Construction Of Nano - Targeted Composite Micelles And Their Anti - Melanoma Based On Polyglutamic Conformational Transformation

The bioactive polymer poly(L-glutamic acid)n-b-poly(D, L-lactic acid)m was synthesized and used to form doxorubicin-loaded hybrid polymeric micelles to treat melanoma. These polymers exhibited pH-responsive changes in conformation, which controlled the diverse functionalities of the micelles. During circulation, poly(L-glutamic acid)n-b-poly(D, L-lactic acid)m protected Tat peptides(Tatp) on the micelles from proteolysis. Under tumor-acidic conditions, polymers with shorter poly(L-glutamic acid) blocks underwent a conformational change to form channels that accelerated the release of doxorubicin. The conformational change also exposed the Tat peptides to tumor cells, thereby promoting cellular internalization of the micelles.Poly(benzyl-L-glutamate)n-b-PLAm (PBGAn-b-PLAm) was then synthesized via ROP of y-benzyl L-glutamate initiated with NH2-terminated poly(D, L-lactide)m. HBr was applied to remove the benzyl group and obtain the final product, PGAn-b-PLAm. DSPE-b-PEG3.5k-Tatp was produced by the reaction between the mateimide group of DSPE-b-PEG3.5k-maleimide and the sulfhydryl group of cysteine-modified Tatp. Yield:91%. The polymers were characterized by1H NMR spectroscopy, fourier transform infrared spectrometer, circular dichroism and gel permeation chromatographyDialysis method was used to prepared h-PMs-1micelles containing DSPE-b-PEG3.5k-Tatp, DSPE-b-PEG2k and PGA5.6k-b-PLA1,8k, and h-PMs-2micelles with DSPE-b-PEG3.5k-Tatp, DSPE-b-PEG2k and PGA12K-b-PLA6K. The conformational transition of PGAn-b-PLAm caused a change in the particle size of h-PMs-1and h-PMs-2when the pH values were varied. At pH6.5, h-PMs-1presented an α-helix conformation in compact form. However, h-PMs-2remained a random coil with a larger diameter than h-PMs-1at pH6.5. The h-PMs-1(2) also exhibited pH-induced changes in zeta potential, which were correlated with the conformational transitions of h-PMs-l(2).Different doxorubicin-loaded hybrid polymeric micelles, D-h-1micelles and D-h-2micelles, were also formed by the dialysis method. The D-h-1micelles with PGA5.6k-b-PLAi,8k were characterized with95±1.72nm (pH7.4) intensity diameter and9.82±1.01wt%of loading efficiency. The D-h-2micelles with PGA12K-b-PLA6K was in165±1.55nm (pH7.4) of intensity diameter and9.92±0.64wt%of loading efficiency. XRD was used to determine whether Dox had been incorporated into the different micelles. D-h-1and D-h-2exhibited similar pH-sensitive release profiles except that the release rate of D-h-1was greater than that of D-h-2at pH6.5. This result correlated well with the conformational transition observed for h-PMs-1(2) going from pH7.4(and9.0) topH5.5.Cellular uptake of these micelles was investigated by flow cytometry and confocal laser scanning microscopy. The results showed that the conformational change of PGA5.6k-b-PLA1.8k contributed to the internalization of different micelles. The shrunken PGA5.6k-b-PLA1.8k exposed Tatp at pH6.5, but the extended PGA5.6k-b-PLA1.8k shielded Tatp to prevent cell penetration at pH7.4. Extended PGA blocks also hid Tatp within micellar shells and protected Tatp from proteolysis at pH7.4.The MTT assay was applied to evaluate the in vitro cytotoxicity of the micelles using A375melanoma cells. The result showed reveals that D-h-1decreased the cytotoxicity of Dox by shielding Tatp at pH7.4, but the cytotoxicity was markedly increased at pH6.5due to the exposure of Tatp. The cytotoxicity of D-h-1at pH6.5was approximately4-fold higher than that at pH7.4due to the efficient cellular uptake under acidic conditions. Enhanced cellular uptake of the micelles induced significant apoptosis of A375melanoma cells in tumor-acidic conditions.A tumor-bearing (human A375melanoma cells) mouse model was developed to test the tumor-suppressing ability of the micelles. D-h-1was the most effective formulation for inhibiting tumor growth and maintaining body weight. The targeting ability of the micelles was observed using near-infrared (NIR) fluorescence imaging. The fluorescence signal of the Di-h-1formulation was substantially stronger than that of Di-h-2and Di-T over24hrs. Most importantly, D-h-1micelles was effectively extending the life of tumor-bearing mice treated with D-h-1to73days after inoculation and lower toxicity to hearts of the model animals. However, micelles with longer poly(L-glutamic acid) blocks did not undergo a conformational change under acidic conditions and performed poorly in both in the evaluations described above.