Poly(Ethylene Glycol) Grafted Amphiphilic Polyphosphazenes: Self-assembly Behavior And Applications In Drug Delivery Systems

In this study,a new series of amphiphilic methoxy-poly(ethylene glycol)graftedpolyphosphazenes with different side groups including glycine ethyl ester(GlyEt),ethyl 4-aminobenzoate(EAB)and Cholesterol (Chol)were synthesized andcharacterized.Differential synthesis strategies of amphiphilic graft polyphosphazeneswere established to achieve controlled hydrophilic ratio of each copolymer withrelatively high product yield.Copolymer structure was characterized by NMR,UV,DSC and GPC.Fluorescence probe was adopted to determine the critical micelle concentration(CMC)of amphiphilic copolymers,and the results revealed that the CMC willdecrease with the increasing hydrophobic content of amphiphilic copolymers.Themorphology of assemblies was observed by TEM,it was found that aggregates withvarious shaps can be formed in aqueous sulution.For the amphipHilic graftpolyphosphazenes that using GlyEt as hydrophobic groups,rod-like micelles wereobserved when copolymer has high GlyEt content,as the weight ratio of hydrophobicgroups decreased,micelles with multiple morphology obtained,where the rods andspherical micelles are coexistent,micelles of copolymer with low GlyEt content are allin spherical shape.For copolymers with EAB as hydrophobic groups,thesecopolymers could self-assemble into distinct aggregates in aqueous solutions. Spherical micelles were observed for the copolymer sample with higher hydrophilicweight fraction.However,when the hydrophilic weight fractions decreased to less than0.50,vesicle-like polymersomes were formed.Noteworthy,micelles derived fromcopolymer with high EAB content and PEG₁₁₀₀as hydrophilic chains are not quitestable,depending on the type of co-solvent and the copolymer concentrations,the shapof aggregates altered between network aand spherical micelles.It was also found thatcholesterol group is quite hydrophobic,cholesterol substituted amphiphilicpolyphosphazenes can not dissolved in water and form micelles until the weightcontent ofhydrophilic PEG side chains reach to a relatively high level.Different hydrophobic drugs were encapsulated into polymeric micelles derivedfrom those copolymers;the size,drug-loading content and entrapment efficiency aredetermined by several factors including physicochemical characteristics of copolymer,structure of drug molecular and the preparation parameters.Drug loading exhibitedconsiderably strong impact on the morphology of micelles derived from GlyEtsubstituted polyphosphazenes:turned the rod-like and spherical drug free micelles intospheres and vesicles respectively.Drug-loaded micelles are quite stable and can befreeze-dried for long-term storage,in vitro release behavior of those drug-loadedmicelles exhibits a sustained release manner and a pH-related drug release wasconfirmed.Water-soluble anti-cancer drug was successfully loaded into the aqueous core ofpolymersome derived from copolymers with EAB as hydrophobic groups,which wasclearly observed by transmission electron microscopy.Drug release profile of thisdrug-loaded polymersome provided linear relationships for Higuchi plotting,indicatingthat Fickian diffusion played an important role during this release period.In vitro cytotoxicity assay revealed that drug-loaded micelles of amphiphilicpolyphosphazenes can effectively suppress proliferation of tumor cells,drug-loadedmicelles based on GlyEt substituted polyphosphazenes have enhanced cytotoxicity on adriamycin-resistant MCF-7 cell line,the 72h IC50 reduced 3 fold comparaed to freedrug formulation;The cytotoxicity against HepG2 cells of drug-loaded polymersomesbased on copolymers with EAB as hydrophobic groups was significantly enhancedcomparaed to free drug formulation,and the 24h IC50 reduced 17 fold by usingpolymersome carrier.Moreover,flow cytometry results revealed that the drug-loaded micelles havehigher cell internalization efficiency,fluorescence intensity of drug in theadriamycin-resistant MCF-7 cells incubate with drug-loaded micelles can be 3 foldhigher than that of free drug parallel,it was also found that smaller size favored theinternalization of micelles by adriamycin-resistant MCF-7 cells,cell up-take efficiencyof drug-loaded micelles with particale size of 147nm is 1.9 fold higher comparaed todrug-loaded micelles with particale size of 279nm.carrageenan-induced acute arthritis was employed to evaluate the therapeutic effectof indomethacin-loaded amphiphilic polyphosphazene micelles,and the resultsindicated that drug-loaded polymeric micelles exhibit more significant therapeuticeffect than that of control group.In summary,the present novel amphiphilic polyphosphazene copolymers have highpotentials on drug delivery applications especially on anti-tumor drug delivery systems.More extensive studies are definitely needed.