The Research Of Podophyllotoxin Nanostructured Lipid Carriers-induced Apoptosis Mechanism In VK2/E6E7 Cells Through Caspase-independent Pathway

BackgroundCondyloma acuminatum(CA)is caused by human papillomavirus(HPV)and are usually transmitted sexually and it often occurs in the anus and external genitalia,and also can infect the other parts of the skin and mucous membranes,such as oral cavity,respiratory tract,esophagus.Condyloma acuminatum is popular in worldwide.Epidemiological survey shows that HPV have extremely high infection rate,75%-80%of sexually active population will have a period in his life of HPV infection.A report form USA found that the cervix,vagina HPV infection rate was 43%in 608 female college students.The HPV infection rate is 1.5 to 2.5%annually between 20 to 24 in the European and American countries,while the United States has 20 million HPV infection currently,and more than 5.5 million newly diagnosed patients each year.According to the statistics showed that there were 702821 new sexually transmitted diseases expcet HIV infrction in 2005 in our country,18.86%was CA,in the third place.The incidence of condyloma acuminatum is 25.4/100000 in 1993,73.53/100000 in 1999,which is 2.89 times compared wirh 1993,according to 26 monitoring stations data.The diseases of the south of our cuntry is more than the north;the ratio of male and female is 0.83:1.The pathogen of condyloma acuminatum is human papillomavirus,belonged to papillomaviridae,current research has confirmed that HPV is the most smallest DNA virus.The HPV is spherical,diameter is about spherical,which is 20 cubic symmetry of nuclear shell structure without peplos.The surface of HPV has 72 capsomeres,there are 8000 base pairs(bp)inside,the molecular weight is 5×106D,88%of which is viral protein.HPV DNA genome is double link shape,covalent closed superhelix structure,open loop structure,linear molecules.HPV is highly host and tissue specificity,human is the only host in nature,which can cause squamous epithelial proliferation and even cancer in human body skin and mucosa.The development of modern molecular biology techniques,HPV has been separated to more than 130 kinds of types,infringement of urogenital system has more than 20 type.Different type of HPV infection can cause different clinical manifestations.CA is mainly caused by HPV-6,11,16,18,31,33,35,39,42,43,44,51,52,53,54.HPV.which is associated with cervical cancer and cervical intraepithelial neoplasia called high risk type,including HPV-16,18,31,33,35,39,45,51,52,56,58,59,68,others associated with CA and Benign lesions called low-risk type.,including HPV-6,11,42,43,44.At present a large number of literature material and basic clinical studies have confirmed that the persistent infection of high-risk type HPV is the main reasons of cervical cancer and cervical intraepithelial lesion(CIN Ⅱ/Ⅲ).The main transmission of CA is sexual contact,indirect contact with infections and mother-to-fetus transmission.The incubation period of CA is generally two weeks to eight months,an average of three months.The main clinical manifestation is pink,gray or beige neoplasm,which can be flat,papillary,cockscomb,or cauliflower shape and no self-conscious symptom usually.Use 5%acetic acid solution daub the lesion,it will become white in 3-5 minutes.The pathological section shows parakeratosis,slight hyperkeratosis,the characteristic of CA is papillomatous hyperplasia,highly acanthosis,epidermis taugmentation andelongation,clear vacuoles in upper-middle-class cells.The treatment principle of CA is to remove visible warts try the best,improve the signs and symptoms,and reduce or prevent recurrence.The main treatment including:①Physical therapy(freeze,carbon dioxide laser treatment,microwave treatment,electrical cauterization,ALA-PDT);②local drug delivery(5%Imiquimod,interferon,5-FU.25%-50%TCA,3%Ftiloxazone);③systematic therapy(Cidofovir);④Chinese Medicine;⑤surgery.Although there are so many methods to treat genital warts,the strong infectivity,long incubation period and often recurrence,always seriously affect the patient’s physical and mental health and family life,so the key to cure this disease and reduce recurrence is eradication of HPV infection.The exact treatment of genital warts how to reduce recurrence 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,the WTO recommend it as the first-line drug for the treatment of HPV infection.There is large mucosa irritation,bad targeting property and short time when POD is used on glans penis,anus,vagina,cervix mucosa,so POD is difficult to treat the latent infection of HPV.The more important is POD is easy to absorb the poisoning,which can cause seriously systemic side effects(such as dizziness,nausea,vomiting,diarrhea,abdominal pain,hands and feet anr not flexible,platelet and white blood cells reduced,kidney failure and liver poisoning,the plant nerve disorder,paralytic ileus,peripheral neuropathy,etc.).So WHO does not recommend applying POD to vagina and cervix,and cannot be applied to the skin in large area.In order to overcome these disadvantages of POD,better play to the treatment of HPV,requires us to improve on its dosage forms,make it can apply to the vagina,cervical mucous membrane condyloma acuminatum and HPV subclinical infection and latent infection.In the field of drug delivery systems,nanoparticle size definition between 1-1000 nm,drugs are dispersed,coating,adsorption on the aggregate particles.According to the different preparation methods,can be made into nanospheres and nanocapsules,etc.Due to the superiority of nanoparticles,the nanopartice drug carrier(NDC)has become an important research direction of medicine in the world.The NDC has these advantages like targeted and slow release,increase drug absorption,increase the biological membrane permeability and reduce the side effects of the drugs.Solid lipid nanopart icles(SLN)was a new type of colloidal drug delivery system since 1990s.SLN has these advantages like good biological compatibility,wide adaptability,suitable for a variety of drug delivery approach,with sustained-release and targeted action,improve the stability of the unstable drug,compared with other delivery systems.But SLN also has disadvantages like low drug loadings,too cold,gelation phenomena and drug precipitationNanostructured lipid carriers(NLC)based on SLN has appeared since 2000,which called the second generation of lipid nanoparticles.The different point of NLC and SLN is the internal structure,NLC is a new type of solid lipid nanoparticles,which based on a percentage of the oil liquid or mixed lipid instead of the solid lipid of SLN.NLC solves the disadvantages of SLN like low drug loadings,poor physical stability.NLC not only have higher drug loadings and better physical stability,and have the same sustained-release as solid lipid nanoparticle.So NLC has good prospects for development as a new type drug carrier preparationOur team has been engaged in POD nanopartice drug carrier study in roder to treat condyloma acuminatum since 1992.We synthesized pro epidermoid podophyllotoxin liposome preparations for the first time in 1997.Then we prepared podophyllotoxin-solid lipid nanoparticle(POD-SLN)successfully.Through animal experiments.we found that podophyllotoxi-liposomes has good skin targeted and sustained release,which increases the drug concentration and bioavailability of POD in the epidermis,and greatly reduces the skin irritation and system toxic of POD.Herefore,we combine the advantages of the NLC on the basis of the long-term studies of our group,used NLC as drug carrier,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 research the podophyllotoxin nanostructured lipid carriers-induced apoptosis mechanism in VK2/E6E7 cells,provides theoretical basis for the clinical application of podophyllotoxin nanostructured lipid carriersObjectTo research the POD-NLC induced apoptosis mechanism of VK2/E6E7 cells.1.Preparation 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.2.The study of the POD-NLC nduced apoptosis mechanism of VK2/E6E7 cells(1)The effect of POD-NLC on apoptosis of VK2E6E7 cells.Four conditions of VK2/E6E7 were used in the experiment:in the presence of 0.25μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of 1μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of controlNLC+caspase inhibitor Z-VAD-FMK,and in media alone.The caspase inhibitor Z-VAD-FMK was first processed for 2h and drugs were processed for 24h,used TUNEL.and observed apoptosis morphology under Inverted Flurescence Microscopy,calculate the rate of apoptosis.The experiment was repeated three times.(2)The effect of POD-NLC on ROS of VK2E6E7 cells.Four conditions of VK2/E6E7 were used in the experiment:in the presence of 0.25μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of 1 μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of controlNLC+caspase inhibitor Z-VAD-FMK,and in media alone.The caspase inhibitor Z-VAD-FMK was first processed for 2h and drugs were processed for 24 h.Carried out in accordance with the ROS kit instructions.The experiment was repeated three times.(3)Different formulations of POD were compared with the express of AIFmRNA in VK2/E6E7 cells.Five conditions of VK2/E6E7 were used in the experiment:in the presence of 0.25μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of 1μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of 1μg/mlPOD+caspase inhibitor Z-VAD-FMK,in the presence of controlNLC+caspase inhibitor Z-VAD-FMK,and in media alone.The caspase inhibitor Z-VAD-FMK was first processed for 2h and drugs were processed for 24h.Collected the total mRNA,tested the express of AIFmRNA.The experiment was repeated three times.(4)The effect of POD-NLC on the express of AIF,cyt-C protein of VK2E6E7 cells.Four conditions of VK2/E6E7 were used in the experiment:in the presence of 0.25μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of 1μg/mlPOD-NLC+caspase inhibitor Z-VAD-FMK,in the presence of controlNLC+caspase inhibitor Z-VAD-FMK,and in media alone.The caspase inhibitor Z-VAD-FMK was first processed for 2h and drugs were processed for 24 h.Collected the total protein,used Western blot to test the express of AIF,cyt-C protein.The experiment was repeated three times.StatisticsStatistical software SPSS 13.0 was used to conduct statistical analysis for all the experimental data.The data was interpreted by X±SD and numbers between the each group were compared using single factor analysis of variance(One-Way ANOVA).Multiple comparisons were addressed by LSD test and heterogeneity of variance was addressed by Dunnett’s T3.When P was<0.05,the difference was statistically significant.Results1.The VK2/E6E7 cells under the optical microscopy showed apoptosis morphology like cell membranes shrinkage,the cells became round,chromatin hyperchromatic.TNEL showed that the apoptosis rate in control group(0±0)%,0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(21.725±1.360)%,lug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(44.403±2.163)%,control NLC with caspase inhibitor Z-VAD-FMK group(15.009±1.507)%,the differences was significant between each group(P<0.05).2.The POD-NLC groups could increase the ROS level.The Flow Cytometer results showed that the ROS in control group(97.667±6.028),0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(102162.000±5005.148),lug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(76456.670±2282.075),control NLC with caspase inhibitor Z-VAD-FMK group(45978.000±2940.915),the differences was significant between each group(P<0.05)3.The POD group and POD-NLC group both could increase the express of AIFmRNA,but there was no significant differences between POD group and POD-NLC group.The express of AIF mRNA in control group(1.000±0.080),0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(1.548±0.037),lug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group(1.650±0.073),lug/mlPOD with caspase inhibitor Z-VAD-FMK group(1.6066±0.061),control NLC with caspase inhibitor Z-VAD-FMK group(1.305±0.024).There was significant differences tbetween control group and othere four groups(P<0.05),but 0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group,lug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group and lug/mlPOD with caspase inhibitor Z-VAD-FMK group had no significant differences(P>0.05)The POD-NLC groups could increase the express of AIF,cyt-C.The Western Blot of AIF,cyt-C showed that in control group,0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group,1ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group,control NLC with caspase inhibitor Z-VAD-FMK group were(0.383±0.027)、(0.533±0.030);(0.388±0.026)、(0.595±0.027);(0.217±0.016)、(0.290±0.027);(0.217±0.016)、(0.207±0.023).The differences of the express of cyt-C were significant between each group(P<0.05).But the express of AIF of 0.25ug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group andlug/mlPOD-NLC with caspase inhibitor Z-VAD-FMK group had no significant differences(P>0.05)Conclusions1.Our study based on the previous research,by improving the preparation process,prepared the POD mass concentration of 0.5%POD-NLC,which applied to the VK2/E6E7 cells in vitro with caspase inhibitor Z-VAD-FMK,could increase the ROS level and induce VK2/E6E7 cells apoptosis.2.The POD and POD-NLC both could increase the express of AIFmRNA,1μg/ml POD and POD-NLC has no difference.Different concentrations of POD-NLC could increase the express of AIF,cyt-C.We demonstrate that POD-NLC might induce VK2/E6E7 cells apoptosis through caspase-indePendent Pathway(POD-NLC→VK2/E6E7 cells→increase ROS→decrease MMP→release AIF、cyt-C→cell apoptosis).