In order to achieve a high density of grafted PEG chains on functional nanoparticles, a 2-step method is proposed that enables the tethering of the PEG chains under theta solvency conditions while preserving their colloidal stability. UV-vis studies were first used to determine the phase transition temperature of PEG chains grafted onto gold nanoparticles, which corresponded to the loss of the colloidal stability of the nanoparticles. In the first step of the process, PEG chains were tethered under good solvency conditions (×1 PBS and room temperature), which avoided problems with colloidal stability. In the second step, the solvent was changed to ×1 PBS supplemented with 0.4 M K2SO4 and the temperature of the solution was raised to 50 °C which was found to correspond to the onset of collapse of the tethered PEG chains. Model studies performed on planar gold surfaces using QCM and XPS showed that the proposed 2-step procedure resulted in a significantly increased density of grafted PEG chains. When applying the process to gold nanoparticles using either PEG–NHS ester or PEG–SH, it was observed that the initial PEG layer obtained in the first step of the process efficiently protected the colloidal nanoparticle suspension from aggregation during the second PEGylation step performed under theta solvency conditions. The dense PEG graft layers created by the 2-step method drastically reduced the non-specific fouling by proteins in vitro and were readily amenable to functionalization by biological ligands. Herceptin antibody was used to target the human breast cancer cell line SK-BR-3. A high extent of binding of nanoparticles to cells was observed, whereas control PEGylated nanoparticles without Herceptin did not bind to cells. The specificity of binding was further confirmed by a competitive binding assay using added free Herceptin in solution. Thus, the proposed procedure using two subsequent additions of PEG chains below and above the phase transition condition can be used to prepare dense PEG layers on solid nanoparticles with a high degree of bio-inertness and immune-specificity.
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