Preparation And In Vitro Evaluation Of Amphiphilic Polyphosphazene As A Carrier Of Doxorubicin Micelles

By changing the malor ratio of hydrophilic and hydrophobic segement, a series of novel amphiphilic graft polyphosphazenes were synthesized via thermal ring-opening polymerization and subsequent two-step substitution reaction of hydrophilic methoxy polyethylene glycol (MPEG) and hydrophobic ethyl tryptophan (EtTrp). FTIR, ¹H NMR and UV spectrophotometer studies confirmed that copolymers were synthesized successfully. Copolymer composition was measured by a UV-visible spectrophotometer. The molar ratio of the segment EtTrp to group PEG was respectively 0.67:1.33, 0.99:1.01 and 1.22: 0.78. Micellization behavior of PEG / EtTrp -PPPs in an aqueous phase was characterized by fluorescence technique, dynamic light scattering (DLS) and transmission electron microscopy (TEM). The critical micelle concentration (CMC) of the graft copolymer in aqueous solution was 0.158, 0.033, 0.020 g/L, and the results revealed that with the hydrophobic content in amphiphilic copolymers increasing, the CMC decreased. The number-averaged particle size of spherical polymeric assemblies . was nearly 20-40nm with a narrow distribution.Antitumor drug doxorubicin (DOX) was physically loaded into micelles prepared by dialysis and O/W emulsion method. Due to the higher loading efficiency, O/W emulsion method was adopted. We optimized the preparation condition by changing the DOX feeding, O/W volume ratio and polymer concentration. In this study, we found that DOX loading efficiency increased with DOX feeding, though entrapment efficiency of DOX reached the highest level at 4:10(weight ratio of DOX/polymer). Also diameters of DOX-loaded micelles increased as DOX loading efficiency increasing. Copolymers with different composition had a nearly the same loading capability, though in the low feeding, we found that copolymer with a high ratio of EtTrp, whose inner core was more hydrophobic, had a high loading efficiency. In vitro release of DOX compounds from the micelles was pH sensitive. With pH shifted from 5.0 ,6.8,7.4 to 8.0, the accumulative release of DOX was respectively 59.2±2.0%, 30.1±1.6%, 23.4±1.3% and 17.7±1.5%. It offered a good opportunity to delivery antitumor drug to tumor tissue with lower release in circulation, and reduced the toxicity to normal tissue. The in vitro release profiles of DOX loaded micelles with different loading efficiency were studied. It was found that DOX loaded micelles with a high loading (22.3%) were released slower than the lower. It was due to the hydrophobic interaction enhanced by DOX loaded in the inner core. Copolymers with different composition had a slightly effect on the release profile of DOX loaded micelles. Micelles constructed by PEG/ EtTrp -PPP-3 released DOX a little slower than that with PEG/ EtTrp -PPP-1 and PEG/ EtTrp -PPP-2.The results of cytotoxicity study using an MTT assay method with Hela cell showed that amphiphilic graft polyphosphazenes (PEG/EtTrp-PPPs) had a good biocompatibility while exposure time had nearly no effect on the cell viability. DOX loaded micelles had nearly the same cell damage compared with unloaded free DOX. Their IC₅₀ were 0.173μg·mL^-1 and 0.180μg·mL^-1 respectively after 48h exposure to Hela cell. MTT assay with Hela and HepG₂ both confirmed that cytotoxicity of DOX and DOX loaded micelles increased with time. DOX loaded micelles had more cytotoxicity to HepG₂ at 72h exposure. Furthermore, DOX loaded micelles had a reversal effect on the DOX resistant MCF-7 (MCF-7/ADM) , the IC₅₀ of free DOX and DOX loaded micelles were 11.506μg·mL and 5.312μg/mL. In summary, these novel amphiphilic copolymers could be used as injectable drug carriers or used in tumor targeted drug delivery systems. However, further study including in vitro and in vivo revolution were under way.