Fast-Curing Injectable Microporous Hydrogel for In Situ Cell Encapsulation.

Injectable hydrogels have previously demonstrated potential as a temporary scaffold for tissue regeneration or as a delivery vehicle for cells, growth factors, or drugs. However, most injectable hydrogel systems lack a microporous structure, preventing host cell migration into the hydrogel interior and limiting spreading and proliferation of encapsulated cells. Herein, an injectable microporous hydrogel assembled from gelatin/gelatin methacryloyl (GelMA) composite microgels is described. Microgels are produced by a water-in-oil emulsion using a gelatin/GelMA aqueous mixture. These microgels show improved thermal stability compared to GelMA-only microgels and benefit from combined photopolymerization using UV irradiation (365 nm) in the presence of a photoinitiator (PI) and enzymatic reaction by microbial transglutaminase (mTG), which together enable fast curing and tissue adhesion of the hydrogel. The dual-crosslinking approach also allows for the reduction of PI concentration and minimizes cytotoxicity during photopolymerization. When applied for in situ cell encapsulation, encapsulated human dermal fibroblasts and human mesenchymal stem cells (hMSCs) are able to rapidly spread and proliferate in the pore space of the hydrogel. This hydrogel has the potential to enhance hMSC anti-inflammatory behavior through the demonstrated secretion of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) by encapsulated cells. Altogether, this injectable formulation has the potential to be used as a cell delivery vehicle for various applications in regenerative medicine.

Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic materials.

Bioinspired from adhesion behaviors of mussels, injectable and thermosensitive chitosan/Pluronic composite hydrogels were synthesized for tissue adhesives and hemostatic materials. Chitosan conjugated with multiple catechol groups in the backbone was cross-linked with terminally thiolated Pluronic F-127 triblock copolymer to produce temperature-sensitive and adhesive sol-gel transition hydrogels. A blend mixture of the catechol-conjugated chitosan and the thiolated Pluronic F-127 was a viscous solution state at room temperature but became a cross-linked gel state with instantaneous solidification at the body temperature and physiological pH. The adhesive chitosan/Pluronic injectable hydrogels with remnant catechol groups showed strong adhesiveness to soft tissues and mucous layers and also demonstrated superior hemostatic properties. These chitosan/Pluronic hydrogels are expected to be usefully exploited for injectable drug delivery depots, tissue engineering hydrogels, tissue adhesives, and antibleeding materials.

Thermally triggered cellular uptake of quantum dots immobilized with poly(N-isopropylacrylamide) and cell penetrating peptide.

Thermally sensitive quantum dots (TSQDs) that exhibit an "on-demand" cellular uptake behavior via temperature-induced "shielding/deshielding" of cell penetrating peptides (CPP) on the surface were fabricated. Poly(N-isopropylacrylamide) (PNIPAAm) (M(w) = 11.5K) and CPP were biotinylated at their terminal ends and co-immobilized on to the surface of streptavidin-coated quantum dots (QDs-Strep) through biotin-streptavidin interaction. The cellular contact of CPP was sterically hindered due to hydrated PNIPAAm chains below the lower critical solution temperature (LCST). In contrast, above the LCST, grafted PNIPAAm chains were collapsed to make CPP moieties resurfaced, leading to increased cellular uptake of QDs. The temperature-controlled "shielding/deshielding" of CPP was further applied for a thermally triggered siRNA delivery system, where biotinylated siRNA was additionally conjugated to the surface of TSQDs. The level of gene silencing was significantly enhanced by increasing temperature above the LCST due to the surface exposure of CPP.

Genetically Programmable Self-Regenerating Bacterial Hydrogels.

A notable challenge for the design of engineered living materials (ELMs) is programming a cellular system to assimilate resources from its surroundings and convert them into macroscopic materials with specific functions. Here, an ELM that uses Escherichia coli as its cellular chassis and engineered curli nanofibers as its extracellular matrix component is demonstrated. Cell-laden hydrogels are created by concentrating curli-producing cultures. The rheological properties of the living hydrogels are modulated by genetically encoded factors and processing steps. The hydrogels have the ability to grow and self-renew when placed under conditions that facilitate cell growth. Genetic programming enables the gels to be customized to interact with different tissues of the gastrointestinal tract selectively. This work lays a foundation for the application of ELMs with therapeutic functions and extended residence times in the gut.

Purification of lysozyme from chicken egg white using nanofiber membrane immobilized with Reactive Orange 4 dye.

Nanofiber membrane chromatography integrates liquid membrane chromatography and nanofiber filtration into a single-step purification process. Nanofiber membrane can be functionalised with affinity ligands for promoting binding specificity of membrane. Dye molecules are a good affinity ligand for nanofiber membrane due to their low cost and high binding affinity. In this study, a dye-affinity nanofiber membrane (P-Chitosan-Dye membrane) was prepared by using polyacrylonitrile nanofiber membrane modified with chitosan molecules and immobilized with dye molecules. Reactive Orange 4, commercially known as Procion Orange MX2R, was found to be the best dye ligand for membrane chromatography. The binding capacity of P-Chitosan-Dye membrane for lysozyme was investigated under different operating conditions in batch mode. Furthermore, desorption of lysozyme using the P-Chitosan-Dye membrane was evaluated systematically. The recovery percentage of lysozyme was found to be ~100%. The optimal conditions obtained from batch-mode study were adopted to develop a purification process to separate lysozyme from chicken egg white. The process was operated continuously using the membrane chromatography and the characteristic of the breakthrough curve was evaluated. At a lower flow rate (i.e., 0.1 mL/min), the total recovery of lysozyme and purification factor of lysozyme were 98.59% and 56.89 folds, respectively.

Novel stereocomplexed sol-gel transition hydrogels prepared from physical packing of self-assembled PEO-PPO-PEO and PPO-PEO-PPO copolymer nanoscale micelles.

A new class of stereocomplex-induced hydrogels was synthesized based on self-assembled PEO-PPO-PEO and PPO-PEO-PPO copolymers having multiple hydroxyl groups in the PPO segment. The copolymers were grafted with enantiomeric oligo(L-lactic acid) or oligo(D-lactic acid) chains using ring opening polymerization onto the PPO segment. WAXS and DSC analysis were used to investigate the stereocomplex formation of the equimolar mixture of the copolymers. The copolymer hydrogels having stereocomplexed crystalline domains as crosslinking loci exhibited significantly altered temperature-sensitive sol-gel transition behaviors, and showed more delayed erosion and sustained protein release behaviors, compared to Pluronic F127 copolymer hydrogels. The current hydrogels are expected to be used as useful injectable hydrogel materials for macromolecular drug delivery.

Bioinspired Nanoparticulate Medical Glues for Minimally Invasive Tissue Repair.

Delivery of tissue glues through small-bore needles or trocars is critical for sealing holes, affixing medical devices, or attaching tissues together during minimally invasive surgeries. Inspired by the granule-packaged glue delivery system of sandcastle worms, a nanoparticulate formulation of a viscous hydrophobic light-activated adhesive based on poly(glycerol sebacate)-acrylate is developed. Negatively charged alginate is used to stabilize the nanoparticulate surface to significantly reduce its viscosity and to maximize injectability through small-bore needles. The nanoparticulate glues can be concentrated to ≈30 w/v% dispersions in water that remain localized following injection. With the trigger of a positively charged polymer (e.g., protamine), the nanoparticulate glues can quickly assemble into a viscous glue that exhibits rheological, mechanical, and adhesive properties resembling the native poly(glycerol sebacate)-acrylate based glues. This platform should be useful to enable the delivery of viscous glues to augment or replace sutures and staples during minimally invasive procedures.

Chitosan-g-hematin: enzyme-mimicking polymeric catalyst for adhesive hydrogels.

Phenol derivative-containing adhesive hydrogels has been widely recognized as having potential for biomedical applications, but their conventional production methods, utilizing a moderate/strong base, alkaline buffers, the addition of oxidizing agents or the use of enzymes, require alternative approaches to improve their biocompatibility. In this study, we report a polymeric, enzyme-mimetic biocatalyst, hematin-grafted chitosan (chitosan-g-hem), which results in effective gelation without the use of alkaline buffers or enzymes. Furthermore, gelation occurs under mild physiological conditions. Chitosan-g-hem biocatalyst (0.01%, w/v) has excellent catalytic properties, forming chitosan-catechol hydrogels rapidly (within 5 min). In vivo adhesive force measurement demonstrated that the hydrogel formed by the chitosan-g-hem activity showed an increase in adhesion force (33.6 ± 5.9 kPa) compared with the same hydrogel formed by pH-induced catechol oxidation (20.6 ± 5.5 kPa) in mouse subcutaneous tissue. Using the chitosan-g-hem biocatalyst, other catechol-functionalized polymers (hyaluronic acid-catechol and poly(vinyl alcohol)-catechol) also formed hydrogels, indicating that chitosan-g-hem can be used as a general polymeric catalyst for preparing catechol-containing hydrogels.

A blood-resistant surgical glue for minimally invasive repair of vessels and heart defects.

Currently, there are no clinically approved surgical glues that are nontoxic, bind strongly to tissue, and work well within wet and highly dynamic environments within the body. This is especially relevant to minimally invasive surgery that is increasingly performed to reduce postoperative complications, recovery times, and patient discomfort. We describe the engineering of a bioinspired elastic and biocompatible hydrophobic light-activated adhesive (HLAA) that achieves a strong level of adhesion to wet tissue and is not compromised by preexposure to blood. The HLAA provided an on-demand hemostatic seal, within seconds of light application, when applied to high-pressure large blood vessels and cardiac wall defects in pigs. HLAA-coated patches attached to the interventricular septum in a beating porcine heart and resisted supraphysiologic pressures by remaining attached for 24 hours, which is relevant to intracardiac interventions in humans. The HLAA could be used for many cardiovascular and surgical applications, with immediate application in repair of vascular defects and surgical hemostasis.

Self-assembled siRNA-PLGA conjugate micelles for gene silencing.

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3' end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA-PLGA hybrid conjugates. siRNA-PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA-PLGA micelles in aqueous solution, stable siRNA-PLGA/LPEI micelles with a size of ~30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA-PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.

Controlled synthesis of PEI-coated gold nanoparticles using reductive catechol chemistry for siRNA delivery.

Development of nano-sized gene delivery vehicles for small interfering RNA (siRNA) delivery is of great importance for their clinical applications such as cancer therapy. Herein, we demonstrate the controlled synthesis of polyethyleneimine (PEI)-coated gold nanoparticles (AuNPs) using catechol-conjugated PEI (PEI-C) for siRNA delivery. Since the conjugated catechol groups are reductive and moderately hydrophobic, PEI-C formed spherical multi-cored micelles in aqueous solution and served as reductive templates for the growth and synthesis of spherical AuNPs with tunable sizes and surface charges. PEI-C was stably anchored on the surface of growing crystal gold seeds with crosslinking, resulting in robust cationic AuNPs. The fabricated PEI-coated AuNPs formed stable complexes with siRNA, and the complexes showed an excellent gene silencing effect in cancer cells. Size and surface charge values of the synthesized AuNPs had a great influence on intracellular uptake and unpacking of siRNA, and the resultant gene silencing efficiency. The PEI-coated AuNPs exhibited an extremely low cytotoxicity due to the reduced density of primary amine groups and the absence of uncomplexed PEI fraction in aqueous solution.

Enzyme-mediated cross-linking of Pluronic copolymer micelles for injectable and in situ forming hydrogels.

A new class of injectable and erodible hydrogels exhibiting highly robust gel strength at body temperature was fabricated by enzyme-mediated cross-linking between Pluronic copolymer micelles. Tyramine-conjugated Pluronic F-127 tri-block copolymers at two terminal ends of polyethylene oxide (PEO) side chains were synthesized and utilized to form self-assembled micelles in aqueous solution. Tyrosinase was employed to convert tyramine-conjugated micelles to highly reactive catechol conjugated micelles that could further cross-link individual Pluronic copolymer micelles to form a highly stable gel structure. The enzyme cross-linked Pluronic hydrogels showed far lower critical gelation concentration, concomitantly showing enhanced gel strength compared to unmodified Pluronic copolymer hydrogels, suitable for sustained delivery of bioactive agents. Rheological studies demonstrated that the enzyme cross-linked hydrogels exhibited a fast and reversible sol-gel transition in response to temperature while maintaining sufficient mechanical strength at the gel state. In situ formed hydrogels were eroded gradually, releasing FITC-labeled dextran in an erosion-controlled manner. Moreover, they showed tissue-adhesive properties due to the presence of unreacted catechol groups in the gel structure. Enzyme cross-linked Pluronic hydrogels could be potentially used for delivery applications of drugs and cells.

Facile synthetic route for surface-functionalized magnetic nanoparticles: cell labeling and magnetic resonance imaging studies.

Currently available methods to stably disperse iron oxide nanoparticles (IONPs) in aqueous solution need to be improved due to potential aggregation, reduction of superparamagnetism, and the use of toxic reagents. Herein, we present a facile strategy for aqueous transfer and dispersion of organic-synthesized IONPs using only polyethylene glycol (PEG), a biocompatible polymer. A library of PEG derivatives was screened, and it was determined that amine-functionalized six-armed PEG, 6(PEG-NH(2)), was the most effective dispersion agent. The 6(PEG-NH(2))-modified IONPs (IONP-6PEG) were stable after extensive washing, exhibited high superparamagnetism, and could be used as a platform material for secondary surface functionalization with bioactive polymers. IONP-6PEG biofunctionalized with hyaluronic acid (IONP-6PEG-HA) was shown to specifically label mesenchymal stem cells and demonstrate MR contrast potential with high r(2) relaxivity (442.7 s(-1)mM(-1)) compared to the commercially available Feridex (182.1 s(-1)mM(-1)).

pH-Sensitive pentablock copolymer nanocapsules as nontoxic and efficient gene carriers.

A biodegradable amphiphilic pentablock copolymer PAE-PCL-PEG-PCL-PAE with a pH-sensitive unit was synthesized for use as a nontoxic, biodegradable carrier for gene delivery by forming nanocapsules entrapping nucleic acid drugs. The PAE block can interact with plasmid DNA to form polyelectrolyte complexes in an acidic environment. At physiological pH, the PAE blocks are deprotonated and form an insoluble skin, resulting in the formation of nanocapsules that encapsulate plasmid DNA. The surface charges of the nanocapsules became almost neutral at pH = 7.4, and their size ranged from 210 to 280 nm. The nanocapsule maintained most of its transfection efficiency even in the presence of serum. These nanocapsules are therefore potential carriers for systemic gene therapy.

Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides.

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics.

Controlled gene-eluting metal stent fabricated by bio-inspired surface modification with hyaluronic acid and deposition of DNA/PEI polyplexes.

A metal stent that could elute plasmid DNA (pDNA) in a controlled manner for substrate-mediated gene transfection was fabricated by first coating with hyaluronic acid (HA) and subsequent deposition of pDNA. To create robust HA coating layer on stainless steel (SS316L) surface, HA was derivatized with dopamine which is a well-known adsorptive molecule involving mussel adhesion process. The HA-coated surface was verified by various analytical techniques and proved to be very hydrophilic and stable, also showing superior biocompatibility in terms of suppressed plasma protein adsorption. For surface loading of pDNA, cationic pDNA/polyethylenimine (PEI) polyplexes were prepared and ionically adsorbed onto the HA-coated SS316L surface. The adsorbed surface exhibited evenly distributed nano-granular topography while the polyplexes maintained the nano-particular morphology. The pDNA was released out in a controlled manner for a period of 10 days with maintaining structural integrity. The dual coated substrate with HA and pDNA/PEI polyplexes exhibited greatly enhanced gene transfection efficiency, when compared to both bare substrate adsorbed with the polyplexes and PEI/pDNA polyelectrolyte multilayers. Dually functionalized stent with HA and pDNA exhibited effective biocompatibility and gene transfection.

Pluronic/polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals for magnetically triggered delivery of siRNA.

Pluronic/polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals (PPMCs) were developed for magnetically triggered delivery of siRNA. The positively charged PPMCs formed stable nanosized polyelectrolyte complexes via electrostatic interactions with negatively charged siRNA-polyethylene glycol conjugate (siRNA-s-s-PEG) that was linked via a cleavable disulfide linkage. PPMC/siRNA-s-s-PEG polyelectrolyte complexes were efficiently taken up by cancer cells upon exposure to a magnet, thereby enhancing intracellular uptake and silencing effect of siRNA. The present study suggests that these novel nanomaterials could be potentially utilized for magnetically triggered delivery of various nucleic acid-based therapeutic agents.

Intracellular delivery of heparin complexed with chitosan-g-poly(ethylene glycol) for inducing apoptosis.

PURPOSE: Chitosan-g-PEG/heparin polyelectrolyte complex micelles were prepared for inducing apoptotic death of cancer cells. MATERIALS AND METHODS: The cytotoxicity of polyelectrolyte complex micelles was evaluated by examining the growth inhibition of mouse melanoma B16F10 cells. Cellular uptake and apoptosis-inducing effect were investigated by confocal laser scanning microscopy and flow cytometric analysis, respectively. RESULTS: The prepared polyelectrolyte complex micelles had a spherical shape with an average diameter of 162.8 +/- 18.9 nm. They were highly stable and well dispersed even in the presence of serum due to the presence of hydrophilic PEG shell layer surrounding the micelles. Moreover, they exhibited significantly higher cytotoxic activity against B16F10 cells compared to heparin or chitosan-g-PEG at the same concentration. The polyelectrolyte complex micelles were internalized by cancer cells to a greater extent than free heparin alone, indicating that the dramatic cell death was attributed to the increased cellular uptake of heparin. The internalized heparin was shown to induce apoptotic death of the cancer cells via a caspase-dependent pathway. CONCLUSIONS: Nanosized and stable chitosan-g-PEG/heparin polyelectrolyte complex micelles were produced by a self-assembly process. The polyelectrolyte complex micelles facilitated the intracellular delivery of heparin, triggered the caspase activation, and consequently promoted apoptotic death of cancer cells.

Thermally triggered intracellular explosion of volume transition nanogels for necrotic cell death.

Here we report a novel class of super-expandable smart nanogels that undergo nano- to micro-scale volume transition in response to temperature change, applicable for thermally triggered death of cancer cells. The nanogels fabricated by light cross-linking between oligo(L-lactic acid)-poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)-oligo(L-lactic acid) and poly(ethylene glycol) grafted poly(L-lysine) exhibited a reversible volume transition from approximately 150 nm at 37 degrees C to approximately 1.4 microm at 15 degrees C. The abrupt and dramatic intracellular volume expansion of the nanogels upon the brief cold-shock treatment severely damaged sub-cellular self-assembled network architectures including cytoskeleton and vesicular membranes and physically ruptured cellular membrane structures, resulting in necrotic cell death. The smart "nanobomb" detonated by external stimuli could be used to effectively induce traumatic death of cells.

Thermo-sensitive and biodegradable hydrogels based on stereocomplexed Pluronic multi-block copolymers for controlled protein delivery.

Injectable sol-gel transition hydrogels based on thermo-sensitive polymers are of great interest as potential biomaterials for sustained delivery of therapeutic molecules. A novel temperature-sensitive and in-situ forming hydrogel system based on Pluronic F127 was developed and evaluated. A series of multi-block Pluronic copolymers linked by d-lactide and l-lactide oligomers with different spacer lengths were synthesized. A pair of multi-block copolymers having the corresponding enantiomeric d- or l-lactide oligomer spacer was blended to form stereocomplexed hydrogels. The resultant physically crosslinked Pluronic hydrogels exhibited significantly altered sol-gel phase transition behaviors with much lower critical gelation concentrations and temperatures, compared to the uncomplexed multi-block or Pluronic homopolymer hydrogels. The stereocomplexed hydrogels also had far increased mechanical strength with high resistance to rapid dissolution in aqueous medium. When human growth hormone (hGH) was incorporated in the strereocomplexed multi-block Pluronic copolymers, hGH was released out in a sustained and zero-order fashion for 13 days by a diffusion/erosion coupled mechanism.