Multi-stage, charge conversional, stimuli-responsive nanogels for therapeutic protein delivery

Abstract

A boronate ester crosslinked zwitterionic nanogel (NGCA) with ATP/pH-sensitivity has been developed with an inverse nanoprecipitation technique to achieve a two-stage charge conversion that responds to tumor extracellular conditions (pH 6.5-6.8) and an intracellular acidic environment (pH 5-6). Cationic cytochrome C (CC), a therapeutic protein, has been encapsulated into NGCA through inverse nanoprecipitation via electrostatic interactions to form protein-loaded nanogel (NGCA-CC). By adjusting the ratio of the amino and carboxyl groups in the nanogels, negatively charged nanogels that are safer under physiological conditions (pH 7.4) can convert their surface charge to positive at tumor extracellular pH, which enhance their cellular uptake efficiency. The citraconic amide formed from citraconic anhydride and amine can be cleaved in the intracellular acidic organelles to expose more amino groups and facilitate endosomal escape. The release of CC is accelerated in the presence of 5 mM ATP or under acidic conditions. Confocal laser scanning microscopy (CLSM) and flow cytometry have shown that NGCA-CC's cell uptake is higher at pH 6.5 than at pH 7.4. MTT and real-time cell analysis (RTCA) have illustrated that there is more toxicity at pH 6.5 than at pH 7.4. The apoptosis process induced by CC was determined by flow cytometry.

N-[2-(Trifluoromethyl)phenyl]maleamic acid: crystal structure and Hirshfeld surface analysis

“…After accumulating into the acidic tumor tissue through the enhanced permeation and retention (EPR) effect, the amide bond slowly cleaves and thus exposes the positive charge, which eventually promotes cell internalization. Therefore, maleamic acids and their derivatives, by virtue of their unique weak acid sensitivity and charge conversion have been widely used as smart carriers to deliver nucleic acids (Meyer et al, 2009), proteins (Zhang et al, 2015;Lee et al, 2007) and drugs (Du et al, 2011;Chen et al, 2015;Han et al, 2015). Simple methods to control the ratio of two positional isomers of mono-substituted maleamic acids and a highly efficient way to synthesize di-substituted maleamic acids have been reported (Su et al, 2017).…”

Zwitterionic poly(amido amine) based nanogels

“…145,161 Surface-charge switching has therefore received interest as a drug delivery mechanism. 28,[209][210][211] Charge conversion of anionic nanoparticles into cationic nanoparticles, greatly enhances their cellular uptake. 145 Du et al reported a PEGdiacrylate-crosslinked 2-aminoethyl methacrylate nanogel, of which the primary amine groups were functionalized with 2,3-dimethylmaleic anhydride.…”

“…74,87,148,310,311 Moreover, the responsive properties of nanogels offer possibilities for triggered release by incorporating enzymatically degradable, 225 bioreducible, 129,312 and pH-labile bonds. 73,74,211,310 Figure 20: Microgel-encapsulated 2-(diethylamino)ethyl methacrylate-co-tert-butyl methacrylate nanogels for oral siRNA-delivery: after degradation of the microgel matrix, fluorescently labeled nanogels were added to murine macrophages, showing cellular uptake (A, cell nuclei are stained in blue, the cell wall in red and the nanogels in green). Delivery of TNF-α knockdown siRNA showed no significant difference between the nanogels and Lipofectamine (B).…”

“…Chen et al prepared a block copolymer via RAFT polymerization consisting of a PEG block and a copolymer of 2-hydroxyethyl methacrylate and a cyclic carbonate monomer. 129 The cyclic carbonate was ring-opened with cystamine and immediately oxidized in the presence of cytochrome C to obtain disulfide crosslinked nanogels Boronic acid bearing polyglycerol nanogels, which were consecutively functionalized with primary amine and dimethylmaleic anhydride, giving them a zwitterionic character with charge conversion properties, have been reported for the delivery of cytochrome C. 211 Cytochrome C was encapsulated via nanoprecipitation and nanogel crosslinking was achieved via boronate ester formation with the polyglycerol backbone. Both studies revealed protein release as well as intracellular release of cytochrome C and the induction of apoptosis.…”

“…Both studies revealed protein release as well as intracellular release of cytochrome C and the induction of apoptosis. 129,211 Enzymes may also be applied as a bioscavenger to remove toxins from the bloodstream. However, due to their short half-life and immunogenicity, application of recombinant enzymes as bioscavengers is not viable, not even after PEG-ylation.…”