Cross-linked Nanogels Research Articles

Supramolecularly Cross-Linked Nanogel by Merocyanine Pendent Copolymer.

Directional dipole-dipole interaction mediated antiparallel dimerization of merocyanine dye (MD) has been explored for maneuvering supramolecular assembly of MD-conjugated flexible macromolecules leading to a cross-linked nanogel. The MD-functionalized copolymer was synthesized by a newly developed organocatalytic transesterification strategy for postpolymerization functionalization of poly(pentafluorophenyl acrylate) (polyPFPA)-based reactive copolymer. Presence of ∼35% pendant MD attached to a coil-like polymer chain leads to spontaneous formation of highly emitting cross-linked nanogel with efficient container property and appreciable stability in toluene owing to strong dimerization propensity among the MD. Considering the significance of MD in the context of nonlinear optics and photovoltaics, these results not only enrich the toolbox for engineering macromolecular assembly, but also open up new possibilities for future organic materials.

Nanoscopic Structural Investigation of Physically Cross-Linked Nanogels Formed from Self-Associating Polymers.

The detailed structure of a nanogel formed by self-association of cholesterol-bearing pullulans (CHPs) was determined by contrast variation small-angle neutron scattering. The decomposition of scattering intensities into partial scattering functions of each CHP nanogel component, i.e., pullulan, cholesterol, and the cross-term between the pullulan and the cholesterol, allows us to investigate the internal structure of the nanogel. The effective spherical radius of the skeleton formed by pullulan chains was found to be 8.1 ± 0.3 nm. In the CHP nanogel, there are about 19 cross-linking points where a cross-linking point is formed by aggregation of trimer cholesterol molecules, and the spatially inhomogeneous distribution of the cross-linking points in the nanogel can be represented by the mass fractal dimension of 2.6. The average radius of gyration of the partial chains can also be determined to be 1.7 ± 0.1 nm by analyzing the extracted cross-correlation between the cross-linker and the tethered polymer chain quantitatively, and the size agrees with the value assuming random distribution of the cross-linkers on the chains. As the result, the complex structure of the nanogels is coherently revealed at the nanoscopic level.

Comprehensive study of serratia peptidase immobilization from Serratia sp. ZF03 onto chitosan nanogels

Abstract Objective In the present work, we have extended the study and immobilized the metalloprotease enzyme in glutaraldehyde cross-linked chitosan nanogels to scrutinize the enzyme’s features including stability over its soluble free form. Method The immobilized metalloprotease was characterized using scanning electron microscopy (SEM), followed by Fourier transform infrared (FTIR) spectroscopy. The enzyme is optimally active at 50°C and pH range of 8.0–10. Results Thermal stability of the enzyme enhanced when immobilized on the nanogel. After 5 min of incubation at 50°C, immobilized enzymes retained 60% of their original activity, while negligible activity (23%) was observed in the case of the free enzyme. Conclusion The results obtained here provide a powerful demonstration of the benefits of taking the glutaraldehyde cross-linked chitosan matrices to enhance metalloprotease stability. The high stability of the immobilized enzyme serves to improve its performance for possible application on the industrial scale.

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Acyclic polyphenylsilsesquioxane: synthesis and properties

New highly functionalized acyclic hyperbranched polyphenylsilsesquioxanes were obtained and transformed into acyclic nonfunctional or cross-linked nanogel particles with specified outer shell.

Reduction- and pH-Sensitive lipoic acid-modified Poly(l-lysine) and polypeptide/silica hybrid hydrogels/nanogels

We report the preparation of biocompatible, dual stimuli-sensitive lipoic acid-modified poly(l-lysine) (PLL-g-Lipo) hydrogels/nanogels by chemical cross-link and polypeptide/silica hybrid hydrogels/nanogels by simply depositing silica in the gel matrix. Hydrogels were prepared by ethylene oxide sterilization through the formation of inter-/intramolecular disulfide cross-link. Their gelation and mechanical properties were dictated by the degree of inter-/intramolecular disulfide cross-link and amphiphilic nature, which was influenced by the chain conformation. These hydrogels exhibited reduction- and pH-sensitive properties as demonstrated by the accelerated drug release upon changing the solution pH from neutral to acidic condition and/or cleaving disulfide bonds. Swelling studies demonstrated that the genipin and disulfide cross-linked nanogels prepared by miniemulsion exhibited redox- and pH-sensitive properties. Silica deposition resulted in hybrid nanogels exhibiting excellent colloid stability. Silica deposition can not only stabilize the gel network but also tune their mechanical properties as well as their payload release and colloidal properties.

Chemically Cross-Linked Hybrid Nanogels of Alginate and PAMAM Dendrimers as Efficient Anticancer Drug Delivery Vehicles.

Proper choice and design of nanocarriers is imperative to achieve the desired therapeutic benefits. Herein, we report a facile methodology for preparation of chemically cross-linked AG-G5 hybrid nanogels of alginate (AG) and G5.0 poly(amidoamine) (PAMAM) dendrimer via carbodiimide chemistry. The rationale behind the formulation of AG-G5 nanogels is to attain effective and sustained delivery of chemotherapeutic agents. Physical entrapment of anticancer drug epirubicin (EPI) within AG-G5 nanogels endows them with therapeutic properties. Analytical techniques such as zeta potential (ζ) measurements, field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) confirm the integration of PAMAM dendrimer with alginate structure. Thermal, swelling studies, and surface area estimations suggest improved stability and porosity of AG-G5 nanogels. Moreover, AG-G5 nanogels exhibited significantly better mechanical properties and pH dependent release of EPI than AG nanogels. Fluorescence microscopy, cell viability assay, cell cycle analysis and FE-SEM imaging indicated apoptosis inducing potential of EPI⊂AG-G5 nanogels by enhanced intracellular EPI accumulation in breast cancer (MCF-7) cells. Overall, our results put forth AG-G5 hybrid nanogels as prospective candidates to achieve enhanced anticancer effects in vitro.

Volume phase transition of electron beam cross-linked thermo-responsive PVME nanogels in the presence and absence of nanoparticles: with a view toward rheology and interactions

We investigate the effect of nanoparticles and radiation dose on interactions in the PVME-based nanogel system and its phase behavior (swelling/deswelling behavior and phase separation mechanism) by rheological and FTIR measurements.

Waterborne physically crosslinked antimicrobial nanogels

Aqueous solutions of poly(ethylene imine) functionalized with decyl- and azetidinium groups form physically cross-linked antimicrobial nanogels.

Preparation and characterisation of gelatin–gum arabic aldehyde nanogels via inverse miniemulsion technique

Gelatin–gum arabic aldehyde nanogels designed by a nanoreactor concept using inverse miniemulsion technique were reported. Stable separate miniemulsions were prepared from gelatin (Gel) and gum arabic aldehyde (GAA). These emulsions were intermixed under sonication to obtain cross-linked nanogels. During fusion, cross-linking occurred between aldehyde groups of GAA and amino groups of gelatin. The concentration of the surfactant and weight fraction of water in the inverse miniemulsion was optimised so as to yield nanogels with controlled particle size. Properties of the nanogels were studied by FT-IR spectroscopy, particle size analysis and XRD. Surface morphology of the nanogels was established by Scanning Electron Microscopy (SEM). SEM and particle size analysis confirmed that nanogels possess spherical morphology with an average diameter of 151±6nm. Hemolysis property of the nanogels was examined and the results indicated that the nanogels were hemocompatible. The in vitro cytotoxicity of the nanogels towards MCF-7 cells was evaluated by MTT assay and the nanogels showed nontoxic behaviour towards the cells. All these studies confirm that these nanogels are potential candidates in applications such as drug and gene delivery.

Competitive binding-accelerated insulin release from a polypeptide nanogel for potential therapy of diabetes

A novel core cross-linked glycopolypeptide nanogel was prepared for glucose-triggered insulin delivery.

Core cross-linked nanogels based on the self-assembly of double hydrophilic poly(2-oxazoline) block copolymers.

The synthesis of poly(2-oxazoline)-based block copolymers consisting of a cationic and a hydrophilic segment is described. The self-assembly of these macromolecules in organic solvents results in the formation of micelles and vesicles, respectively, depending on the solvent used. To transfer the systems into water, cross-linking using glutaraldehyde was applied, followed by the consumption of excessive aldehyde functions by either diethylamine or 6-aminofluorescein (6AF). The cross-linked assemblies were analyzed regarding their size and shape by electron microscopy and light scattering methods, as well as for their chemical composition by solid state NMR spectroscopy. 6AF associated samples were examined with respect to their absorption and fluorescence behavior in aqueous environment, revealing an alkaline microenvironment within the presented nanostructures. The toxicity of the systems against mouse fibroblast cell line L929 was examined by the XTT assay and was found to be insignificant for concentrations of up to 2.5 mg mL-1. Flow cytometry and fluorescence microscopy analysis revealed an efficient concentration and time dependent cellular uptake of the nanogels.

The effect of polymer nanostructure on diffusion of small molecules using tryptophan as a FRET probe

The amino acid L-tryptophan has been converted into a polymerizable monomer which has been incorporated into a range of cross-linked polymeric nanogels prepared by emulsion polymerization. By using time-lapse fluorescence spectroscopy the diffusion time of a small molecule Forster resonance energy transfer (FRET) pair, dansyl amide, into the central nanogel domain has been monitored through the decreased emission of the L-tryptophan FRET donor. In this initial study it has been found that diffusion of the small molecule into the nanogels is affected by altering the synthetic parameters (cross-linking density and co-monomer hydrophobicity). When increasing the cross-linking density of the nanogels the uptake diffusion time increased, while increasing the hydrophobicity of the co-monomer (and consequently lowering the glass-transition temperature (Tg)) caused a decrease in the diffusion time.

Dendrimer-Assisted Formation of Fluorescent Nanogels for Drug Delivery and Intracellular Imaging

Although, in general, nanogels present a good biocompatibility and are able to mimic biological tissues, their unstability and uncontrollable release properties still limit their biomedical applications. In this study, a simple approach was used to develop dual-cross-linked dendrimer/alginate nanogels (AG/G5), using CaCl2 as cross-linker and amine-terminated generation 5 dendrimer (G5) as a cocrosslinker, through an emulsion method. Via their strong electrostatic interactions with anionic AG, together with cross-linker Ca(2+), G5 dendrimers can be used to mediate the formation of more compact structural nanogels with smaller size (433 ± 17 nm) than that (873 ± 116 nm) of the Ca(2+)-cross-linked AG nanogels in the absence of G5. Under physiological (pH 7.4) and acidic (pH 5.5) conditions, the sizes of Ca(2+)-cross-linked AG nanogels gradually decrease probably because of their degradation, while dual-cross-linked AG/G5 nanogels maintain a relatively more stable structure. Furthermore, the AG/G5 nanogels effectively encapsulate the anticancer drug doxorubicin (Dox) with a loading capacity 3 times higher than that of AG nanogels. The AG/G5 nanogels were able to release Dox in a sustained way, avoiding the burst release observed for AG nanogels. In vitro studies show that the AG/G5-Dox NGs were effectively taken up by CAL-72 cells (a human osteosarcoma cell line) and maintain the anticancer cytotoxicity levels of free Dox. Interestingly, G5 labeled with a fluorescent marker can be integrated into the nanogels and be used to track the nanogels inside cells by fluorescence microscopy. These findings demonstrate that AG/G5 nanogels may serve as a general platform for therapeutic delivery and/or cell imaging.

Studying the activity of the MacMillan catalyst embedded within hydrophobic cross-linked polymeric nanostructures

The immobilization of the MacMillan catalyst within a unique hydrophobic environment created by a lightly cross-linked nanogel structure and its resulting catalytic activity is reported.

In Situ Forming Reduction-Sensitive Degradable Nanogels for Facile Loading and Triggered Intracellular Release of Proteins

In situ forming reduction-sensitive degradable nanogels were designed and developed based on poly(ethylene glycol)-b-poly(2-(hydroxyethyl) methacrylate-co-acryloyl carbonate) (PEG-P(HEMA-co-AC)) block copolymers for efficient loading as well as triggered intracellular release of proteins. PEG-P(HEMA-co-AC) copolymers were prepared with controlled Mn of 9.1, 9.5, and 9.9 kg/mol and varying numbers of AC units per molecule of 7, 9 and 11, respectively (denoted as copolymer 1, 2, and 3) by reversible addition-fragmentation chain transfer copolymerization. These copolymers were freely soluble in phosphate buffer but formed disulfide-cross-linked nanogels with defined sizes ranging from 72.5 to 124.1 nm in the presence of cystamine via ring-opening reaction with cyclic carbonate groups. The sizes of nanogels decreased with increasing AC units as a result of increased cross-linking density. Dynamic light scattering studies showed that these nanogels though stable at physiological conditions were rapidly dissociated in response to 10 mM dithiothreitol (DTT). Interestingly, FITC-labeled cytochrome C (FITC-CC) could be readily loaded into nanogels with remarkable loading efficiencies (up to 98.2%) and loading contents (up to 48.2 wt.%). The in vitro release studies showed that release of FITC-CC was minimal under physiological conditions but significantly enhanced under reductive conditions in the presence of 10 mM DTT with about 96.8% of FITC-CC released in 22 h from nanogel 1. In contrast, protein release from 1,4-butanediamine cross-linked nanogels (reduction-insensitive control) remained low under otherwise the same conditions. MTT assays showed that these nanogels were nontoxic to HeLa cells up to a tested concentration of 2 mg/mL. Confocal microscopy results showed that nanogel 1 delivered and released FITC-CC into the perinuclei region of HeLa cells following 8 h incubation. CC-loaded reductively degradable nanogels demonstrated apparently better apoptotic activity than free CC as well as reduction-insensitive controls. These in situ forming, surfactant and oil-free, and reduction-sensitive degradable nanogels are highly promising for targeted protein therapy.

Synthesis of Nanogel-Protein Conjugates.

The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics.

Synthesis of new enzymatically degradable thermo-responsive nanogels

Two new families of thermo-responsive and enzymatically degradable nanogels were synthesized by batch emulsion polymerization of N-vinylcaprolactam (VCL) with dextran methacrylates (Dex-MA) with different degrees of substitution (DS). The first family was prepared using different amounts of Dex-MA with high DS forming highly cross-linked nanogel particles with the typical thermal behavior of PVCL-based nanogels: below the volume phase transition temperature (VPTT) nanogel particles were swollen and above it they were collapsed. After their enzymatic degradation with dextranase, nanogel particles swelled due to the cleavage of some glucopyranosyl bonds of dextran, but preserved their identity. On the other hand, the second family was prepared using different amounts of Dex-MA with low DS forming slightly cross-linked nanogel particles with an anomalous thermal behavior. Surprisingly, above the VPTT of the nanogel particles monodisperse interparticle reversible aggregates were formed. In addition, after their enzymatic degradation, a release of reducing sugars together with an intense de-swelling due to the fragmentation of the nanogel structure was observed. Both nanogel families could be suitable for drug delivery in tissues or organs where dextranase is present.

Bio-Derived Poly(γ-Glutamic Acid) Nanogels as Controlled Anticancer Drug Delivery Carriers

We have developed a novel type of polymer nanogel loaded with anticancer drug based on bio-derived poly(gamma- glutamic acid) (gamma-PGA). gamma-PGA is a highly anionic polymer that is synthesized naturally by microbial species, most prominently in various bacilli, and has been shown to have excellent biocompatibility. Thiolated gamma-PGA was synthesized by covalent coupling between the carboxyl groups of gamma-PGA and the primary amine group of cysteamine. Doxorubicin (Dox)-loaded gamma-PGA nanogels were fabricated using the following steps: (1) an ionic nanocomplex was formed between thiolated gamma-PGA as the negative charge component, and Dox as the positive charge component; (2) addition of poly(ethylene glycol) (PEG) induced hydrogen-bond interactions between thiol groups of thiolated gamma-PGA and hydroxyl groups of PEG, resulting in the nanocomplex; and (3) disulfide crosslinked gamma-PGA nanogels were fabricated by ultrasonication. The average size and surface charge of Dox-loaded disulfide cross-linked gamma-PGA nanogels in aqueous solution were 136.3 +/- 37.6 nm and -32.5 +/- 5.3 mV, respectively. The loading amount of Dox was approximately 38.7 microgram per mg of gamma-PGA nanogel. The Dox-loaded disulfide cross-linked gamma-PGA nanogels showed controlled drug release behavior in the presence of reducing agents, glutathione (GSH) (1- 10 mM). Through fluorescence microscopy and FACS, the cellular uptake of gamma-PGA nanogels into breast cancer cells (MCF-7) was analyzed. The cytotoxic effect was evaluated using the MTT assay and was determined to be dependent on both the concentration and treatment time of gamma-PGA nanogels. The bio-derived gamma-PGA nanogels are expected to be a well-designed delivery carrier for controlled drug delivery applications.

Gallic acid loaded disulfide cross-linked biocompatible polymeric nanogels as controlled release system: synthesis, characterization, and antioxidant activity

In this article, a sustained release formulation of the antioxidant gallic acid (GA) is presented in the form of glutathione responsive disulfide cross-linked poly(ethylene glycol)-based nanogels synthesized via aqueous inverse miniemulsion using atom transfer radical polymerization. The particle size was found to be in the range from 227 ± 51.78 to 573.3 ± 207.2 nm at three drug loading levels achieved i.e. 6.6, 14.26, and 18.29 wt.% of the nanogels with loading efficiency in the range of 60–70%. The release profile of the GA studied at three drug loading levels suggested a controlled release and the nanogels were capable of scavenging radicals and retained the antioxidant activity. The GA-loaded nanogels were found to be biocompatible on human cervical cancer cell lines (HeLa). DCFH-DA (2,7-dichlorofluorescin diacetate) assay evidenced that the nanogels were capable of scavenging the reactive oxygen species in cellular environment.