The entropy-driven affinity of trivalent (in)organic arsenicals for closely spaced dithiols has been exploited to develop a novel route to peptide/protein-polymer conjugation. A trivalent arsenous acid (As(III)) derivative (1) obtained from p-arsanilic acid (As(V)) was shown to readily undergo conjugation to the therapeutic peptide salmon calcitonin (sCT) via bridging of the Cys(1)-Cys(7) disulfide, which was verified by RP-HPLC and MALDI-ToF-MS. Conjugation was shown to proceed rapidly (t < 2 min) in situ and stoichiometrically through sequential reduction-conjugation protocols, therefore exhibiting conjugation efficiencies equivalent to those reported for the current leading disulfide-bond targeting strategies. Furthermore, using bovine serum albumin as a model protein, the trivalent organic arsenical 1 was found to demonstrate enhanced specificity for disulfide-bond bridging in the presence of free cysteine residues relative to established maleimide functional reagents. This specificity represents a shift toward potential orthogonality, by clearly distinguishing between the reactivity of mono- and disulfide-derived (vicinal or neighbors-through-space) dithiols. Finally, p-arsanilic acid was transformed into an initiator for aqueous single electron-transfer living radical polymerization, allowing the synthesis of hydrophilic arsenic-functional polymers which were shown to exhibit negligible cytotoxicity relative to a small molecule organic arsenical, and an unfunctionalized polymer control. Poly(poly[ethylene glycol] methyl ether acrylate) (PPEGA480, DPn = 10, Mn,NMR = 4900 g·mol(-1), Đ = 1.07) possessing a pentavalent arsenic acid (As(V)) α-chain end was transformed into trivalent As(III) post-polymerization via initial reduction by biological reducing agent glutathione (GSH), followed by binding of GSH. Conjugation of the resulting As(III)-functional polymer to sCT was realized within 35 min as indicated by RP-HPLC and verified later by thermodynamically driven release of sCT, from the conjugate, in the presence of strong chelating reagent ethanedithiol.
This study reports a novel nanoparticle system with simple and modular one-step assembly, which can respond intelligently to biologically relevant variations in pH. Importantly, these particles also show the ability to induce escape from the endosomal/lysosomal compartments of the cell, which is integral to the design of efficient polymeric delivery systems. The nanoparticles were formed by the nanoprecipitation of pH-responsive poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and poly(2-(diethylamino)ethyl methacrylate)-b-poly(ethylene glycol) (PDEAEMA-b-PEG). Rhodamine B octadecyl ester perchlorate was successfully encapsulated within the hydrophobic core of the nanoparticle upon nanoprecipitation into PBS at pH 8. These particles disassembled when the pH was reduced below 6.8 at 37 °C. Cellular experiments showed the successful uptake of the nanoparticles into the endosomal/lysosomal compartments of 3T3 fibroblast cells. The ability to induce escape from the endosomes was demonstrated by the use of calcein, a membrane-impermeable fluorophore. The modular nature of these particles combined with promising endosomal escape capabilities make these dual component PDEAEMA nanoparticles useful for drug and gene delivery applications.
SHIP is able to distinguish between nanoparticles that are associated on the outer cell membrane from nanoparticles that are internalized. This study demonstrates the assay can be used to probe the kinetics of nanoparticle internalization and the mechanisms by which the nanoparticles are taken up by cells. This information is fundamental for engineering more effective nanoparticle delivery systems. The SHIP assay is a simple and a high-throughput technique that could have wide application in therapeutic delivery research.
Engineering the properties of nanoparticles to limit non-specific cellular interactions is critical for developing effective drug delivery systems. Differences between interactions with cultured cells and human blood highlights the need for appropriate assays.
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