Summary of cross‐linking reactions of complementary groups.
With the development of chemical synthetic strategies and available building blocks, in situ‐forming hydrogels have attracted significant attention in the biomedical fields over the past decade. Due to their distinct properties of easy management and minimal invasiveness via simple aqueous injections at target sites, in situ‐forming hydrogels have found a broad spectrum of biomedical applications including tissue engineering, drug delivery, gene delivery, 3D bioprinting, wound healing, antimicrobial research, and cancer research. The objective of this chapter is to provide a comprehensive review of updated research methods in chemical synthesis of in situ‐forming cross‐linking hydrogel systems and their diverse applications in the biomedical fields. This chapter concludes with perspectives on the future development of in situ‐forming hydrogels to facilitate this multidisciplinary field.
- chemical cross‐linking
- free radical polymerization
- in situ‐forming hydrogel
- biomedical applications
- hydrophilic polymers
Hydrogels are a class of three‐dimensional (3D) cross‐linked polymeric structures capable of holding large amounts of water or biological fluids in their swollen state . The first report of water‐swollen cross‐linking polymer network of 2‐hydroxylethyl methacrylate and ethylene glycol dimethacrylate for applications of contact lenses was published by Wichterle and Lim in 1960 . Due to their high water content and structure similarity to natural extracellular matrix (ECM) as well as their biodegradability and low immunogenicity, hydrogels have gained considerable interest in biomedical and pharmaceutical fields over the past few decades, especially with the development of a wide variety of chemical building blocks and various synthetic strategies in polymer chemistry, organic chemistry, and bioconjugation chemistry. Up to date, hydrogels have been applied in a broad spectrum of biomedical applications including tissue engineering, 3D bioprinting, drug delivery, gene delivery, would healing, antimicrobial research, and cancer research . Among these applications,
With regard to biomedical applications,
There are two strategies to synthesize
The most used natural polymers for
2. Chemistry of
in situ‐forming cross‐linking hydrogels
Since hydrogels are simply hydrophilic polymer networks cross‐linked in some fashion to produce an elastic structure, any strategy that can produce a cross‐linked network could be used for
To be suitable for biomedical applications, it is preferred that the cross‐linking occurs in aqueous and mild conditions. The reactions should not damage the cells or biofunctional molecules in the hydrogel matrix. Reactions employed should not generate toxic side products and should not require high temperatures or heavy metal catalysts that are toxic to the cells .
Chemical cross‐linking is the most common and highly efficient method for the formation of
There are two commonly used strategies to prepare chemically cross‐linked hydrogels which are illustrated in Figures 1 and 2 . The first strategy is called “3D polymerization,” which is achieved by polymerization of hydrophilic molecules, such as acrylates and vinylic monomers, in the presence of multifunctional cross‐linkers. The drawback of 3D polymerization is the significant amount of unreacted monomers and other small molecules, which could be toxic and have to be removed by extensive purification processes. The second strategy is to directly crosslink hydrophilic polymers so that extensive purification can be avoided as there are not as many toxic small molecules remained in the system. Water‐soluble polymers such as PAA, PVA, PEG, PAM, and polysaccharides are commonly used systems for biomedical and pharmaceutical applications due to their nontoxicity and biocompatibility .
2.1. Cross‐linking by free radical polymerization
Free radical polymerization is the most commonly used cross‐linking strategy for hydrogel synthesis due to its advantages over other polymerization methods. First, it is highly reactive, which results in polymers of high molecular weights and cross‐linking density. Second, free radical polymerization tolerates a variety of functional groups and it occurs in mild conditions, even in aqueous conditions. This makes it a facile approach for cross‐linking hydrogel synthesis . Free radical polymerization can be further classified as homopolymerization, copolymerization, and multipolymer interpenetrating polymeric hydrogels by hydrogel composition .
2.1.1. Hydrogels by homopolymerization
Homopolymeric hydrogels are hydrogel systems originated from polymerization of a single monomer species in the presence of an initiator and a cross‐linker. For example, homopolymerization of
2.1.2. Hydrogels by copolymerization
Copolymerization hydrogels are synthesized by polymerization of two or more different monomers with at least one hydrophilic component. Depending on the structure of the polymer chain, copolymers are further classified into random, block, or alternating copolymers. A recent report described the synthesis of pH temperature dual stimuli‐responsive smart hydrogel for drug release. In this research, PEG was reacted with methyl ether methacrylate to afford methacrylate terminated PEG, which copolymerized with
2.1.3. Hydrogels of interpenetrating polymer networks
Interpenetrating polymer networks (IPNs) and semi‐interpenetrating polymer networks (semi‐IPNs) have emerged as innovative biomaterials for biomedical applications. IPNs are a family of hydrogels that contain two independent hydrogel components with each component being a cross‐linked hydrogel from synthetic and/or natural polymers. Semi‐IPNs contain two independent hydrogels with one being cross‐linked hydrogel and the other noncross‐linked hydrogel . The purpose of two hydrogel components is to provide tuned physical properties or stimuli responsiveness. For example, Reddy et al. fabricated cyclotriphosphazene‐based IPN hydrogels through free radical polymerization of mono (methacryloyl‐2‐ethoxy)‐pentakis(
2.2. Cross‐linking by chemical reaction of complementary groups
|1||Click reaction||Alkyne + azide||Cu(I), aqueous||[23–25]|
|Alkyne + azide||aqueous, 37 °C|||
|Oxanorbornadiene + azide||aqueous, 37 °C||[22, 27]|
|Cyclooctyne + azide||aqueous, 37 °C||[28–31]|
|2||Michael addition||Maleimide + thiol||aqueous, 37 °C||[15, 36]|
|Vinyl sulfone + thiol||aqueous, 37 °C|||
|Acrylate + thiol||aqueous, 37 °C|
|Methacrylate + thiol||aqueous, 37 °C|
|3||Thiol‐ene/yne coupling||Alkene + thiol||aqueous, 37 °C||[38–43]|
|Norbornene + thiol||Photo, radical|
|Alkyne + thiol|
|4||Diels‐Alder reaction||Furan + maleimide||aqueous, 37 °C||[44, 45]|
|Tetrazine + norbornene||aqueous, 37 °C|||
|Tetrazine + ||aqueous, 37 °C||[47, 48]|
|5||Disulfide formation/exchange||Thiol‐thiol||aqueous, H2O2||[49, 50]|
|Pyridyl disulfide + thiol||aqueous, 37 °C||[52, 53]|
|6||Epoxide coupling||Epoxide + amine||aqueous, 37 °C||[54, 55]|
|Epoxide + hydroxyl||acidic or basic|||
|Diepoxide + amine||aqueous, carbon black|||
|7||Schiff‐base formation||Aldehyde + amine||aqueous, 37 °C|||
|Aldehyde + hydrazide||aqueous, 37 °C|||
|Aldehyde + hydroxylamine||aqueous, 37 °C||[60, 61]|
|8||Condensation||Amine or hydroxyl + acid derv.||aqueous, 37 °C|||
|Amine or hydroxyl + isocyanate||DMSO, 35 °C|||
|Boronic acid + amine/hydroxyl||aqueous, pH 4.8|||
|Genipin coupling||Amines + genipin||0.1 M acetic acid, 4 °C|||
|9||Staudinger‐ligation||Azide + ester derivative of triphenylphosphine||aqueous, BaCl2, 37 °C|||
|Tetrazole‐photoclick||Alkene + tetrazole||aqueous, UV light|||
|Quadricyclane‐ligation||Quadricyclane + Ni bis(dithiolene)||aqueous, pH 4.5|||
2.2.1. Click reactions
Click reactions refer to the cycloaddition of azide and alkyne to form a linkage through a triazole ring. It was proposed by Sharpless and co‐workers in 2001 as copper‐catalyzed azide‐alkyne cycloaddition (CuAAC), aiming for efficient chemical synthesis with minimized byproducts and purification effort . Over the past decade, click chemistry has gained significant application in a broad range of chemical synthesis of small molecules, polymers, dendrimers, biomacromolecules, and bioconjugation. Click chemistry has been widely used in cross‐linking hydrogels due to its high selectivity and efficiency without generating by‐products in aqueous conditions, plus the bioorthogonality of the components without interaction with the environment of biological or biomedical systems . To date, click reactions of different versions have been developed not only to build materials that are biologically compatible, highly functional and organized in structure, but also to produce highly complex patterns of biofunctionalities within a single cellular scaffold . For example, HA‐based hydrogels were cross‐linked by CuAAC reaction to produce a thermo‐responsive hydrogel with tailorable mechanical properties [23, 24]. Kaga et al. fabricated “clickable” hydrogels using dendron polymer‐based triblock polymers .
To avoid the toxicity of copper, metal‐free click chemistry has been developed to eliminate heavy metal residue in hydrogel matrix. Truong et al. synthesized chitosan‐PEG hydrogels by copper‐free azide‐alkyne click reaction . Chitosan and HA‐based hydrogel were cross‐linked after functionalization by oxanorbornadiene and azide [22, 27] (Table 1, entry 1). Another good example of metal‐free click reaction is strain‐promoted azide‐alkyne cycloaddition (SPAAC) that involves a difluorinated cyclooctyne moiety. Due to the ring strain and the electron‐withdrawing difluoride, the alkyne functionality is greatly activated for a cycloaddition without a catalyst . This reaction has been shown to be very efficient with high chemoselectivity even for
2.2.2. Michael addition
Michael addition is a 1,4‐addition of nucleophiles to α,β‐unsaturated ketones or esters. It occurs in high efficiency under aqueous conditions without any side products, making it a suitable approach for cross‐linking in hydrogel synthesis. The common nucleophiles are macromolecules that are functionalized with multiple terminal amine or thiol groups, which cross‐link with electrophilic macromolecules functionalized with alkene groups with adjacent electron withdrawing groups, such as vinyl sulfone, acrylate, or methacrylate . Surfactants can be used to promote the kinetics of the Michael addition when the nucleophilic and the electrophilic macromolecules exhibit significant differences in hydrophilicity .
Like the cross‐linking of alkyne and azide in SPAAC, the cross‐linking kinetics of Michael addition depends greatly on the electron deficiency of the alkenes. Our research group has systematically studied the gelation kinetics and mechanical property of poly(amidoamine) (PAMAM) dendrimer‐HA cross‐linking hydrogels. PAMAM dendrimers are a family of synthetic polymers with well‐defined structures and ample surface groups for conjugation of bioactive functionalities. They are widely used as a platform to deliver bioactive molecules into biological systems due to their water solubility, nontoxicity and nonimmunogenicity . The combination of PAMAM and HA allows for easy chemical modification of dendrimer structures to modulate the physical and mechanical properties of the cross‐linking hydrogel. In this research, PAMAM dendrimers are functionalized with maleimide, vinyl sulfone, acrylic, methacrylic, and a normal alkene group. When alkene functionalized PAMAM dendrimers are cross‐linked with thiolated HA at experimental concentration, the gelation time displayed a large range from 8 seconds to 18 hours, and modulus from 36 to 183 Pa depending on the alkene group attached to the dendrimer. 1H NMR study revealed that the gelation time is governed by the electron deficiency of alkenes . Introduction of a RGD peptide in hydrogel greatly enhanced the cell attachment, viability, and proliferation of both bone marrow stem cells and human umbilical vein endothelial cells .
2.2.3. Thiol‐ene/yne coupling
Thiol‐ene reaction is a radical‐mediated mechanism at room temperature and in aqueous conditions even in the presence of biological cargos such as proteins or cells, thus making it a good technique for hydrogel cross‐linking. Unlike the traditional free radical polymerization, the radical thiol‐ene reactions are relatively not oxygen sensitive . The radicals that initiate the thiol‐ene reaction can be generated using thermal, oxidation‐reduction, or photochemical process based on initiator selection . Fairbanks et al. devised photoinitiated thiol‐ene reaction between four‐armed PEG tetra‐norbornene and dicysteine‐terminated peptide to form
2.2.4. Diels‐Alder reaction
The Diels‐Alder reaction is a robust cross‐linking strategy for biopolymer‐based hydrogels as it is rapid, efficient, versatile, and selective. It proceeds with high efficiency in aqueous conditions for hydrogel cross‐linking or covalent immobilization of functional biomolecules. The most commonly employed functional groups for Diels‐Alder cross‐linking are furan and maleimide groups. As an example, furan and maleimide functionalized HA were cross‐linked in 2‐(
2.2.5. Disulfide formation/exchange
Disulfide bonds are usually formed from oxidation of thiol groups. For example, thiolated HA can be chemically synthesized with varied degrees of thiolation and cross‐linked through oxidation in the air or using hydrogen peroxide . In another report, HA and gelatin were chemically modified using 3,3'‐dithiobis(propionic hydrazide) followed by treatment of dithiothreitol. The thiol derivatives of HA and gelatin bearing thiol groups were mixed to form a disulfide cross‐linking hydrogel in the presence of hydrogen peroxide. The hydrogel can be degraded by hyaluronidase . Zhang et al. synthesized elastin‐like polypeptide hydrogels for wound repair by disulfide bond cross‐linking in the presence of ultraviolet (UV) light .
Macromolecules with pyridyl disulfide can react with thiol functionalized polymer through disulfide exchange, eliminating pyridine‐2‐thione as a by‐product. Kannan et al. developed PAMAM dendrimer‐PEG hydrogels for the sustained release of amoxicillin through disulfide exchange of a pyridyl disulfide functionalized PAMAM dendrimer generation 4 and an eight‐armed thiolated PEG . Similarly, an HA‐based cleavable hydrogel was synthesized by cross‐linking pyridyl disulfide functionalized HA with PEG dithiol . The disulfide exchange reaction has been found to exhibit fast cross‐linking kinetics and cytocompatibility as hydrogels can be synthesized in minutes under physiological pH in the presence of many cell types, with tunable rheological and physical properties. The limitation of the disulfide cross‐linking approach is that the hydrogels may exhibit low stability by degradation especially in the presence of hyaluronidase or reducing agents such as glutathione .
2.2.6. Epoxide coupling
Water‐soluble epoxides are highly reactive electrophiles that readily react with nucleophiles such as amines, alcohols, and even carboxylic acids, and the reactions are not oxygen sensitive. Due to the difference in nucleophilicity, the reaction rate is fast with amines and slow with alcohols. PEG diepoxide, 1,2,3,4‐diexpoxbutane, and 1,4‐butandiol diepoxide are commonly used epoxide sources . HA was reported to react with diepoxide to form cross‐linking hydrogels under either basic or acidic conditions [54, 55]. However, epoxides may suffer from some degree of hydrolysis under basic conditions. Binetti and others cross‐linked PVA with PEG diglycidylether (PEGDGE) through epoxide coupling to form PVA/PEG hydrogels for injectable nucleus replacement . Calvert et al. made epoxy hydrogels as hydrogel sensors for glucose by epoxide coupling of PEGDGE and Jeffamine in aqueous conditions .
2.2.7. Schiff‐base reaction
A Schiff base is usually achieved by reaction of amines, hydrazides, or hydroxylamines with aldehydes or ketones to form an imine, hydrazone, or oxime linkage. Schiff‐base formation can occur in aqueous conditions without using extra chemicals or catalysts. It also displays controllable reaction rates depending on the pH. Therefore, it becomes a facile approach to produce
2.2.8. Cross‐linking by other reactions
Other chemical reactions suitable for hydrogel cross‐linking include condensation reactions that involve hydroxyl or amino groups reacting with carboxylic acid derivatives such as activated esters or isocyanates [33, 62], and genipin coupling through which nucleophiles such as amine or alcohol‐containing polymers react with a natural cross‐linker genipin . Reactants that contain a photo‐activated functional group can be cross‐linked by photoirradiation . The reaction between boronic acid and amine or hydroxyl group has been employed as a cross‐linking approach to make pH responsive hydrogels .
2.2.9. Cleavable cross‐linking
It is worth noticing that the stability of cross‐linking is not always preferable in some applications when the encapsulated payload in hydrogel needs to be released at the target site. In that case, reversible cross‐linking with labile linkages can be introduced so that the hydrogel is broken down in response to stimuli and the cargo can be released for function. The most common cleavable cross‐links are cleavable to pH, photo, redox, enzyme, etc. Interested readers should refer to relevant articles for details [33, 66].
2.2.10. Bioorthogonal chemistry
Despite the various cross‐linking strategies for synthesis of different hydrogel networks and immobilization of functional cues within a cellular scaffold, techniques are needed to introduce different functionalities in specific locations and at different times to produce spatiotemporally complex and yet well‐defined biochemical cues in hydrogels for specific applications or studies . For that purpose, a strategy has been established to use two or more orthogonal reactions in a sequential fashion with the first reaction to form the cross‐linking hydrogel and the second or later reactions to introduce biochemical functionalities. Figure 3 illustrates how bioorthogonal strategy increases spatiotemporal diversity in hydrogel networks.
The reactions employed in bioorthogonal chemistry must be nontoxic to cells. In addition, the reaction should be selective and the fidelity of the reaction should not be affected by other functionalities present. Among the cross‐linking reactions for
In spite of all the recent advancements, bioorthogonal strategy is still open for expansion in chemical synthesis of biomaterials for specific applications. Staudinger ligation, tetrazole photoclick reaction, and quadricyclane ligation are new orthogonal reactions that are potentially useful for synthesizing
2.3. Cross‐linking by enzyme catalysis
Enzymes can be used to cleave or establish a chemical bond with greater efficiency than other methods. Due to their short reaction times and specificity, enzymes have been used for catalytic cross‐linking to form hydrogels without interference with other chemical functional groups in macromolecules. Enzymatic cross‐linking occurs under mild reaction conditions such as an aqueous environment, a neutral pH, or mild temperatures . The most studied enzymes to prepare hydrogels are horseradish peroxidase, transglutaminase, tyrosinase, phosphopantetheinyl transferase, and lysyl oxidase . Themolysin, galactosidase, and esterase have also been used to prepare
Overall, enzyme catalyzed reactions are a new approach in hydrogel formation. The reactions provide exceptional control over hydrogel formation, promoting higher complexity, noncytotoxicity and noninvasiveness. Despite the major advantages of enzymatic reactions, the challenges of this approach include instability of some of the enzymes and the insufficient mechanical properties of the gels formed. Interested readers may refer to more recent reports for a comprehensive review [76, 77].
3. Biomedical applications of
in situ‐forming cross‐linking hydrogels
Hydrogels are suitable for biomedical applications due to their favorable properties such as biocompatibility, structural similarity to ECM, and biodegradability. Among the hydrogel family,
3.1. Tissue engineering
The objective of tissue engineering is the fabrication of living parts for the body due to the tremendous need for organs and tissues . Hydrogels have been widely used in tissue engineering because their properties are similar to those of natural ECM, which is essential for these purposes . Many strategies employ material scaffolds to engineer tissues. These scaffolds serve as a synthetic ECM to provide a 3D architecture for cells and direct the growth and formation of desired tissues. Those scaffolds can be used for space filling agents, bioactive molecule delivery, and cell/tissue delivery.
Hydrogels made from natural polysaccharides are ideal scaffolds, as they resemble the native ECM of tissues which are comprised of various glycosaminoglycans. An injectable electroactive and antioxidant hydrogel based on a tetra‐aniline functional copolymer and α‐CD has exhibited excellent biodegradation, cell proliferation, and regeneration properties in tissue engineering .
One major drawback of hydrogels is the lack of mechanical strength; hence, maintaining and improving the mechanical integrity of the processed scaffolds has become a key issue regarding 3D hydrogel structures . Many researchers focus on the development of hydrogels using synthetic biomaterials that may have enhanced mechanical properties. Schuurman et al. prepared gelatin‐methacrylamide (GelMA) hydrogels with tunable mechanical properties by manipulating the cross‐linking parameters . Hu et al. designed and synthesized a library of supramolecular hydrogels inspired by collagen. Those hydrogels exhibited promising potential for tissue engineering as they mimic properties of collagen . Readers may refer to reviews of hydrogel scaffolds for tissue engineering [13, 76].
Lack of vascular networks in engineered tissues thicker than 200 μm puts a limitation on the nutrition of encapsulated cells. Therefore, advances in tissue engineering
3.2. 3D Bioprinting
3D bioprinting technologies enable the automated biofabrication of cell‐laden constructs through the layer‐by‐layer deposition of biochemicals (termed as “bioinks”) both
The full implementation of bioprinting strongly depends on the development of novel biomaterials exhibiting fast‐cross‐linking kinetics, appropriate printability, cell‐compatibility, and biomechanical properties. Different biomaterials have been used in 3D bioprinting, such as alginate , HA, PEG , gelatin, collagen , and thermo‐responsive biodegradable polyurethane . Various cells have been printed on those materials, such as tumor cells, neural cells, and stem cells [88‒91]. Hsieh et al. investigated two thermo‐responsive water‐based biodegradable polyurethane dispersions (PU1 and PU2) . The stiffness of the hydrogel could be easily fine‐tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation and showed excellent proliferation and differentiation in 25‒30% PU2 hydrogels. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermo‐responsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering.
Photo‐cross‐linkable hydrogels are attractive materials for bioprinting as they provide fast polymerization under cell‐compatible conditions and exceptional spatiotemporal control over the gelation process. Photo‐cross‐linkable GelMA and PEG dimethacrylate (PEGDA) were used as example hydrogels to demonstrate the feasibility and effectiveness of the approach. An array of GelMA/PEG hydrogels encapsulating human periodontal ligament stem cells (PDLSCs) with a gradient of material composition was printed, and the responses of human PDLSCs in the hydrogel array were investigated. The approach may be helpful for human PDLSCs‐ECM screening and other cell‐ECM systems. Please refer to the review for most recent developments on 3D bioprinting of photo‐cross‐linkable biodegradable hydrogels for tissue engineering .
3.3. Drug delivery
The use of hydrogels in drug delivery applications has been a significant subject in recent research due to the unique physical properties of hydrogels, the biodegradability, and the changes in gel structure according to environmental stimuli such as temperature, pH, and/or ionic strength. Their highly porous structure can easily be tuned by controlling the density of cross‐links in the gel matrix and the affinity of the hydrogels for the aqueous environment in which they are formed. Their porosity also permits the loading of drugs into the gel matrix and subsequent drug release through the gel network . The hydrogel systems have been developed for delivery of biomolecules ranging from small molecular drugs to large biomacromolecules such as nucleic acids, peptides, and proteins [93, 94]. Readers are directed to some recent reviews on drug delivery [95, 96].
There are several important areas in the field of hydrogels for drug delivery. First, development of
Second, the development of strategies to increase the loading rate and capacity and to control the release of drugs from the hydrogel is an important area of interest. There are generally two ways to load drugs into hydrogels, e.g., soaking formed hydrogels in drug solution and forming hydrogels in the presence of drugs. Both ways have their limitations and often result in a modest amount of drugs loaded into hydrogels. Ray et al. prepared an IPN hydrogel based on PVA networking with PAA (PVA‐co‐PAA)/NaCl microspheres. The hydrogels were loaded with diltiazem hydrochloride (DL) and showed comparatively higher DL entrapment (79%), better control over DL release up to 24 h, and were more effective in reducing blood pressure to 40.1% . Josef et al. investigated a composite gel system formulated from microemulsions (ME) embedded in alginate hydrogels. These hydrogels appeared to be a promising drug delivery system since they were capable of loading several hydrophobic compounds with a wide range of aqueous solubility and exhibited a release of 6‒8 hours in water .
Third, there is a great interest in the development of stimuli‐responsive hydrogels that are sensitive to temperature, pH, sugar, ionic strength, etc. These hydrogels are good candidates for controlled delivery systems that would release drugs to match a patient's physiological needs at the proper time and/or site . Zhou et al. synthesized a series of pH‐temperature dual stimuli‐responsive copolymers . Altering the temperature and the pH values of the environment could effectively control the release of the model drug. Li et al. synthesized pH and glucose dually responsive injectable hydrogels through the dynamic covalent imine bond and phenylboronate ester based on phenylboronic modified chitosan and oxidized dextran . The rapid gelation and biocompatible cross‐linking chemistry were appropriate for the incorporation of drug molecules and cells by
3.4. Gene delivery
Gene delivery via hydrogels provides a fundamental tool for a variety of clinical applications including regenerative medicine, gene therapy for inherited disorders, and drug delivery . Hydrogels serve the purpose of gene delivery by preserving activity of viral or nonviral vectors and shielding vectors from any host immune response. Hydrogels can also be injectable and environmentally responsive. Therefore, hydrogels hold great promise for gene delivery. There are two major areas that attracted great attention, vectors and biomaterial delivery system optimization.
Hydrogels used in gene delivery often need higher strength for extended use in order for internalization and transgene expression to occur. A hydrogen bonding strengthened hydrogel was prepared by radical copolymerization of PEG methacrylated β‐CD and 2‐vinyl‐4,6‐diamino‐l,3,5‐triazine monomer . Kidd et al. investigated the delivery of lentiviral gene therapy vectors from fibrin hydrogels containing hydroxyapatite nanoparticles that can interact with both fibrin and the lentivirus . The interaction of the hydroxyapatite with the fibrin may stabilize the hydrogels that will influence the rate of cell infiltration and vector release. The interaction of hydroxyapatite with lentiviral particles can enhance and localize gene transfer within the hydrogel. These studies demonstrate the potential of fibrin hydrogels to serve as a material support for regenerative medicine and as a vehicle for the localized delivery of lentiviral vectors
By choosing the proper vector and biomaterial system, gene delivery can be controlled for improved transgene expression. The interactions between scaffolds and vectors should be optimized so that vectors are adequately retained, but undergo dissociation, which enable vectors to interact with nearby cells and internalize . The diffusion rate must be balanced to maintain spatial localization of gene transfer. The addition of interconnected macropores into the hydrogels can further increase the probability that infiltrating cells will internalize vectors and thereby improve transgene expression.
3.5. Wound healing
Wound healing is a complex process that implies equilibrium between inflammatory and vascular activity in the connective tissue and epithelial cells. The regenerative process needs the assistance of important elements to activate the natural processes of angiogenesis, activation of growth factors, and regeneration in a well‐structured and biomimetic sequential process . Hydrogel wound dressing has been widely researched because hydrogels promote wound healing by moisture retention to maintain a homeostatic environment. Ajji et al. prepared hydrogel wound dressings composed of PVP, PEG, and agar . The gel fraction increased with increasing PVP, and decreased with increasing PEG. The hydrogel dressings could also be considered a good barrier against microbes. Reyes‐Ortega et al. reported a new system based on the sequential release of two complementary bioactive components for application in the healing of compromised wounds . The internal layer was a highly hydrophilic and biodegradable film loaded with the proangiogenic, anti‐inflammatory, and antibacterial peptide, proadrenomedullin N‐terminal 20 peptide for release. The more stable and less hydrophilic external layer was loaded with resorbable nanoparticles of bemiparin to promote the activation of growth factors and to provide a good biomechanical stability and controlled permeability of the bilayer dressing. This system demonstrated high efficacy of the early steps of the regenerative process in the wound site.
Traditional hydrogel dressings are inconvenient in applications as they need some degree of expertise and cause pain during changes. The thermo‐sensitive hydrogels avoid the necessity of repeated and complicated application. Lee et al. investigated the ability of a thermo‐sensitive hydrogel made of a triblock copolymer, PEG‐PLGA‐PEG, with TGF‐β1 to treat the wound surface . Results showed that the thermo‐sensitive hydrogel provided excellent wound dressing activity and delivered plasmid TGF‐β1 to promote wound healing in a diabetic mouse model. Hassan et al. developed a stem cell hydrogel system, in which the hADSCs were encapsulated
Supramolecular hydrogels are formed by noncovalent cross‐linking of polymeric chains in water and can be developed specifically for biomedical applications . Supramolecular hydrogels prepared by incorporating uranium chelating agents to eliminate uranium ions from the radionuclides contaminated wound sites of mice . d‐Glucosamine‐based supramolecular hydrogels assist wound healing and prevent the formation of scars .
3.6. Antimicrobial hydrogels
The infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, and parasites are still a public health problem despite the major development in health care and medical technology. Treatment with conventional antibiotics of infectious diseases often leads to the development of antibiotic resistance . Recently, a new strategy to treat infectious diseases has been developed using antimicrobial hydrogels. The hydrogels act on the entire cellular membrane, which leads to cell membrane rupture, followed by a leakage of cytoplasmic contents and cell death. Different types of antimicrobial hydrogels have been developed in recent research.
Some hydrogels possess antimicrobial properties that include natural and synthetic polymeric hydrogels, and peptide‐based hydrogels. Mohamed et al. prepared hydrogels by chitosan cross‐linked with different amounts of pyromellitimide benzoyl thiourea moieties . The hydrogels were extremely porous and exhibited a higher antibacterial activity and antifungal activity. The swelling ability of hydrogels and their antimicrobial activity increased with cross‐linking density. Peng et al. developed novel cellulose‐based hydrogels that showed superabsorbent property, high mechanical strength, good biocompatibility, and excellent antimicrobial efficacy against
Despite the tremendous ability of antimicrobial hydrogels in breaking down multidrug resistant microbes, the interactions between antimicrobial polymers and microbial cell membranes are nonspecific which, in most cases, cause mammalian cell death above certain concentrations . One solution is to combine antibiotics and antimicrobial hydrogels so that less antimicrobial hydrogel is used and the associated toxicity is minimized.
Another type of interesting hydrogels contains antimicrobial metal nanoparticles. The use of silver ions and silver nanoparticles in hydrogels has obtained substantial advances in wound treatment . The silver nanoparticles supported within PVA/cellulose acetate/gelatin was successfully synthesized. The hydrogels have antimicrobial activity against various fungi and bacteria . The toxicity of silver and other metal salts is a disadvantage for this type of hydrogels. Efforts have been made to reduce the toxicity.
3.7. Cancer research
Hydrogels have been used in cancer research among many other applications. However, many drugs are hydrophobic and cannot be efficiently loaded and released from hydrogels. There are two ways to improve the loading and releasing, incorporating hydrophobic domains into hydrogels and introducing nanoparticles which encapsulate hydrophobic compounds .
Recent trends have indicated significant and growing interest in developing nanocomposite hydrogels (NCH) for various biomedical applications. NCH are hydrated polymeric networks, cross‐linked with each other and/or with nanostructures . Some of the nonocarriers have been successfully incorporated in gel networks, such as carbon nanotubes, ME, dendrimers, metal, ceramic, and polymeric nanoparticles [99, 117]. The NCH has nanocarrier stabilization, shape regulation, improved composite viscoelasticity, and mechanical properties with optimized drug release kinetics on top of the conventional hydrogel characteristics . Abdel‐Bar et al. developed a cisplatin ME hydrogel for controlled cisplatin release and improved cytotoxicity with decreased side effects . The NCH containing nano‐sized carriers allowed a zero order drug release for 14 days and enhanced cytotoxicity. The higher animal survival rate and lower tissue toxicities proved the decreased toxicity of cisplatin nanocomposite compared to its solution. This system could help in achieving better outcomes and quality of life during use of chemotherapy for cancer treatment by intraperitoneal administration.
Another great interest of hydrogels in cancer research focuses on the cancer cell invasion in hydrogels. Fisher et al. studied the HA‐based cross‐linked hydrogels in breast cancer cell invasion . The results showed that increased crosslink density correlates with decreased breast cancer cell invasion whereas incorporation of enzyme‐cleavable sequences within the peptide cross‐linker enhances invasion. This study provides a platform that recapitulates variable tissue properties and elucidates the role of the microenvironment in cancer cell invasion by independently tuning the mechanical and chemical environment of ECM mimetic hydrogels. Zhang et al. created covalently cross‐linked hydrogel materials through a rapid reaction at the gel‐liquid interface . The interfacial cross‐linking was then used to encapsulate prostate cancer cells. The cells obtained 99% viability, proliferated readily, and formed aggregated clusters. Such
4. Conclusions and perspectives
In this chapter, we have discussed recent progress in polymerization, various strategies for cross‐linking of natural and synthetic biopolymers for preparation of
From a chemistry point of view, the reactions selected should occur in an aqueous environment under mild reaction conditions without damaging the encapsulated biofunctional molecules and cells. The chemicals used for cross‐linking such as monomers, initiators, cross‐linkers, and catalysts should be carefully selected to minimize toxicity to cells. Attention should also be paid to biocompatibility between polymers and incorporated bioactive species such as cells and proteins. Introduction of reactive functional groups to hydrogel materials will surely promote the cross‐linking of hydrogels, but the active groups may also show off‐target reactivity to incorporated bioactive species or cells. For example, amino groups and thiol groups on proteins can react with vinyl sulfone and acrylate groups used in Michael addition, aldehyde groups used in Schiff‐base formation, or diepoxide groups in coupling reaction for hydrogel cross‐linking. These undesired reactions may damage proteins, reduce drug efficacy, or induce immunogenicity. Therefore, reactions are preferred to be highly efficient and selective for cross‐linking so that all groups intended for cross‐linking are reacted, but no undesired reactions occur between the hydrogel and incorporated biofunctionalities or cells.
Because of the widespread biomedical applications, research on
In general, a deeper understanding of material properties for the development of
This research is financially supported by the NSF EPSCoR RII Track 1 cooperative agreement awarded to the University of South Carolina (NSF EPSCoR Cooperative Agreement number EPS‐0903795).
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