| Objective:In order to further evaluate the properties of the PDMP hydrogel composite. Then, the particle size and morphology, drug-loading, entrapment efficiency, cytotoxicity and in vitro drug release profile of MPEG-PCL/PTX micelles were determined. In addition, the prepared PDMP hydrogel composite was analyzed by rheological assay. Subsequently, the A549cell mouse model was established and then treated. Tumor nodules and survival time of tumor-bearing mice were examined. Furthermore, immunohistochemistry of tumor tissues in each group was conducted to confirm the therapeutic effects. Methods:This study is completed in two parts. The first part (in vitro):the preparation of MPEG-PCL copolymers、PECE copolymers、MPEG-PCL/PTX micelles and PDMP hydrogel composite, the drug loading (DL) and encapsulation efficiency (EE) were determined in triplicate by reverse-phase High Performance Liquid Chromatography (RP-HPLC) with a C18column (4.6mm×250mm-5Pm, Grace Analysis column). The particle size distribution of prepared blank micelles and MPEG-PCL/PTX micelles were measured by Malvern Nano-ZS90laser particle size analyzer at25℃. Atomic force microscope (Veeco Multimode Nanoscope Ⅲa controllor) was used to observe the surface morphology of MPEG-PCL/PTX micelles. In vitro drug release behaviors of PTX from MPEG-PCL/PTX micelles were studied by a dialysis method. In vitro cytotoxicity of the samples including blank MPEG-PCL, PTX micelles was determined by standard MTT assays using A549cells. Rheological measurements of the PECE copolymer and PDMP hydrogel composite were carried out by using an AR2000ex rheometer. The second part (in vivo):The48tumor-bearing mice were randomly divided into four groups (each group was12, half of the mice were observed the survival time, the remained for immunohistochemistry):(a) mice treated with100ul normal saline (NS),(b) mice treated with100ul blank thermosensitive hydrogel (blank PECE),(c) mice treated with100ul paclitaxel (5mg/Kg) and cisplatin (2mg/Kg)(free PTX+DDP),(d) mice treated with100ul the PDMP hydrogel composite (PECE/DDP+MPEG-PCL/PTX, PDMP). Drugs were intratumorally injected at the first day. Tumor diameters were measured every3days, and tumor volume was calculated using the formula:Tumor size=(length)×(width)×(width)/2. Half of the animals (n=6) in each group were sacrificed after treatment for10days, tumor tissues were harvested and fixed in4%formaldehyde for immunohistochemistry. And the remained animals in each group were used for further observation. Results:1、According to the obtained results from HPLC measurements, the drug-loading (DL%) and entrapment efficiency (EE%) were of3.96±0.03%and99.18±0.65%, respectively.2、The average particle size of the micelles was20.1nm, and it was monodisperse and had a very narrow particle size distribution. The diameter of the polymeric micelles observed by AFM was in good agreement with the results of particle size analysis.3、The experiments showed only about64%of initial loading drugs amount released from the MPEG-PCL/PTX micelles in14days, which showed a much slower cumulative release rate compared with the free paclitaxel.4、MPEG-PCL/PTX micelles showed well lower cytotoxicity than free paclitaxel when the concentration of PTX micelles was higher than8ug/mL (P<0.05), however, MPEG-PCL/PTX micelles showed the same cytotoxicity with free paclitaxel when the concentration was lower than8ug/mL (P>0.05). The cytotoxicity of MPEG-PCL/PTX micelles increased with the increase of action time (P<0.05), and the cytotoxicity of MPEG-PCL/PTX micelles had the time-dependent behavior. Meanwhile, MPEG-PCL copolymer showed very low cytotoxicity and could be regarded as a safe drug delivery carrier.5、The PDMP hydrogel composite existed obvious thermal sensitivity which was in good agreement with the results of the blank PECE hydrogel.6> the PDMP hydrogel composite group (255.58±25.22) is more effective in suppressing tumors growth compared with the free DDP+PTX group(330.54±25.94, P<0.05), the blank PECE group(513.33±50.96, P<0.05), or NS group(522.70±50.34, P<0.05), whereas the blank PECE group did not show any anti-tumor activity. The median survival time in PDMP hydrogel composite group (53days) is significantly longer compared with the free DDP+PTX group (40days, P<0.05), the blank PECE group (26days, P<0.05), and NS group (25days, P< 0.05).7、The percentage of Ki-67positive cells in PDMP-treated mice group was significantly lower (21.68±4.53%) than that in free DDP+PTX group (40.64±4.37%, P<0.05), blank PECE group (87.16±4.97%, P<0.05), or NS group (89.24±3.96%, P<0.05), respectively. The percentage of MVD area of tumor tissues from PDMP-treated mice group (0.71±0.31%) was dramatically lower compared with free DDP+PTX group (1.78±0.47%, P<0.05), PECE group (4.59±0.59%, P<0.05), or NS group (4.72±0.65%, P<0.05). No significant difference between the NS and blank PECE groups (P>0.05) was observed. Conclusion:1、The PDMP hydrogel composite showed very low cytotoxicity and could be regarded as an ideal safe drug delivery carrier.2、 The MPEG-PCL/PTX micelles showed a much slower cumulative release rate and well drug loading、encapsulation efficiency, and it was monodisperse and had a very narrow particle size distribution. Meanwhile, the cytotoxicity of MPEG-PCL/PTX micelles had the time-dependent behavior.3、The PDMP hydrogel composite existed obvious thermal sensitivity which was in good agreement with the results of the blank PECE hydrogel.4、Compared with free DDP+TX group, PECE group, or NS group, PDMP-treated mice group significantly inhibit tumor growth and prolong survival time of mice. The immunohistochemical results showed the mechanisms of anti-tumor activity may be suppressing cell proliferation and antiangiogenesis. |
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