| Nano-materials and technology developed rapidly in recent years. its development in the field of medicine diagnosis and treatment of diseases was important, especially in the field of anti-tumor study. It’s development in clinical treatment of cancer has a broad and very attractive prospect. PAMAM dendrimers used as a drug delivery system was non-toxic, no-immunogenic and biodegradable. PAMAM dendrimers have shown great advantages as carriers for gene delivery and anticancer therapies.In order to enhance the tumor site targetting ability andmaintain the dendrimer inherent affinity with the tumor cells, an pH-sensitive PAMAM complexes modified with lactose were prepared with PAMAM dendrimers and LA-PEG-b-PSD in this paper.Lactose -poly (ethylene glycol)-poly (methacryloyl sulfadimethoxine) diblock copolymer (LA-PEG-b-PSD) were synthesized to produce a novel pH sensitive polymer with a mono-galactoside moiety. The chemical structure was characterized by IR, NMR and GPC. The solubility in water of LA-PEG-b-PSD was investigated, the solubility of PSD was 10μg/mL.An increase in the solubility was observed in LA-PEG-b-PSD, and this increase was affected by the the molecular weight of PEG. Moreover, when the pH of the solution higher than 7.0, the solubility f LA-PEG-b-PSD increased significantly. The apparent pKa values of SD, PSD, PEG-b-PSD and LA-PEG-b-PSD were investigated by potentiometric titration. the pKa of PSD, PEG-b-PSD and LA- PEG-b-PSD was about 6.97,7.04 and 6.95, respectively, which is close to the acidic conditions at the tumor sites.The LA-PEG-b-PSD/PAMAM complexes were prepared with LA-PEG-b-PSD carrying a negative charge and PAMAM dendrimers carrying a positive charge. The DOX was encapsulated in PAMAM by a hydrophobic interaction. The effect ofζpotential, charge ratio (+/-), molar ratio and ionic strength on the preparation of LA-PEG-b-PSD/PAMAM complexes was studied. The morphology of LA-PEG-b-PSD/PAMAM complexes was observed using TEM and the particle size was 50nm. The drug release study in vitro indicated that the release behavior was affected by the pH value.The MTT assay showed that the blank LA- PEG-b-PSD/PAMAM complexes have lower cytotoxicity against HepG2 cells and Hela cells. The hemolysis experiments using rabbit red blood cells indicated that all the formulations were not hemolysis.The LA- PEG-b-PSD/PAMAM/DOX complexes showed higher cytotoxicity against HepG2 cells. Their growth-inhibiting activity was higher than PEG-b-PSD/PAMAM/DOX complexes and PAMAM/DOX at pH 7.4 after 48h incubation, and this advantage was not observed at pH 6.5. The flow cytometry and the confocal laser scanning microscopy were also used to study the pH sensitive and targeting ability of LA- PEG-b-PSD/PAMAM/DOX complexes, the results indivated that the lactose moiety and PSD can enhance the liver tumor cells targetting ability of LA- PEG-b-PSD/PAMAM/DOX complexes. To study the intracellular kineties behavior,the HPLC methord was established to determine the concentration of DOX in HepG2 cells.Using H22 tumor-bearing mice, the anti-tumor efficacy of DOX in different formulations was evaluated. All of the treatment groups produced a significant anti-tumor effect compared with the physiological saline control group (P< 0.05). The treatment with LA-PEG-b-PSD/PAMAM/DOX complexes and PEG-b-PSD/PAMAM/DOX complexes inhibited primary tumor growth compared with other control formulations, the relative tumor inhibition was 80.87% and 66.07% separately.The results indicated that the pH sensitive complexes exhibited better antitumor activity than PAMAM/DOX group and PEG-PAMAM group, furthermore, the LA-PEG-b-PSD/PAMAM/DOX complexes showed higher anti-tumor efficacy than non-lactose PEG-b-PSD/PAMAM/DOX complexes. The pathology of the tumor was investigated to anti-tumor effect of drugs. The expression level of the caspase-8 and caspase-3 was measured by biochemical kits, the results show that the contents of caspase-8 and caspase-3 in the tumor of LA-PEG-b-PSD/PAMAM/DOX complexes group were higher.To test the targetability in KM mice bearing H22 cells, LA-PEG-b-PSD/PAMAM/DOX complexes was prepared. It was found that the DOX distribution was changed after treated with LA-PEG-b-PSD/PAMAM/DOX complexes. The DOX concentration in tumor treated with LA-PEG-b-PSD/PAMAM/DOX complexes was significantly greater than PEG-PAMAM/DOX, the ratio of Te was 2.91. Results suggested the targetability was enhanced when using LA-PEG-b-PSD/PAMAM/DOX complexes. The pharmacokinetics parameters of LA-PEG-b-PSD/PAMAM/DOX complexes and other formulations were studied in wastar rat. For LA-PEG-b-PSD/PAMAM/DOX complexes, PEG-b-PSD/PAMAM/DOX complexes, PEG-PAMAM/DOX, PAMAM/DOX and DOX, the t1/2 value were 4.59±1.68 h,6.86±1.09 h,8.66±1.78 h, 2.31±0.74 h,2.67±1.42h; AUC0-24为271.34±52.29μg·h/mL,433.12±57.47±ng·h/mL, 576.52±67.21 h,191.18±34.56 h,125.26±33.21 h, respectively. |
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