms of POD that can be applied to the vagina,cervix and other mucous for the treatment of HPV subclinical infection and latent infection nanopartice drug carrier（NDC）refers to a kind of drug delivery systems that Constituted by drugs and biomedical materials,the particle size is1-1000nn range.,it can improve the physical and chemical properties of drugs,reduce the side effects of drugs,achieve targeted drug delivery,controlled release.Solid lipid nanopart icles（SLN）is a new colloidal drug carrier systems that extensive studied in 1990s,Compared to other carrier systems,it has a higher physical stability,not easy to be degraded,better biological tissue compatibility,targeting,controlled release.But SLN also has its disadvantages including limited drug loading capacity,drug leakage during storage,Water dispersions moisture content is too high.Nanostructured lipid carriers（NLC）is the second-generation lipid nanoparticles developed from solid lipid nanoparticles（SLN）after 2000 year.NLC is a mixture of solid lipid and liquid lipid,and thus it has a unique nano-structure to improve the drug loading.NLC has both the characteristics of SLN and overcome the disadvantage of of the SLN,Meanwhile its mass production and low cost technology has been fully developed,it has advantage in biodegradability and long-term stability.Therefore it is a very promising new drug carrier.The team led by Professor Zeng Kang has long been engaged in the new formulation of podophyllotoxin.we found that POD on nano-drug carriers had good skin targeting and were able to eliminate HPV infection.genital warts of mucous membranes and Latent infection targeted therapy less at home and abroad on the and HPV,but abroad anti-HIV mucosal targeting nano-formulations reported,Therefore,we combine the advantages of the NLC on the basis of the long-term studies of our group,and in the support of the National Natural Science fund and the key projects of Foundation and the Education Department of Guangdong Province,we used modified low temperature curing-emulsion evaporation to preopare drug concentration of 0.5%of podophyllotoxin nano lipid carrier,applied it to VK2/E6E7,We studied the the podophyllotoxin nano lipid carrier the VK2/E6E7 cell proliferation inhibition and apoptosis,so as to open up a new way of widening the podophyllotoxin in the clinical range of applications,provide a reference for basic and clinical research of new nano drug carriers.Object1.In order to expand the scope of application of podophyllotoxin preparations,prepared by the mass concentration of podophyllotoxin 0.5%podophyllotoxin nanolipid carrier,and to inspect the physical and chemical properties of the agents2.To observe the POD-NLC inhibiting the proliferationthe and apoptosis induction of VK2/E6E7Methods1.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 curing-emulsion evaporation method using glycerin monostearate as solid lipid material,caprylic/capric triglyceride as liquid lipid material and poloxamer 188 as mixed surfactant.The sample was characterized by appearance,particle size,encapsulation efficiency and stability:the particle size was measured by Malvern particle size analyzer,the encapsulation efficiency was detected by high performance liquid chromatography（HPLC）and the stability was determined by its appearance.2.The effect of POD-NLC on proliferation and apoptosis of immortalized human Vaginal epithelial cells.（1）The effect of POD-NLC on proliferation of VK2/E6E7Set the POD-NLC concentration gradient for 0.0005ug/ml,0.005ug/ml,0.05ug/ml,0.5ug/ml,5ug/ml,After VK2/E6E7 was treated with POD-NLC for 24h,48h,72hrespectivelyby CCk8 on the inhibition rates,then enzyme-linked immunizing appearance was used to detect absorbance of each hollow at 450 nm wavelength,and calculate inhibition rates.The experiment was repeated three times,at each concentration set 3 complex empty.（2）Different formulations of POD were compared with VK2/E6E7 cell growth inhibitionThe POD and POD-NLC concentration gradients were set to 0.0005,0.005,0.05,and 0.5 μg/mL after treatment of VK2/E6E7 with POD and POD-NLC for 24,48,and 72 h using CCk8.Enzyme-linked immunizing appearance was used to detect the absorbance of each hollow at 450 nm,and inhibition rates were calculated afterwards.The two inhibitory rates were compared against each other.The experiment was repeated three times,and,at each concentration set,three complexes were empty.（3）Different formulations of POD were compared with VK2/E6E7 cell cycle arrestThree conditions of VK2/E6E7 were used in the experiment:in the presence of POD（0.05 ug/mL）,in the presence of POD-NLC（0.05 ug/mL）,and in media alone.The VK2/E6E7 cells were processed for 24 and 48 h and then centrifuged for harvesting.Harvested cells were placed in 70%ethanol,stained by PI,and then subjected to flow cytometry（FCM）.（4）Comparison of effects of different formulations of POD on VK2/E6E7 apoptosisThree conditions of VK2/E6E7 were used in the experiment:in the presence of POD（0.05 ug/mL）,in the presence of POD-NLC（0.05 ug/mL）,and in media alone.VK2/E6E7 cells were processed for 24 and 48 h to observe the morphology of apoptotic cells using an inverted microscope.The cell nuclear morphology of apoptotic cells was observed under a fluorescence microscope after Hoechst33242 staining.Apoptosis was detected using an Annexin V-FITC/PI kit.Cells were stained according to the instructions provided in the kit.Finally,the cells were subjected to FCM.（4）Comparison of different formulations of POD on VK2/E6E7 apoptosisThere are three groups VK2/E6E7 in experiment,respectively in the presence of POD（0.05ug/ml）,POD-NLC（0.05ug/ml）or media alone,processing VK2/E6E7 cells 24h,48h,①to observe the morphology of apoptosis by Inverted microscope ②Observed cell nuclear morphology of apoptotic under fluorescence microscope after Hoechst33242 staining of cells,③to detect poptosis by the Annexin V-FITC/PI kit,according to the kit instructions to stain cells,then detected by flow cytometry（FCM）.StatisticsStatistical software SPSS 13.0 was used to conduct statistical analysis for all the experimental data.The data was interpreted by±S and numbers between the two groups were compared using two independent samples t test（Independent-Samples T Test）.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.Results1.The prepared POD-NLC suspension was homogeneous colloidal dispersion system that translucent light blue opalescent.The morphologies of the prepared POD-NLC observed by scanning electron microscope（SEM）were spherical or ellipsoidal and evenly distributed.the average particle size of prepared POD-NLC suspension were 180 ± 20nm detected by Malvern particle size analysis.the PH was 6.20±0.04 by PH meter.The average encapsulation efficiency was 82.9±2%by HPLC.There were no evident change in appearance,and no stratification,sedimentation and drug crystallization at 4℃ for 6 months.There were no floc sedimentation,stratification in three months at room temperature,but there were a small amount of precipitation stratified after 6 months.All of the samles have no mildew spots.2.POD-NLC significantly inhibited the growth of VK2/E6E7 cells which depended on its concentration and the culture-duration,in vitro.The inhibition effect enhances as the concentration of the POD-NLC increase in the same Condition.under the specific concentration（0.05,0.5 μg/ml）,inhibition effect enhances as prolongation of the exposed time.3.The VK2/E6E7 cells were treated for 48 hours with POD and POD-NLC respectively in the following concentration gradient 0.0005μg/ml,0.005μg/ml,0.05μg/ml,0.5μg/ml and5μg/ml.The inhibition rates of POD and POD-NLC on VK2/E6E7 proliferation were compared at each concentration.There was no difference between POD and POD-NLC group at 0.0005μg/ml and 0.05μg/ml.When the concentration was 0.005μg/ml,the inhibition rates of POD group was significantly higher than POD-NLC group（0.332±0.116%vs.0.104±0.052%P<0.05）.The inhibition rates of POD-NLC groups were significantly higher than POD groups when the concentrations were 0.5μg/ml and 5μg/ml（0.813 ± 0.095%vs 0.632±0.043%,P<0.05;0.993 ± 0.002 vs 0.645±0.048,P<0.01）.The inhibition rates of POD-NLC increased with the treated concentration from 0.0005μg/ml to 5μg/ml,and the inhibition rate was 99%at 5μg/ml.The inhibition rates of POD increased with the treated concentration from 0.0005μg/ml to 0.05μg/ml,but when concentrations were more than 0.05μg/ml the inhibition rates didn’t increase any more.When the treated time of 5μg/ml POD extended from 24 hours to 72 hours,the inhibition rates had no difference statistically and the highest inhibition rate of POD was 68.9%at 5μg/ml for 72 hours.The inhibition rate was 98.4%,when 5μg/ml POD-NLC treated for 24 hours,and the inhibition rate was persistently more than 98%from 24 hours to 72 hours.4.Compared with control,VK2/E6E7 cells treated with POD and POD-NLC for 48 hours were accumulated predominantly in G2/M phases.There was obvious difference that POD-NLC caused more cells arrest in the G2/M phase,compared with POD.POD-NLC had super inhibition of cell cycle activity than POD.5.Both the POD and POD-NLC-treated cells showed apoptotic morphology including cell membrane shrinkage,cell shedding,chromatin condensation,and broken massive nucleus.When detected using Annexin V-FITC apoptosis kit,the apoptosis rate of POD group was increased by 9.48%compared with control,and the POD-NLC group increased by 7.51%.There were no difference between POD and POD-NLC group.Conclusions1.It is able to prepare POD-NLC by emulsification-evaporation.Physicochemical property of POD-NLC corresponded with the requests of NDC.2.The inhibition of POD-NLC on VK2/E6E7 proliferation was in a time and concentration manner.POD-NLC induce VK2/E6E7 apoptosis and arrest at G2/M phase like POD,and POD-NLC had super inhibition of cell cycle activity than POD. |
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