Supramolecularly-Cross-Linked Nanogel Assemblies for On-Demand, Ultrasound-Triggered Chemotherapy.

Stimulating the release of small nanoparticles (NPs) from a larger NP via the application of an exogenous stimulus offers the potential to address the different size requirements for circulation versus penetration that hinder chemotherapeutic drug delivery. Herein, we report a size-switching nanoassembly-based drug delivery system comprised of ultrasmall starch nanoparticles (SNPs, ∼20-50 nm major size fraction) encapsulated in a poly(oligo(ethylene glycol) methyl ether methacrylate) nanogel (POEGMA, ∼150 nm major size fraction) cross-linked via supramolecular PEG/α-cyclodextrin (α-CD) interactions. Upon heating the nanogel using a non-invasive, high-intensity focused ultrasound (HIFU) trigger, the thermoresponsive POEGMA-CD nanoassemblies are locally de-cross-linked, inducing in situ release of the highly penetrative drug-loaded SNPs. HIFU triggering increased the release of nanoassembly-loaded DOX from 17 to 37% after 3 h, a result correlated with significantly more effective tumor killing relative to nanoassemblies in the absence of HIFU or drug alone. Furthermore, 1.5× more total fluorescence was observed inside a tumor spheroid when nanoassemblies prepared with fluorophore-labeled SNPs were triggered with HIFU relative to the absence of HIFU. We anticipate this strategy holds promise for delivering tunable doses of chemotherapeutic drugs both at and within a tumor site using a non-invasive triggering approach.

Microgels and Nanogels for the Delivery of Poorly Water-Soluble Drugs.

While microgels and nanogels are most commonly used for the delivery of hydrophilic therapeutics, the water-swollen structure, size, deformability, colloidal stability, functionality, and physicochemical tunability of microgels can also offer benefits for addressing many of the barriers of conventional vehicles for the delivery of hydrophobic therapeutics. In this review, we describe approaches for designing microgels with the potential to load and subsequently deliver hydrophobic drugs by creating compartmentalized microgels (e.g., core-shell structures), introducing hydrophobic domains in microgels, leveraging host-guest interactions, and/or applying "smart" environmentally responsive materials with switchable hydrophobicity. In particular, the challenge of promoting hydrophobic drug loading without compromising the inherent advantages of microgels as delivery vehicles and ensuring practically relevant release kinetics from such structures is highlighted, with an eye toward the practical translation of such vehicles to the clinic.

A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout.

Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4 days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6 nM (0.37 µg mL-1 ) in saliva-spiked PEDv samples. Clinical evaluation of this biosensor with 12 porcine saliva samples demonstrated a diagnostic sensitivity of 83 % and specificity of 100 % with a concordance value of 92 % at an analysis time of one hour.

Effect of Reaction Media on Grafting Hydrophobic Polymers from Cellulose Nanocrystals via Surface-Initiated Atom-Transfer Radical Polymerization.

Hydrophobic polymer-grafted cellulose nanocrystals (CNCs) were produced via surface-initiated atom-transfer radical polymerization (SI-ATRP) in two different solvents to examine the role of reaction media on the extent of surface modification. Poly(butyl acrylate)-grafted CNCs were synthesized in either dimethylformamide (DMF) (D-PBA-g-CNCs) or toluene (T-PBA-g-CNCs) alongside a free polymer from a sacrificial initiator. The colloidal stability of unmodified CNCs, initiator-modified CNCs, and PBA-g-CNCs in water, DMF, and toluene was evaluated by optical transmittance. The enhanced colloidal stability of initiator-modified CNCs in DMF led to improved accessibility to initiator groups during polymer grafting; D-PBA-g-CNCs had 30 times more grafted chains than T-PBA-g-CNCs, determined by thermogravimetric and elemental analysis. D-PBA-g-CNCs dispersed well in toluene and were hydrophobic with a water contact angle of 124° (for polymer grafts > 13 kDa) compared to 25° for T-PBA-g-CNCs. The cellulose crystal structure was preserved, and individual nanoparticles were retained when grafting was carried out in either solvent. This work highlights that optimizing CNC colloidal stability prior to grafting is more crucial than solvent-polymer compatibility to obtain high graft densities and highly hydrophobic CNCs via SI-ATRP.

High-Throughput Synthesis, Analysis, and Optimization of Injectable Hydrogels for Protein Delivery.

The development of in situ-gelling hydrogels that can enable prolonged protein release is increasingly important due to the emergence of a growing number of protein-based therapeutics. Herein, we describe a high-throughput strategy to fabricate, characterize, and subsequently optimize hydrazone-cross-linked in situ-gelling hydrogels for protein delivery. Hydrogels are fabricated using an automated high-throughput robot to mix a variety of thermoresponsive, nonthermoresponsive, charged, neutral, naturally sourced, and synthetic polymers functionalized with hydrazide or aldehyde groups, generating in situ-gelling hydrogels with well-defined compositions within a 96-well plate. High-throughput characterization strategies are subsequently developed to enable on-plate analysis of hydrogel swelling, mechanics, degradation, transparency, and protein (ovalbumin) release kinetics that yield results consistent with those collected using traditional bulk hydrogel analysis techniques. Dynamic regression and latent variable modeling are then applied to fit performance statistics to the collected data set; subsequently, numerical optimization is used to identify mixtures of precursor polymers that exhibit targeted combinations of minimal burst release, maximum total protein release, minimum release rate, and maximum transparency (the latter of particular relevance for ophthalmic protein delivery applications). Given the rapid throughput of the protocols developed (i.e., 126 hydrogels can be synthesized and screened in quadruplicate within hours), this approach offers particular promise for accelerating the identification of injectable hydrogel compositions relevant for both protein delivery as well as other biomedical applications for which clearly predefined materials properties are required.

Dynamically Cross-Linked Self-Assembled Thermoresponsive Microgels with Homogeneous Internal Structures.

The internal morphology of temperature-responsive degradable poly(N-isopropylacrylamide) (PNIPAM) microgels formed via an aqueous self-assembly process based on hydrazide and aldehyde-functionalized PNIPAM oligomers is investigated. A combination of surface force measurements, small angle neutron scattering (SANS), and ultrasmall angle neutron scattering (USANS) was used to demonstrate that the self-assembled microgels have a homogeneously cross-linked internal structure. This result is surprising given the sequential addition process used to fabricate the microgels, which was expected to result in a densely cross-linked shell-diffuse core structure. The homogeneous internal structure identified is also significantly different than conventional microgels prepared via precipitation polymerization, which typically exhibit a diffuse shell-dense core structure. The homogeneous structure is hypothesized to result from the dynamic nature of the hydrazone cross-linking chemistry used to couple with the assembly conditions chosen that promote polymer interdiffusion. The lack of an internal cross-linking gradient within these degradable and monodisperse microgels is expected to facilitate more consistent drug release over time, improved optical properties, and other potential application benefits.

Single-Step Reactive Electrospinning of Cell-Loaded Nanofibrous Scaffolds as Ready-to-Use Tissue Patches.

A reactive electrospinning strategy is used to fabricate viable and proliferative cell-loaded nanofibrous hydrogel scaffolds in a single step using an all-aqueous approach. In situ gelling and degradable hydrazone-cross-linked poly(oligo ethylene glycol methacrylate)-based hydrogel nanofibrous networks can be produced directly in the presence of cells to support long-term cell viability, adhesion, and proliferation, in contrast to bulk hydrogels of the same composition. Furthermore, the capacity of the gel nanofibers to retain bound water maintains this high cell viability and proliferative capacity following a freeze/thaw cycle without requiring any cryoprotectant additives, ideal properties for ready-to-use functional tissue patches.

Patterning of Structurally Anisotropic Composite Hydrogel Sheets.

Compositional and structural patterns play a crucial role in the function of many biological tissues. In the present work, for nanofibrillar hydrogels formed by chemically cross-linked cellulose nanocrystals (CNC) and gelatin, we report a microextrusion-based 3D printing method to generate structurally anisotropic hydrogel sheets with CNCs aligned in the direction of extrusion. We prepared hydrogels with a uniform composition, as well as hydrogels with two different types of compositional gradients. In the first type of gradient hydrogel, the composition of the sheet varied parallel to the direction of CNC alignment. In the second hydrogel type, the composition of the sheet changed orthogonally to the direction of CNC alignment. The hydrogels exhibited gradients in structure, mechanical properties, and permeability, all governed by the compositional patterns, as well as cytocompatibility. These hydrogels have promising applications for both fundamental research and for tissue engineering and regenerative medicine.

Autonomously Self-Adhesive Hydrogels as Building Blocks for Additive Manufacturing.

We report a simple method of preparing autonomous and rapid self-adhesive hydrogels and their use as building blocks for additive manufacturing of functional tissue scaffolds. Dynamic cross-linking between 2-aminophenylboronic acid-functionalized hyaluronic acid and poly(vinyl alcohol) yields hydrogels that recover their mechanical integrity within 1 min after cutting or shear under both neutral and acidic pH conditions. Incorporation of this hydrogel in an interpenetrating calcium-alginate network results in an interfacially stiffer but still rapidly self-adhesive hydrogel that can be assembled into hollow perfusion channels by simple contact additive manufacturing within minutes. Such channels withstand fluid perfusion while retaining their dimensions and support endothelial cell growth and proliferation, providing a simple and modular route to produce customized cell scaffolds.

Enhanced Mechanical Properties in Cellulose Nanocrystal-Poly(oligoethylene glycol methacrylate) Injectable Nanocomposite Hydrogels through Control of Physical and Chemical Cross-Linking.

While injectable hydrogels have several advantages in the context of biomedical use, their generally weak mechanical properties often limit their applications. Herein, we describe in situ-gelling nanocomposite hydrogels based on poly(oligoethylene glycol methacrylate) (POEGMA) and rigid rod-like cellulose nanocrystals (CNCs) that can overcome this challenge. By physically incorporating CNCs into hydrazone cross-linked POEGMA hydrogels, macroscopic properties including gelation rate, swelling kinetics, mechanical properties, and hydrogel stability can be readily tailored. Strong adsorption of aldehyde- and hydrazide-modified POEGMA precursor polymers onto the surface of CNCs promotes uniform dispersion of CNCs within the hydrogel, imparts physical cross-links throughout the network, and significantly improves mechanical strength overall, as demonstrated by quartz crystal microbalance gravimetry and rheometry. When POEGMA hydrogels containing mixtures of long and short ethylene oxide side chain precursor polymers were prepared, transmission electron microscopy reveals that phase segregation occurs with CNCs hypothesized to preferentially locate within the stronger adsorbing short side chain polymer domains. Incorporating as little as 5 wt % CNCs results in dramatic enhancements in mechanical properties (up to 35-fold increases in storage modulus) coupled with faster gelation rates, decreased swelling ratios, and increased stability versus hydrolysis. Furthermore, cell viability can be maintained within 3D culture using these hydrogels independent of the CNC content. These properties collectively make POEGMA-CNC nanocomposite hydrogels of potential interest for various biomedical applications including tissue engineering scaffolds for stiffer tissues or platforms for cell growth.

Properties of Poly(ethylene glycol) Hydrogels Cross-Linked via Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC).

A series of poly(ethylene glycol) (PEG) hydrogels was synthesized using strain-promoted alkyne-azide cycloaddition (SPAAC) between PEG chains terminated with either aza-dibenzocyclooctynes or azide functionalities. The gelation process was found to occur rapidly upon mixing the two components in aqueous solution without the need for external stimuli or catalysts, making the system a candidate for use as an injectable hydrogel. The mechanical and rheological properties of these hydrogels were found to be tunable by varying the polymer molecular weight and the number of cross-linking groups per chain. The gelation times of these hydrogels ranged from 10 to 60 s at room temperature. The mass-based swelling ratios varied from 45 to 76 at maximum swelling (relative to the dry state), while the weight percent of polymer in these hydrogels ranged from 1.31 to 2.05%, demonstrating the variations in amount of polymer required to maintain the structural integrity of the gel. Each hydrogel degraded at a different rate in PBS at pH = 7.4, with degradation times ranging from 1 to 35 days. By changing the composition of the two starting components, it was found that the Young's modulus of each hydrogel could be varied from 1 to 18 kPa. Hydrogel incubation with bovine serum albumin showed minimal protein adsorption. Finally, a cell cytotoxicity study of the precursor polymers with 3T3 fibroblasts demonstrated that the azide- and strained alkyne-functionalized PEGs are noncytotoxic.

An Injectable Hydrogel Prepared Using a PEG/Vitamin E Copolymer Facilitating Aqueous-Driven Gelation.

Hydrogels have been widely explored for biomedical applications, with injectable hydrogels being of particular interest for their ability to precisely deliver drugs and cells to targets. Although these hydrogels have demonstrated satisfactory properties in many cases, challenges still remain for commercialization. In this paper, we describe a simple injectable hydrogel based on poly(ethylene glycol) (PEG) and a vitamin E (Ve) methacrylate copolymer prepared via simple free radical polymerization and delivered in a solution of low molecular weight PEG and Ve as the solvent instead of water. The hydrogel formed immediately in an aqueous environment with a controllable gelation time. The driving force for gelation is attributed to the self-assembly of hydrophobic Ve residues upon exposure to water to form a physically cross-linked polymer network via polymer chain rearrangement and subsequent phase separation, a spontaneous process with water uptake. The hydrogels can be customized to give the desired water content, mechanical strength, and drug release kinetics simply by formulating the PEGMA-co-Ve polymer with an appropriate solvent mixture or by varying the molecular weight of the polymer. The hydrogels exhibited no significant cytotoxicity in vitro using fibroblasts and good tissue compatibility in the eye and when injected subcutaneously. These polymers thus have the potential to be used in a variety of applications where injection of a drug or cell containing depot would be desirable.

Temperature-Induced Assembly of Monodisperse, Covalently Cross-Linked, and Degradable Poly(N-isopropylacrylamide) Microgels Based on Oligomeric Precursors.

A simple, rapid, solvent-free, and scalable thermally driven self-assembly approach is described to produce monodisperse, covalently cross-linked, and degradable poly(N-isopropylacrylamide) (PNIPAM) microgels based on mixing hydrazide (PNIPAM-Hzd) and aldehyde (PNIPAM-Ald) functionalized PNIPAM precursors. Preheating of a seed PNIPAM-Hzd solution above its phase transition temperature produces nanoaggregates that are subsequently stabilized and cross-linked by the addition of PNIPAM-Ald. The ratio of PNIPAM-Hzd:PNIPAM-Ald used to prepare the microgels, the time between PNIPAM-Ald addition and cooling, the temperature to which the PNIPAM-Hzd polymer solution is preheated, and the concentration of PNIPAM-Hzd in the initial seed solution can all be used to control the size of the resulting microgels. The microgels exhibit similar thermal phase transition behavior to conventional precipitation-based microgels but are fully degradable into oligomeric precursor polymers. The microgels can also be lyophilized and redispersed without any change in colloidal stability or particle size and exhibit no significant cytotoxicity in vitro. We anticipate that microgels fabricated using this approach may facilitate translation of the attractive properties of such microgels in vivo without the concerns regarding microgel clearance that exist with other PNIPAM-based microgels.

Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

Tuning gelation time and morphology of injectable hydrogels using ketone-hydrazide cross-linking.

Injectable, covalently in situ forming hydrogels based on poly(N-isopropylacrylamide) have been designed on the basis of mixing hydrazide-functionalized nucleophilic precursor polymers with electrophilic precursor polymers functionalized with a combination of ketone (slow reacting) and aldehyde (fast reacting) functional groups. By tuning the ratio of aldehyde:ketone functional groups as well as the total number of ketone groups in the electrophilic precursor polymer, largely independent control over hydrogel properties including gelation time (from seconds to hours), degradation kinetics (from hours to months), optical transmission (from 1 to 85%), and mechanics (over nearly 1 order of magnitude) can be achieved. In addition, ketone-functionalized precursor polymers exhibit improved cytocompatibility at even extremely high concentrations relative to polymers functionalized with aldehyde groups, even at 4-fold higher functional group densities. Overall, increasing the ketone content of the precursor copolymers can result in in situ-gellable hydrogels with improved transparency and biocompatibility and equivalent mechanics and stimuli-responsiveness while only modestly sacrificing the speed of gel formation.

Designing injectable, covalently cross-linked hydrogels for biomedical applications.

Hydrogels that can form spontaneously via covalent bond formation upon injection in vivo have recently attracted significant attention for their potential to address a variety of biomedical challenges. This review discusses the design rules for the effective engineering of such materials, and the major chemistries used to form injectable, in situ gelling hydrogels in the context of these design guidelines are outlined (with examples). Directions for future research in the area are addressed, noting the outstanding challenges associated with the use of this class of hydrogels in vivo.

A Nanocomposite Dynamic Covalent Cross-Linked Hydrogel Loaded with Fusidic Acid for Treating Antibiotic-Resistant Infected Wounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with high levels of morbidity and is considered a difficult-to-treat infection, often requiring nonstandard treatment regimens and antibiotics. Since over 40% of the emerging antibiotic compounds have insufficient solubility that limits their bioavailability and thus efficacy through oral or intravenous administration, it is crucial that alternative drug delivery products be developed for wound care applications. Existing effective treatments for soft tissue MRSA infections, such as fusidic acid (FA), which is typically administered orally, could also benefit from alternative routes of administration to improve local efficacy and bioavailability while reducing the required therapeutic dose. Herein, we report an antimicrobial poly(oligoethylene glycol methacrylate) (POEGMA)-based composite hydrogel loaded with fusidic acid-encapsulating self-assembled polylactic acid-b-poly(oligo(ethylene glycol) methyl ether methacrylate) (PLA-POEGMA) nanoparticles for the treatment of MRSA-infected skin wounds. The inclusion of the self-assembled nanoparticles (380 nm diameter when loaded with fusidic acid) does not alter the favorable mechanical properties and stability of the hydrogel in the context of its use as a wound dressing, while fusidic acid (FA) can be released from the hydrogel over ∼10 h via a diffusion-controlled mechanism. The antimicrobial studies demonstrate a clear zone of inhibition in vitro and a 1-2 order of magnitude inhibition of bacterial growth in vivo in an MRSA-infected full-thickness excisional murine wound model even at very low antibiotic doses. Our approach thus can both circumvent challenges in the local delivery of hydrophobic antimicrobial compounds and directly deliver antimicrobials into the wound to effectively combat methicillin-resistant infections using a fraction of the drug dose required using other clinically relevant strategies.

Injectable and Dynamically Crosslinked Zwitterionic Hydrogels for Anti-Fouling and Tissue Regeneration Applications.

A zwitterionic injectable and degradable hydrogel based on hydrazide and aldehyde-functionalized [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS) precursor polymers that can address practical in vivo needs is reported. Zwitterion fusion interactions between the zwitterionic precursor polymers create a secondary physically crosslinked network to enable much more rapid gelation than previously reported with other synthetic polymers, facilitating rapid gelation at much lower polymer concentrations or degrees of functionalization than previously accessible in addition to promoting zero swelling and long-term degradation responses and significantly stiffer mechanics than are typically accessed with previously reported low-viscosity precursor gelation systems. The hydrogels maintain the highly anti-fouling properties of conventional zwitterionic hydrogels against proteins, mammalian cells, and bacteria while also promoting anti-fibrotic tissue responses in vivo. Furthermore, the use of the hydrogels for effective delivery and subsequent controlled release of viable cells with tunable profiles both in vitro and in vivo is demonstrated, including the delivery of myoblasts in a mouse skeletal muscle defect model for reducing the time between injury and functional mobility recovery. The combination of the injectability, degradability, and tissue compatibility achieved offers the potential to expand the utility of zwitterionic hydrogels in minimally invasive therapeutic applications.

Injectable superparamagnets: highly elastic and degradable poly(N-isopropylacrylamide)-superparamagnetic iron oxide nanoparticle (SPION) composite hydrogels.

Injectable, in situ-gelling magnetic composite materials have been fabricated by using aldehyde-functionalized dextran to cross-link superparamagnetic nanoparticles surface-functionalized with hydrazide-functionalized poly(N-isopropylacrylamide) (pNIPAM). The resulting composites exhibit high water contents (82-88 wt.%) while also displaying significantly higher elasticities (G' >60 kPa) than other injectable hydrogels previously reported. The composites hydrolytically degrade via slow hydrolysis of the hydrazone cross-link at physiological temperature and pH into degradation products that show no significant cytotoxicity. Subcutaneous injections indicate only minor chronic inflammation associated with material degradation, with no fibrous capsule formation evident. Drug release experiments indicate the potential of these materials to facilitate pulsatile, "on-demand" changes in drug release upon the application of an external oscillating magnetic field. The injectable but high-strength and externally triggerable nature of these materials, coupled with their biological degradability and inertness, suggest potential biological applications in tissue engineering and drug delivery.

Injectable polysaccharide hydrogels reinforced with cellulose nanocrystals: morphology, rheology, degradation, and cytotoxicity.

Injectable hydrogels based on carboxymethyl cellulose and dextran, reinforced with rigid rod-like cellulose nanocrystals (CNCs) and aldehyde-functionalized CNCs (CHO-CNCs), were prepared and characterized. The mechanical properties, internal morphology, and swelling of injectable hydrogels with unmodified and modified CNCs at various loadings were examined. In all cases, gelation occurred within seconds as the hydrogel components were extruded from a double-barrel syringe, and the CNCs were evenly distributed throughout the composite, as observed by scanning and transmission electron microscopy. When immersed in purified water or 10 mM PBS, all CNC-reinforced hydrogels maintained their original shape for more than 60 days. The maximum storage modulus was observed in hydrogels with 0.250 wt % of unmodified CNCs and 0.375 wt % of CHO-CNCs. CHO-CNCs acted as both a filler and a chemical cross-linker, making the CHO-CNC-reinforced hydrogels more elastic, more dimensionally stable, and capable of facilitating higher nanoparticle loadings compared to hydrogels with unmodified CNCs, without sacrificing mechanical strength. No significant cytotoxicity to NIH 3T3 fibroblast cells was observed for the hydrogels or their individual components. These properties make CNC-reinforced injectable hydrogels of potential interest for various biomedical applications such as drug delivery vehicles or tissue engineering matrices.