Enhanced Photodynamic Killing Effect To Ovarian Cancer Cells Using Folate Receptor-Targeted Nanocarrier System

The morbidity of ovarian cancer ranks number two of gynecologic malignant tumor, and the mortality of ovarian cancer is number one. Ovarian cancer is asymptomatic and lack of detections, which has reached stage Ⅲ-Ⅳ when it is diagnosed. Though the surgery and chemomedicine have been improved, the mortality of late stage ovarian cancer is still high. Therefore, it is hot to seek a new adjuvant therapy to improve treatment results and life quality. Photodynamic therapy (PDT) is a new selective tumor treatment, which has applied to tumor treatment to combine with other methods. The core of PDT is photosensitizers (Ps). The first generation of Ps is mixture with high toxicity, and they changed to pure compounds (Hypocrellin B). The third generation of Ps, which is modified with moleculars, can improve tumor-targeted ability. Nanoparticle technology has been developed quickly in the 21th century. The nanoparticle delivery system can improve effect of drugs and reduce toxicity, which has great advantages compared with traditional methods. Folate receptors are overexpressed on many tumors, which can apply to modifying nanocarriers to achieve targeted treatment. Therefore, this study is based on these developments to prepare nanoparticles to carry Ps. This research will investigate amphiphilic copolymers polyethylene glycol and polylactic acid (PEG-PLA) and PEG-PLA combined with folate (FA-PEGPLA), to prepare PEG-PLA and FA-PEG-PLA nanoparticles entrapping HB and detect their properties. It will also investigate the folate-target effects and lethal effects of FA-PEG-PLA in HO8910 and SKOV3.Part 1 Preparation and detetion of folic acid targeted nanoparticle carriersObjective:To prepare PEG-PLA and FA-PEG-PLA nanoparticles entrapping HB, and to detect their properties.Methods:Using rotary evaporation dialysis method to prepare HB-PEG-PLA and HB-FA-PEG-PLA nanoparticles. Transmission electron microscope and dynamic light scattering were used to measure the diameter of nanoparticles. Two kinds of nanoparticles solution were mixed with equal methanol. The filtered solution was measured with liquid chromatography to obtain encapsulation efficiency and drug loading capacity. Solution was converted to powder using vacuum freeze drying methods. Two kinds of powders were made to store.Results:Two kinds of nanoparticles entrapping HB are made. They are spherical and good dispersible. Their size distributions are consistent. The average particle size of PEG-PLA and FA-PEG-PLA are 156.9 nm and 174.0 nm, respectively. Encapsulation efficiency and drug loading capacity of PEG-PLA and FA-PEG-PLA are96%、1.28%, and 78.1%、1.04%, respectively. Water solution of nanoparticle powders doesn’t destroy their shape.Conclusions:FA-PEG-PLA and PEG-PLA nanoparticles are successfully made. They are stable. Their capsulation efficiency and drug loading capacity are suitable for other study.Part 2 Enhanced Photodynamic Killing Effect to Ovarian Cancer Cells using Folate Receptor-Targeted Nanocarrier System Objective:To investigate target effects of HB-FA-PEG-PLA to two cell lines, and lethal effect of HB-FA-PEG-PLA to two cell lines.Methods:Designing primers of folic acid receptors and using quantitative PCR to detect folate receptor expression of these two cell lines. Nanoparticles were prepared to incubate together with the HO8910 cells in different incubation time and folate concentration to observe the fluorescence intensity using fluorescence microscope. Obtain survival rate of HO8910 of different nanoparticles, light energy and HB concentration using MTT methods. Obtain survival rate of SKOV3 and HO8910 in different nanoparticles and HB concentration using MTT methods. Apoptosis of two cell lines after PDT were detected using flow cytometry after dying Annexin-V-FITC/PI.Results:1. Quantitative PCR showed the expression of folate receptor of HO8910 were significantly higher than those of SKOV3 (P<0.01).2. To some extent, the longer time the two cell lines were incubated with nanoparticles, the higher of intensity of fluorescence (P<0.05). When HO8910 compares with SKOV3, intensity of fluorescence of FA-PEG-PL A is higher in nono-folate(99.104±2.979vs 94.651±2.173,P=0.027),5% folate (107.502±1.992vs 98.427±1.369, P<0.01) and 10% folate (111.038±5.528 vs 102.416±2.493, P=0.013). Group of 10% compared with 5% in HO8910 have no difference (111.0378±5.528 vs 107.502±1.992, P=0.215), which is high in SKOV3 (102.416±2.493 vs 98.427±1.369, P<0.01). The fluorescence intensity doesn’t increase with more folate. Ten percent folate is suit for other study because of high fluorescence intensity.3. After receiving PDT with light enery of 2 J/cm2 and HB concentration of 2 ug/ml, the relative sutvival rate of HB-FA-PEG-PLA is lower than HB (0.222±0.027 vs 0.457±0.030, P<0.01) and HB-PEG-PLA (0.222±0.027 vs 0.344±0.066, P=0.010). This condition is ideal. The relative sutvival rate of HO8910 with HB-FA-PEG-PLA is lower than SKOV3 in concentration of 2 ug/ml (0.113±0.038 vs 0.376±0.091, P<0.001)3 ug/ml (0.086±0.027 vs 0.284±0.099, P=0.022) and 4 ug/ml (0.056±0.011 vs 0.120±0.067, P=0.007). The lethal effects of HB-FA-PEG-PLA are obvious in high folate receptor group.4. The same as HB, the percentage of apoptosis cells of PEG-PLA and FA-PEG-PLA are high, which is the main mode of klling. Early apoptosis of PEG-PLA and FA-PEG-PLA are the same in HO8910 (24.232±1.225 vs 23.324±4.512, P=0.753). In SKOV3, early apoptosis of FA-PEG-PLA are higher than PEG-PLA (17.284±1.935 vs 10.744±1.253, P=0.008). Early apoptosis of HO8910 is higher than SKOV3 in HB-FA-PEG-PLA group (24.232±1.225 vs 17.284±1.935,P=0.006).Conclusion:Folate can achive targeted effets. More nanoparticles can enter cells with folate receptors.The killing ability of FA-PEG-PLA to HO8910 is higher than SKVO3, for high expression of folate receptors. The same as HB, PEG-PLA and FA-PEG-PLA can lead to apoptosis of cells. The effect of HB-FA-PEG-PLA is good to cells with high folate receptors.