Different Insight into Amphiphilic PEG-PLA Copolymers: Influence of Macromolecular Architecture on the Micelle Formation and Cellular Uptake

One constrain in the use of micellar carriers as drug delivery systems (DDSs) is their low stability in aqueous solution. In this study “tree-shaped” copolymers of general formula mPEG-(PLA)_n (<i>n</i> = 1, 2 or 4; mPEG = poly­(ethylene glycol) monomethylether 2K or 5K Da; PLA = atactic or isotactic poly­(lactide)) were synthesized to evaluate the architecture and chemical composition effect on the micelles formation and stability. Copolymers with mPEG/PLA ratio of about 1:1 wt/wt were obtained using a “core-first” synthetic route. Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), and Zeta Potential measurements showed that mPEG_2K-(PD,LLA)₂ copolymer, characterized by mPEG chain of 2000 Da and two blocks of atactic PLA, was able to form monodisperse and stable micelles. To analyze the interaction among micelles and tumor cells, FITC conjugated mPEG-(PLA)_<i>n</i> were synthesized. The derived micelles were tested on two, histological different, tumor cell lines: HEK293t and HeLa cells. Fluorescence Activated Cells Sorter (FACS) analysis showed that the FITC conjugated mPEG_2K-(PD,LLA)₂ copolymer stain tumor cells with high efficiency. Our data demonstrate that both PEG size and PLA structure control the biological interaction between the micelles and biological systems. Moreover, using confocal microscopy analysis, the staining of tumor cells obtained after incubation with mPEG_2K-(PD,LLA)₂ was shown to be localized inside the tumor cells. Indeed, the mPEG_2K-(PD,LLA)₂ paclitaxel-loaded micelles mediate a potent antitumor cytotoxicity effect

Raman spectroscopy of different cell lines.

<p>Raman spectra for control and stressed cancer cell lines (Mel 59c (A), Mel 42a (B), Mel 103b(C) and 293T(D)) with standard deviation error bar. The spectra were performed before and after mechanical stress with micropump.</p

Principal component analysis.

<p>PCA analysis on control and stress cells for various cell lines; Mel 42a, Mel 59c, Mel 103b and 293T. a) PC1 vs. PC2, b) PC2 vs. PC3 and c) PC1 vs. PC3.</p

Protein concentration in different cell lines.

<p>Normalized area of band centred at around 1440 (top) and 1670 cm⁻¹ (down) for all the cell lines, showing the variation of protein concentration and relative α-helix content over the cell surface.</p

Increased NK susceptibility on mechanical stressed tumor cells.

<p>NK cell recognition of different tumor cell targets at different E/T (effector/target) ratio: 59c, 42a, 66b (melanoma cell lines), 293T (kidney carcinoma) and IM9 (lymphoblastoidcell lines) before (grey) and after (black) mechanical stress. The Mel 42a, Mel 66b, fibroblasts cells (panels B, E, and F) were treated with the micropump, the Mel 59c, IM9, 293 T cells (panels A, C and D) were stressed with the shock waves. As healthy target cells, in this case fibroblasts are shown. Representative experiments are reported for each cell type. Panels G and H show the statistics derived from three different functional assays, using NK lymphocytes as effectors cells (E) and IM9 and Melanoma cells as targets (T). The IM9 target cells were treated with the shock waves (panel G: n = 3, p = 0.0325), while the Melanoma target cells were stressed with the micropump (panel H, n = 3, p = 0.0186). E/T ratio 12/1, p<0.05.</p

Western Blotting of MHC class I expression on the supernatants of treated samples: Tumor (A) and healthy cells (B) were analysed before and after mechanical stress by shock waves.

<p>MHC-I has molecular weight of 45 kDa. (C) Membrane incubated with Ponceau S red staining solution, as loading controls.</p