Gold nanoparticles co-functionalized with poly(ethylene glycol and receptor mediated endocytosis peptide for cellular uptake

摘要

Introduction: A clinically useful AuNP must evade the human immune system, target the cancer cell, initiate uptake and deliver either a payload and/or a radiosensitizer. Therefore it is necessary to co-functionalize the AuNP surface with a range of moieties by creating either a mixed monolayer or by using a bifunctiona! polyethylene glycol (PEG) linker. While both methods have been successfully used in a range of in-vitro and animal studies, there have been no studies which directly compare the two approaches. This work builds upon previous studies carried out at Ulster University which investigated AuNP synthesis, the influence of PEG capping on stability, payload release and co-functionalization with pepticles for uptake and endosomal escape. The linker methodology has the potential to enhance bioavailability and increase the amount of functional agent attached. This research compares the two methodologies by synthesizing and characterizing PEG and receptor mediated endocytosis (RME) peptide co-functionalized AuNPs, prepared using both approaches. Stability, non-specific protein adsorption and cellular uptake were studied. Materials and Methods: AuNP with an average diameter of ～16nm were prepared by the Turkevich method. These were then functionalized with either mono functional (SH-PEG, Sigma Aldrich) or bifunctional thiol terminated PEG (SH-PEG-SGA, JenKem). Once PEGylated, the AuNP conjugate was then co-functionalized with RME (Biomatik) by either binding the RME directly to the AuNP surface using the cysteine unit or by attaching the peptide to the PEG using the NHS ester group on the bifunctional PEG. The AuNPs were characterized by DLS, UV/Vis, FTIR. TEM and Gel Electrophoresis. The stability of various systems was studied and protein adsorption was investigated by foetal calf serum (FCS), while payload release and cellular intemalization were investigated across a range of cell lines. Results and Discussion: Attachment was confirmed by a combination of FTIR, gel electrophoresis and OLS with the addition of PEG yielding an AuNP increase in size from ～16nm to ～40nm. Subtle differences were apparent in relation to non-specific protein adsorption; gel electrophoresis showed some changes in migration profile after incubation with fetal calf serum (FCS). Preliminary cell line results have indicated that citrate capped AuNPs enter the cells in abundance, however PEGylation blocks uptake. This can be recovered somewhat by the addition of RME. The preliminary results have not shown any significant difference in the uptake of the mixed monolayer in contrast to the linker, this is an unexpected result however can be hypothesized due to the arrangement of the moieties in 'islands' on the surface. Conclusion: While there appears to be no significant difference in the uptake for either surface arrangement, subtle differences are apparent in the ability to resist protein adsorption. It was found that PEG rendered both arrangements stable up to 1M NaCl however inhibited uptake. This could be recovered to some degree for both approaches by co-functionalization with RME. This led to the preliminary conclusion that the optimum surface methodology may be a combination of both arrangements to create a mixed monolayer and PEG linker complex. Future work includes the influence of peptide quantity on uptake, surface topography and the addition of a targeting moiety.