Polyethyleneimine coated nanogels for the intracellular delivery of RNase A for cancer therapy

The aim of this study was to deliver ribonuclease A (RNase A) intracellularly using dextran nanogels for cancer treatment. To this end, positively charged RNase A was electrostatically loaded in anionic dextran nanogels with an average size of 205 nm, which were prepared by an inverse mini-emulsion technique. To chemically immobilize the loaded protein in the nanogels and prevent its unwanted release in the extracellular environment, the protein was covalently linked to the nanogel network via disulfide bonds, which are cleavable in the reductive cytosolic environment. A high loading efficiency and loading content of RNase A (75% and 20%, respectively) were obtained. Coating of the nanogels with the cationic polymer polyethyleneimine reversed the zeta potential of nanogels from -31.6 mV to +7.6 mV. The nanogels showed a fast and triggered release of RNase in the presence of glutathione. Negatively charged RNase A loaded nanogels did not show cytotoxicity, likely due to their limited cellular uptake. In contrast, PEI coated RNase A loaded nanogels showed high uptake by MDA-MB 231 breast cancer cells and exhibited a concentration-dependent cytotoxic effect by apoptosis. The results demonstrate that PEI coated nanogels are promising nano-carriers for intracellular protein delivery, encouraging further evaluation of this formulation in preclinical models

Cationic synthetic long peptides-loaded nanogels : An efficient therapeutic vaccine formulation for induction of T-cell responses

Recent studies have shown a high potency of protein-based vaccines for cell-mediated cancer immunotherapy. However, due to their poor cellular uptake, efficient immune responses with soluble protein antigens are often not observed. As a result of superior cellular uptake, nanogels loaded with antigenic peptides were investigated in this study as carrier systems for cancer immunotherapy. Different synthetic long peptides (SLPs) containing the CTL and CD4+ T-helper (Help) epitopes were synthesized and covalently conjugated via disulfide bonds to the polymeric network of cationic dextran nanogels. Cationic nanogels with a size of 210 nm, positive zeta potential (+24 mV) and high peptide loading content (15%) showed triggered release of the loaded peptides under reducing conditions. An in vitro study demonstrated the capability of cationic nanogels to maturate dendritic cells (DCs). Importantly, covalently SLP-loaded nanogels adjuvanted with poly(I:C) showed superior CD8+ T cell responses compared to soluble peptides and nanogel formulations with physically loaded peptides both in vitro and in vivo. In conclusion, covalently SLPs-loaded cationic nanogels are a promising system to provoke immune responses for therapeutic cancer vaccination

Polyethyleneimine coated nanogels for the intracellular delivery of RNase A for cancer therapy

The aim of this study was to deliver ribonuclease A (RNase A) intracellularly using dextran nanogels for cancer treatment. To this end, positively charged RNase A was electrostatically loaded in anionic dextran nanogels with an average size of 205 nm, which were prepared by an inverse mini-emulsion technique. To chemically immobilize the loaded protein in the nanogels and prevent its unwanted release in the extracellular environment, the protein was covalently linked to the nanogel network via disulfide bonds, which are cleavable in the reductive cytosolic environment. A high loading efficiency and loading content of RNase A (75% and 20%, respectively) were obtained. Coating of the nanogels with the cationic polymer polyethyleneimine reversed the zeta potential of nanogels from -31.6 mV to +7.6 mV. The nanogels showed a fast and triggered release of RNase in the presence of glutathione. Negatively charged RNase A loaded nanogels did not show cytotoxicity, likely due to their limited cellular uptake. In contrast, PEI coated RNase A loaded nanogels showed high uptake by MDA-MB 231 breast cancer cells and exhibited a concentration-dependent cytotoxic effect by apoptosis. The results demonstrate that PEI coated nanogels are promising nano-carriers for intracellular protein delivery, encouraging further evaluation of this formulation in preclinical models

Native chemical ligation for cross-linking of flower-like micelles

\u3cp\u3eIn this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM-co-HPMA-Cys)-PEG-P(NIPAM-co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM-co-HPMA-ETSA)-PEG-P(NIPAM-co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number (N\u3csub\u3eagg\u3c/sub\u3e) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.\u3c/p\u3

Native Chemical Ligation for Cross-Linking of Flower-Like Micelles

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM-co-HPMA-Cys)-PEG-P(NIPAM-co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM-co-HPMA-ETSA)-PEG-P(NIPAM-co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number (Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.</p

Native Chemical Ligation for Cross-Linking of Flower-Like Micelles

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications