| BackgroundThe application of nanotechnology for disease diagnosis,treatment,and prevention is now often referred to as nanomedicine by the National Institutes of Health.Research into the targeted pharmaceutical nanocarriers is at the forefront of projects in nanomedicine.When a drug is suitably encapsulated within the carriers or deposited in subsurface,in nanoparticulate form,it can be specifically delivered to the selected target site,released in a controlled way and protected from undergoing premature degradation.This results in higher therapeutic efficacy and dramatically minimizes systemic side effects and toxicity.Solid lipid nanoparticles（SLN） is thought as the next generation on the targeted durg delivery nanosystems with solid lipid as durg carriers,diameters ranging between 50 and1000 nanometers,which has the advantages of high physical stability, slow speed of durg leakage and low toxicity etc.Thus,SLN is a pormising durg delivery system due to its good physiology compatibility,controlled release,easy,and reasonably cheap to prepare;and specifically accumulate in required sites in the body.Genital warts（condyloma acuminatum,CA） are one of the most frequent sexually transmitted diseases worldwide,occurring at incidence rates of 0.6%-1.2%in men and women aged 20-24 years.They represent overt clinical infection with human papillomavirus（HPV）.It is estimated that 20 million Americans are currently infected with HPV,and more than 5.5 million new cases are diagnosed annually.Epidemiologic studies suggest that 75%of all sexually active people will become infected with HPV at some point during their lifetimes.Moreover,HPV infection is associated with cervical cancer,anal cancer,ulvar cancers etc.A wide variety of treatments are in use,but failure of treatment and recurrence after initial clearance are seen with all treatments.Podophyllotoxin is a lignan,naturally occurring in roots and rhizomes of Podophyllum emodi Wall var. Podophyllotoxin itself is used in the treatment of genital warts by WHO,but its application is restrained due to its high toxicity and side effect on the human body.Due to their unique size-dependent properties,solid lipid nanoparticles are expected to offer the possibility to improve new dosage form of podophyllin and develop more effective therapy for genital warts and related diseases.Therefore,under the financial assistance provided by guangdong provinceal science and technology programme,podophyllotoxin-loaded solid lipid nanoparticles were prepared by a modified solvent evaporation method and then proliferation and apoptosis of human keratinocyte treated by POD-SLN were investigated to provide new materials in the nanometer range with many potential applications in clinical medicine and research.Purpose1.To develop the formulation and characterize the physicochemical properties of podophyllotoxin-loaded solid lipid nanoparticles（POD-SLN）.2.To study the effects of POD-SLN on proliferation and apoptosis of human keratinocyte and observe the cellular uptake of POD-SLN in human keratinocyte.Method1.Podophyllotoxin-loaded SLNs preparation and characterization.Based on the optimized results of single-factor and orthogonal design, podophyllotoxin-loaded SLNs were prepared by a modified solvent evaporation method and freeze-drying procedure.The morphology was examined by transmission electron microscope,particle size and zeta potential,drug entrapment efficiency of POD were determined by Particle-diameter-analysator and High-performance of liquid chromatography respectively,and long-term physical stability of the SLNs were investigated in detail.2.The methods of culturing human keratinocyte in vitro and its biological feature Keratinocytes isolated from healthy human foreskin were incubated in a humidified 5%carbon dioxide atmosphere at 37℃.Cool digestion and dermal scratching were used to improve the method of culture.KCs were then identified by their morphological features under light microscope,transmission electron microscope and immuno-histochemistry.3.Effects of POD-SLN on proliferation,apoptosis and the cellular uptake of human keratinocytes in vitro.（1） Cells were grown in a humidified 5%carbon dioxide atmosphere at 37℃on a 96-well microplate,with each well containing 4000 cells immersed in 100uL of 10% fetal bovine serum,and 1%penicillin/streptomycin.The cells were allowed to adhere for overnight.Then the cells were incubated with POD-SLN at various concentrations （0.1-1000μg/L） for 6h、12h、24h、48h.Following incubation,20uL MTT labeling mixture was added,to each well.The microplate was incubated for a further 4h.A Benchmark microplate reader with a 570nm optical filter was utilized to measure the absorbance of each well.（2） Cells were grown in a humidified 5%carbon dioxide atmosphere at 37℃on a 96-well microplate,with each well containing 4000 cells immersed in 100uL of 10% fetal bovine serum,and 1%penicillin/streptomycin.The cells were allowed to adhere for overnight.The cells were then incubated with free POD and POD-SLN at various concentrations（0.1-1000μg/L） for 48h.A colorimetric MTT assay system was utilized to determine the proliferation of the free PODand POD-SLN on human keratinocytes.（3） The cells were incubated with free POD,blank SLN or POD-SLN for 24h,the percentage changes of apoptotic cells and cell cycle were examined by flow cytometry（FCM）,respectively.（4） Morphological changes of human keratinocytes treated by free POD,blank SLN or POD-SLN at the concentration of 10μg/L for 24h were observed with transmission electron microscope.（5） The cells were incubated with free POD,blank SLN or POD-SLN at the concentration of 10μg/L for 24h,apoptosis of cells were examined by invert fluorescence microscope. （6） Cell uptake of free POD and POD delivered by solid lipid nanoparticles was confirmed using confocal laser scanning microscopy（CLSM）.4.Statistics and analysis:Statistic software SPSS10.0 was used for statistic analysis. Measurement data were expressed with（?）±S.Independent—Samples T test was used for significant test with two groups.One-way Annova analysis and Repeated measure was used for significant test with 3 groups or more than 3 groups,Bonferroni test was used when the variance was homogeneity and Tamhane’sT2 was used when the variance was not homogeneity.Differences were significant if P≤0.05.Result1.The prepared suspension of SLN containing POD exhibited translucent,even quality and the light milk external appearance.The formulated SLNs were found to be relatively uniform in size 87.2±10.29nm,with zeta potential 25.27±0.78mv.The average drug entrapment efficiency of POD in the nanoparticles was 83.24%±2.44%. The mean particle sizes and zeta potential of POD-loaded SLN after re-dissolving of the freeze-dried powders were 113.58±5.40nm and 23.73±1.92mv respectively. POD-SLN displayed spherical or elliptical in shape and was stable.2.A large amount of human keratinocytes could be obtained after 7 days,KCs displayed typical morphology when observed by phase-contrast microscopy,electron microscope,and immunohistochemical staining.3.After treated by different concentrations（0.1-1000μg/L） POD-SLN for 6,12,24 and 48 h respectively,there were obvious changes of human keratinocytes proliferation in a concentration-and time-dependent manner.Blank SLN had no effect on the proliferation of human KCs.4.After treated by different concentrations（0.1-1000μg/L） POD-SLN or POD for 48 h, there were obvious changes of human keratinocytes proliferation.At the same concentration, the effect of POD-SLN on anti-proliferation was better than that of POD.5.Both POD-SLN and POD could significantly induce the apoptosis of human kerat-inocyte. After treated with 10μg/L POD-SLN or POD for 24h,the total apoptotic rates was 81.40%±8.01%and 59.65%±5.24%,respectively.Cell cycle analysis revealed that both POD-SLN and POD blocked human keratinocytes in G2-M phase. 6.After treated with 10μg/L POD-SLN or POD for 24h,human keratinocytes showed typically morphological changes of apoptotic phase.7 Confocal laser canning microscopy showed that POD as free drug or bond to SLN was accumulated as red staining in the cell nucleus of human keratinocyte.Conclusion1.This formulation and technology are stable and practical.,the quantity is more ideal.2.POD-SLN can significantly inhibit the proliferation of human keratinocytes in a concentration-and time-dependent manner and induce the apoptosis of human keratinocytes in vitro,the inhibitory effect was better than that of POD.3.The effect of POD-SLN on anti-proliferation and induce the apoptosis of human keratinocytes was better than that of POD.POD as free drug or bond to SLN was accumulated as red staining in the cell nucleus of human keratinocyte. |
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