In situ forming injectable hydrogels for drug delivery and wound repair

Abstract

Hydrogels have been utilized in regenerative applications for many decades because of their biocompatibility and similarity in structure to the native extracellular matrix. Initially, these materials were formed outside of the patient and implanted using invasive surgical techniques. However, advances in synthetic chemistry and materials science have now provided researchers with a library of techniques whereby hydrogel formation can occur in situ upon delivery through standard needles. This provides an avenue to minimally invasively deliver therapeutic payloads, fill complex tissue defects, and induce the regeneration of damaged portions of the body. In this review, we highlight these injectable therapeutic hydrogel biomaterials in the context of drug delivery and tissue regeneration for skin wound repair.

Keywords: Hydrogel; Injectable; Regeneration; Skin wounds.

Figure 1. Skin biology and wound recovery

(A) Skin is composed of three primary layers. The outermost layer, the epidermis, functions as a protective barrier and limits the interaction of underlying tissues with damaging pathogens, high energy UV radiation, and external forces. Below the epidermis, the dermis imparts mechanical integrity onto the tissue while facilitating higher order functions including oxygen exchange, temperature regulation, sweat production, nerve signaling, and hair growth. Finally, subcutaneous tissue serves as a depot for stored fats and anchors the more superficial layers of skin to underlying bone and muscle. (B) Upon injury, the skin progresses through a complex yet finely regulated wound healing response. Immediately following damage, platelets from the blood stream accumulate at the wound surface and form a fibrin clot which halts blood loss and simultaneously protects the injured area from foreign species. Macrophages and neutrophils are next recruited to the area and degrade pathogens to prime the area for recovery, while hypoxic signaling factors induce the formation of new vasculature. The wound is then healed through successive iterations of cellular migration, ECM maturation, and myofibroblast driven epidermal closure. (C) When wounds become chronic the normal healing response is impaired and proper regeneration can take months or even years to occur. Chronicity is driven by an imbalance between the rates of immune cell mediated degradation and the regeneration of functional ECM. Injectable hydrogel biomaterials offer an attractive option for treating chronic wounds because of their inherent ability to fill the wound defect while mimicking natural ECM, both by providing a scaffold for tissue ingrowth and by controlling the availability of regenerative signaling molecules.

Figure 2. Key considerations for injectable hydrogel design

(A) Over the past several decades numerous chemistries and material processing techniques have been utilized for the production of injectable hydrogels. These materials can be delivered either as liquid precursors which are crosslinked into stable gels through environmental triggers at the site of injection or as deformable solids which can withstand injection shear forces and re-anneal in the wound bed. In-situ annealing triggers include temperature, pH, light intensity, and concentration of ionic species and enzymes. (B) Hydrogel scaffolds function as regenerative templates that provide a suitable substrate for cellular ingrowth while matching the physiochemical properties of the native ECM. Matching scaffold stiffness to the surrounding tissue and optimizing its rate of degradation ensures that infiltrating cells remain viable, maintain their desired phenotype, and coordinate their response over the entirety of the wound healing process. Scaffolds can also be functionalized to provide sites for cellular binding or protein adsorption, enabling spatial control over the density of seeded cells as well as the availability of cytokines and growth factors. (C) Injectable hydrogels can also be designed to serve as depots for the controlled release of therapeutic compounds. Here, the release profile of compounds can be controlled through parameters such as polymer mesh size, polymer affinity for the target molecule, or rates of polymer degradation. Additionally, therapeutic compounds can be covalently linked to the polymer mesh by cleavable anchoring groups for controlled release upon exposure to external signals.

Figure 3. Overview of non-covalent gelation mechanisms

Polymer backbones can be reversibly crosslinked into hydrogel networks through several methods that take advantage of interchain affinity. These crosslinking reactions include (A) self-organization of amphiphilic micelles at high temperature, (B) polymer bridging via multivalent ions, and (C) hierarchical self-assembly of short peptides. Source: Adapted with permission from Refs [13], [51], & [81].

Figure 4. Material rearrangement can induce changes to cellular phenotype

(A) Chaudhuri et al. synthesized ionically linked alginate hydrogels with identical elastic moduli but varying rates of stress relaxation by simultaneously modulating the molecular weight of crosslinked alginate and incorporating PEG spacers into the polymer backbone. (B) Fibroblasts encapsulated in rapidly stress relaxing hydrogels exhibited increased spreading due to their ability to dynamically rearrange the surrounding scaffold. (C) Interestingly, the rate of stress relaxation also influenced the efficiency with which cultured mesenchymal stem cells differentiated into different lineages. Here, stem cells grown on stiff substrates with high rates of stress relaxation had a tendency to mature towards an osteogenic phenotype, whereas stem cells grown on slowly relaxing soft substrates efficiently differentiated towards a more adipogenic phenotype. (D) Stress relaxing materials have also been designed using other chemistries. In one example Rodell et al. combined guest-host complexation with covalent crosslinking to form stiff stress relaxing hydrogels which showed promise as regenerative scaffolds for tissue engineering applications. The routine consideration of stress relaxation rates in hydrogel scaffolds may aid the design of new materials which provide a greater degree of control and uniformity in response for both pre-seeded and infiltrating cell populations. Source: Adapted with permission from Refs: [79] & [135].

Figure 5. Application of light improves spatial control of hydrogel formation and drug release

(A) Polymers decorated with radical generating functional groups can form hydrogels in the presence of photoinitiators and appropriate wavelengths of light. (B,C) When combined with specialized projection techniques or photomasks these systems can be utilized to form complex 3-dimensional geometries or induce localized binding and release of therapeutic payloads. (D) De Forest and coworkers demonstrated the utility of such light mediated controlled release schemes by utilizing orthogonal photoactivated chemistries for the binding and subsequent release of full length proteins. Here hydrogels were pre-formed and decorated with photocaged alkoxyamine groups. When illuminated with UV light through patterned photomasks, photocages were selectively degraded. Subsequent addition of aldehyde functionalized proteins allowed spatial patterning of bioactive molecules. Introduction of o-nitrobenzyl esters into protein linkers allowed for subsequent photorelease upon exposure to similar UV irradiation. (E,F) To highlight this technology, multiphoton laser scanning lithography and photolithography demonstrated spatial control over photorelease of fluorescently tagged proteins in 3D and 2D respectively. Source: Adapted with permissions from Refs [103], [104], [107], & [110].

Figure 6. Sustained release of drugs from covalently bound hydrogel networks can aid wound repair

(A) Zhang and coworkers encapsulated 1,4-DPCA, a potent inhibitor of prolyl hydroxylases into an injectable hydrogel for sustained release in vivo. Inhibition of prolyl hydroxylases stabilizes HIF-1α, a central molecule in many regenerative processes, and allows for examination of its contribution to the wound healing process. (B) The drug eluted continuously from the material over the course of several days. (C) Mice were ear punched and treated with either gel alone (G₀) or gel loaded with 2 mg/mL 1,4 DPCA (G_d). At day 35, after three successive injections of gel, wound healing was visibly improved for G_d treated wounds over G₀ controls. (D) Histological analysis of regenerating ear at day 35 showed more complete wound closure for G_d treated wounds along with a greater number of mesenchymal cells in the regenerating bridge when compared with G₀ controls. Further analysis (not shown) demonstrated that HIF-1α blockage using siRNA reverted this regenerative improvement, supporting the hypothesis that upregulation of HIF-1α is sufficient to improve regenerative outcomes. Source: Adapted with permission from Ref [120].

Figure 7. Cryogels form porous scaffolds with shape memory properties

(A) Cryogels are formed when polymeric precursors are mixed with radical initiators and subsequently frozen in aqueous solution. This process confines the dissolved solutes to grain boundaries surrounding frozen water crystals where polymerization takes places. After thawing, the material forms a sponge-like hydrogel. (B) Cryogels exhibit shape memory properties. The hydrated materials can collapse to a fraction of their size as they are injected through standard needles, but subsequently rapidly revert to their pre-formed geometry after injection is complete. Cryogelation is easily adapted to form hydrogels of many different (C) sizes and (d) shapes. Source: Adapted with permission from Ref [38].

Figure 8. Microporous annealed particle hydrogels decouple tissue integration from material degradation

(A) In the microporous annealed particle (MAP) gel system, microfluidically generated hydrogel beads are collected, washed and subsequently annealed via peptide functional groups present on their surface. This forms a microporous scaffold that allows cellular infiltration without degradation of the bonds that hold the material together. (B) Microgels are small enough to be injected through a standard syringe, and can conform to the shape of their injection cavity after annealing. (C) MAP hydrogels were evaluated for their effectiveness as regenerative scaffolds in a murine wound healing model, and demonstrated improved rates of wound closure versus no treatment, non-annealed microgels, and physically matched non-porous controls. Source: Adapted with permission from Ref [37].