Intracellular Delivery of DNA and Enzyme in Active Form Using Degradable Carbohydrate-Based Nanogels

The facile encapsulation of biomolecules along with efficient formulation and storage makes nanogels ideal candidates for drug and gene delivery. So far, nanogels have not been used for the codelivery of plasmid DNA and proteins due to several limitations, including low encapsulation efficacy of biomolecule of similar charges and the size of cargo materials. In this study, temperature and pH sensitive carbohydrate-based nanogels are synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization technique and are studied in detail for their capacity to encapsulate and codeliver plasmid DNA and proteins. The temperature sensitive property of nanogels allows the facile encapsulation of biomaterials, while its acid-degradable profile allows the burst release of biomolecules in endosomes. Hence these materials are expected to serve as efficient vectors to deliver biomolecules of choice either alone or as codelivery system. The nanogels produced are relatively monodisperse and are around 30–40 nm in diameter at 37 °C. DNA condensation efficacy of the nanogels is dependent on the hydrophobic property of the core of the nanogels. The DNA–nanogel complexes are formed by the interaction of carbohydrate residues of nanogels with the DNA, and complexes are further stabilized with linear cationic glycopolymers. The DNA-nanogels complexes are also studied for their protein loading capacity. The degradation of the nanogels and the controlled release of DNA and proteins are then studied <i>in vitro</i>. Furthermore, the addition of a nontoxic, cationic glycopolymer to the nanogel–DNA complexes is found to improve the cellular uptake and hence to improve gene expression

Cationic Galactose-Conjugated Copolymers for Epidermal Growth Factor (EGFR) Knockdown in Cervical Adenocarcinoma

Glycopolymers of statistical and block configurations were synthesized from 2-lactobionamidoethyl methacrylamide (LAEMA) and 2-aminoethyl methacrylamide hydrochloride (AEMA) by the reversible addition–fragmentation chain transfer (RAFT) polymerization. These cationic glycopolymers were found to form very stable polyplexes with EGFR siRNA as determined by dynamic light scattering and agarose gel electrophoresis. The polyplexes revealed to be very stable even in the presence of serum proteins. Transfection studies of the glycopolymer-EGFR siRNA polyplexes were achieved in HeLa cells to determine the EGFR knockdown efficiency, cellular uptake and cytotoxicity. In this study, the block copolymer with the shortest AEMA segment was the most effective in EGFR gene silencing, however this block copolymer revealed to be slightly more toxic as compared to the statistical copolymers studied at higher w/w ratios. In addition, gene silencing of up to 80–85% was achieved with this low-molecular-weight block copolymer

Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers

Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition–fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver

Study of Bacterial Adhesion on Biomimetic Temperature Responsive Glycopolymer Surfaces

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen responsible for diseases such as bacteremia, chronic lung infection, and acute ulcerative keratitis. <i>P. aeruginosa</i> induced diseases can be fatal as the exotoxins and endotoxins released by the bacterium continue to damage host tissues even after the administration of antibiotics. As bacterial adhesion on cell surfaces is the first step in bacterial based pathogen infections, the control of bacteria–cell interactions is a worthwhile research target. In this work, thermally responsive poly­(<i>N</i>-isopropylacrylamide) [P­(NIPAAm)] based biomimetic surfaces were developed to study the two major bacterial infection mechanisms, which is believed to be mediated by hydrophobic or lectin–carbohydrate interactions, using quartz crystal microbalance with dissipation. Although, a greater number of <i>P. aeruginosa</i> adhered to the NIPAAm homopolymer modified surfaces at temperatures higher than the lower critical solution temperature (LCST), the bacterium–substratum bond stiffness was stronger between <i>P. aeruginosa</i> and a galactose based P­(NIPAAm) surface. The high bacterial adhesion bond stiffness observed on the galactose based thermally responsive surface at 37 °C might suggest that both hydrophobic and lectin–carbohydrate interactions contribute to bacterial adhesion on cell surfaces. Our investigation also suggests that the lectin–carbohydrate interaction play a significant role in bacterial infections

Temperature-Responsive Hyperbranched Amine-Based Polymers for Solid–Liquid Separation

Temperature-responsive hyperbranched polymers containing primary amines as pendent groups have been synthesized for solid–liquid separation of kaolinite clay suspension. The effects of temperature, polymer charge density, and polymer architecture on particle flocculation have been investigated. Suspensions treated with the temperature-responsive amine-based hyperbranched polymers showed remarkable separation of the fine particles at a low polymer dosage of 10 ppm and at testing temperatures of 40 °C. In comparison to other polymers studied (linear and hyperbranched homopolymers and copolymers), the temperature-responsive amine-based hyperbranched copolymers showed better particle flocculation at 40 °C, as evidenced by the formation of a thinner sediment bed without compromising the amount of clay particles being flocculated. This superior solid–liquid separation performance can be explained by the hydrophobic interaction of PNIPAM segments on particle surfaces or the capture of additional free particles or small floc due to the exposure of buried positive charges (because of the phase separation of the hydrophilic amines and hydrophobic PNIPAM part) at temperatures above the lower critical solution temperature (LCST)

Ultrafast Derived Self-Healable, Reprocessable Polyurethane Elastomer Based on Dynamic “Electrophilic Substitution (ES)-Click” Chemistry

Conventional self-healable polyurethanes (PUs) based on the furan–maleimide combination take several hours to prepare and require an elevated temperature to endow self-healing characteristics via Diels–Alder (DA) chemistry. In this work, furan end-capped triarm PU prepolymers (FAPUs) were prepared using polycaprolactone triol, 4,4′-methylene bis(phenyl isocyanate) (MDI), and furfuryl alcohol in the presence of a tin(II) catalyst. Cross-linked FAPUs were accomplished within 10 s under ambient conditions after reaction with bis-1,2,4-triazoline-3,5-dione (bis-TAD) via ES-Click chemistry. Structural elucidation of the synthesized prepolymer and ES-cross-linked FAPUs was carried out by 1H NMR and FTIR analyzes. Differential scanning calorimetric (DSC) analysis revealed that TAD-derived FAPU elastomers were thermoreversible at 110 °C and room temperature via ES-Click chemistry, and the thermoreversibility of FAPUs was confirmed via solution reprocessability. The self-healing behavior of PUs was monitored under an optical microscope, by scanning electron microscopy, and by tensile measurement. Unlike pristine prepolymer with a tensile strength of σ = 0.1 N/mm2, TAD-derived FAPU1 polymer showed a significant tensile strength of σ = 34.68 N/mm2 with healing efficiency (Hσ = 83%) without using any additive. The surface microhardness and depth penetration of FAPUs improved significantly after cross-linking with bis-TAD and retained their properties even after healing. Similarly, the resultant TAD-derived PUs had improved surface hydrophobicity compared to pristine PU prepolymers as supported by AFM analysis. These ES-Click-derived PU polymer materials showed significant mechanical, good self-healing, and hydrophobic characteristics and will be potential materials for advanced coatings, adhesives, and paint applications

Well-Defined Cationic <i>N</i>‑[3-(Dimethylamino)propyl]methacrylamide Hydrochloride-Based (Co)polymers for siRNA Delivery

Cationic glycopolymers have shown to be excellent candidates for the fabrication of gene delivery devices due to their ability to electrostatically interact with negatively charged nucleic acids and the carbohydrate residues ensure enhanced stability and low toxicity of the polyplexes. The ability to engineer the polymers for optimized compositions, molecular weights, and architectures is critical in the design of effective gene delivery vehicles. Therefore, in this study, the aqueous reversible addition–fragmentation chain transfer polymerization (RAFT) was used to synthesize well-defined cationic glycopolymers with various cationic segments. For the preparation of cationic parts, <i>N</i>-[3-(dimethylamino)­propyl]­methacrylamide hydrochloride (DMAPMA·HCl), water-soluble methacrylamide monomer containing tertiary amine, was polymerized to produce DMAPMA·HCl homopolymer, which was then used as macroCTA in the block copolymerization with two other methacrylamide monomers containing different pendant groups, namely, 2-aminoethyl methacrylamide hydrochloride (AEMA) (with primary amine) and <i>N</i>-(3-aminopropyl) morpholine methacrylamide (MPMA) (with morpholine ring). In addition, statistical copolymers of DMAPMA.HCl with either AEMA or MPMA were also synthesized. All resulting cationic polymers were utilized as macroCTA for the RAFT copolymerization with 2-lactobionamidoethyl methacrylamide (LAEMA), which consists of the pendent galactose residues to achieve DMAPMA·HCl-based glycopolymers. From the in vitro cytotoxicity study, the cationic glycopolymers showed better cell viabilities than the corresponding cationic homopolymers. Furthermore, complexation of the cationic polymers with siRNA, cellular uptake of the resulting polyplexes, and gene knockdown efficiencies were evaluated. All cationic polymers/glycopolymers demonstrated good complexation ability with siRNA at low weight ratios. Among these cationic polymer-siRNA polyplexes, the polyplexes prepared from the two glycopolymers, P­(DMAPMA₆₅-<i>b</i>-LAEMA₁₅) and P­[(DMAPMA₆₅-<i>b</i>-MPMA₆₃)-<i>b</i>-LAEMA₁₆], showed outstanding results in the cellular uptake, high EGFR knockdown, and low post-transfection toxicity, suggesting the great potential in siRNA delivery of these novel glycopolymers

Ultrafast Derived Self-Healable, Reprocessable Polyurethane Elastomer Based on Dynamic “Electrophilic Substitution (ES)-Click” Chemistry

Conventional self-healable polyurethanes (PUs) based on the furan–maleimide combination take several hours to prepare and require an elevated temperature to endow self-healing characteristics via Diels–Alder (DA) chemistry. In this work, furan end-capped triarm PU prepolymers (FAPUs) were prepared using polycaprolactone triol, 4,4′-methylene bis(phenyl isocyanate) (MDI), and furfuryl alcohol in the presence of a tin(II) catalyst. Cross-linked FAPUs were accomplished within 10 s under ambient conditions after reaction with bis-1,2,4-triazoline-3,5-dione (bis-TAD) via ES-Click chemistry. Structural elucidation of the synthesized prepolymer and ES-cross-linked FAPUs was carried out by 1H NMR and FTIR analyzes. Differential scanning calorimetric (DSC) analysis revealed that TAD-derived FAPU elastomers were thermoreversible at 110 °C and room temperature via ES-Click chemistry, and the thermoreversibility of FAPUs was confirmed via solution reprocessability. The self-healing behavior of PUs was monitored under an optical microscope, by scanning electron microscopy, and by tensile measurement. Unlike pristine prepolymer with a tensile strength of σ = 0.1 N/mm2, TAD-derived FAPU1 polymer showed a significant tensile strength of σ = 34.68 N/mm2 with healing efficiency (Hσ = 83%) without using any additive. The surface microhardness and depth penetration of FAPUs improved significantly after cross-linking with bis-TAD and retained their properties even after healing. Similarly, the resultant TAD-derived PUs had improved surface hydrophobicity compared to pristine PU prepolymers as supported by AFM analysis. These ES-Click-derived PU polymer materials showed significant mechanical, good self-healing, and hydrophobic characteristics and will be potential materials for advanced coatings, adhesives, and paint applications

Ultrafast Derived Self-Healable, Reprocessable Polyurethane Elastomer Based on Dynamic “Electrophilic Substitution (ES)-Click” Chemistry

Conventional self-healable polyurethanes (PUs) based on the furan–maleimide combination take several hours to prepare and require an elevated temperature to endow self-healing characteristics via Diels–Alder (DA) chemistry. In this work, furan end-capped triarm PU prepolymers (FAPUs) were prepared using polycaprolactone triol, 4,4′-methylene bis(phenyl isocyanate) (MDI), and furfuryl alcohol in the presence of a tin(II) catalyst. Cross-linked FAPUs were accomplished within 10 s under ambient conditions after reaction with bis-1,2,4-triazoline-3,5-dione (bis-TAD) via ES-Click chemistry. Structural elucidation of the synthesized prepolymer and ES-cross-linked FAPUs was carried out by 1H NMR and FTIR analyzes. Differential scanning calorimetric (DSC) analysis revealed that TAD-derived FAPU elastomers were thermoreversible at 110 °C and room temperature via ES-Click chemistry, and the thermoreversibility of FAPUs was confirmed via solution reprocessability. The self-healing behavior of PUs was monitored under an optical microscope, by scanning electron microscopy, and by tensile measurement. Unlike pristine prepolymer with a tensile strength of σ = 0.1 N/mm2, TAD-derived FAPU1 polymer showed a significant tensile strength of σ = 34.68 N/mm2 with healing efficiency (Hσ = 83%) without using any additive. The surface microhardness and depth penetration of FAPUs improved significantly after cross-linking with bis-TAD and retained their properties even after healing. Similarly, the resultant TAD-derived PUs had improved surface hydrophobicity compared to pristine PU prepolymers as supported by AFM analysis. These ES-Click-derived PU polymer materials showed significant mechanical, good self-healing, and hydrophobic characteristics and will be potential materials for advanced coatings, adhesives, and paint applications

Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release

Reversible addition–fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly­(2-methacryloyloxyethyl phosphorylcholine), poly­(MPC) <i>macro</i>RAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly­(MPC) shell and thermoresponsive core consisting of poly­(methoxydiethylene glycol methacrylate) (poly­(MeODEGM) and poly­(2-aminoethyl methacrylamide hydrochloride) (poly­(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h