Heparin-based and heparin-inspired hydrogels: size-effect, gelation and biomedical applications

Chao He a, Haifeng Ji a, Yihui Qian a, Qian Wang a, Xiaoling Liu a, Weifeng Zhao *a and Changsheng Zhao *ab
aCollege of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China. E-mail: zhaoscukth@163.com; zhaochsh70@163.com
bNational Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China

Received 11th October 2018 , Accepted 4th January 2019

First published on 5th January 2019


Heparin is the highest negatively charged biomolecule, which is a polysaccharide belonging to the glycosaminoglycan family, and its role as a regulator of various proteins, cells and tissues in the human body makes it an indispensable macromolecule. Heparin-based hydrogels are widely investigated in various applications including implantation, tissue engineering, biosensors, and drug-controlled release due to the 3D-constructs of hydrogels. However, heparin has supply and safety problems because it is usually derived from animal sources, and has the clinical limitations of bleeding and thrombocytopenia. Therefore, analogous heparin-mimicking polymers and hydrogels derived from non-animal and/or totally synthetic sources have been widely studied in recent years. In this review, the progress and potential biomedical applications of heparin-based and heparin-inspired hydrogels are highlighted. We classify the forms of these hydrogels by their size including macro-hydrogels, injectable hydrogels, and nano-hydrogels. Then, we summarize the various fabrication strategies for these hydrogels including chemical covalent bonding, physical conjugation, and the combination of chemical and physical interactions. Covalent bonding includes free radical polymerization of vinyl-containing components, amide bond formation reaction, Michael-type addition reaction, click-chemistry, divinyl sulfone crosslinking, and mussel-inspired coating. Hydrogels physically conjugated via host–guest interaction, electrostatic interaction, hydrogen bonding, and hydrophobic interaction are also discussed. Finally, we conclude with the challenges and future directions for the fabrication and the industrialization of heparin-based and heparin-inspired hydrogels. We believe that this review will attract more attention toward the design of heparin-based and heparin-inspired hydrogels, leading to future advancements in this emerging research field.


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Chao He

Chao He obtained his BS degree in Polymer Processing Engineering in 2012, and then obtained his PhD degree in Biomedical Engineering in 2017 under the supervision of Prof. Changsheng Zhao from the College of Polymer Science and Engineering, Sichuan University. He is currently a lecturer in Sichuan University. His current research work focuses on the blood and cell compatibility of polymeric hydrogels, and GO-based blood-contacting materials for hemopurification.

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Haifeng Ji

Haifeng Ji obtained his BS degree in Polymer Processing Engineering in 2016, from the College of Materials Science and Engineering, Sichuan University. In June 2016, he became a PhD candidate in Biomedical Engineering, College of Polymer Science and Engineering, Sichuan University under the supervision of Prof. Changsheng Zhao. His research interests include the blood compatibility of polymeric membranes and hydrogels, bacterial eradication of polymeric microspheres, and blood toxin eradication of carbon-based materials.

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Weifeng Zhao

Prof. Weifeng Zhao received his BS degree in Polymer Science & Engineering in 2009 and then obtained his PhD degree in Biomedical Engineering in 2014 from Sichuan University. He did another PhD study at KTH Royal Institute Technology from 2012 to 2015, and received that degree in 2015. Subsequently, Dr Zhao joined the College of Polymer Science and Engineering, Sichuan University and as an Associate Professor in 2016. Dr Zhao's research interests are mostly focused on the development of bio- and blood-compatible polymers and functional hydrogels and he is also dedicated to the design of hemoperfusion adsorbents.

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Changsheng Zhao

Prof. Changsheng Zhao received his BS degree in Polymer Materials from Sichuan University in 1991 and then obtained his PhD degree in Biomedical Engineering from Sichuan University in 1998. From 2001 to 2003, he did research work in Hokkaido University (Japan) as a postdoctoral research fellow. Subsequently, he joined Sichuan University and became a professor in 2004. Prof. Zhao's research interests are mostly focused on the development of hemodialysis materials with bioactive and anticoagulant properties, he is also dedicated to the design of heparin-inspired hydrogels and composite biomaterials for tissue engineering and regeneration medicine.


1. Introduction

Hydrogels are a class of polymer materials with hydrophilic groups, which are swollen but insoluble in water since they have crosslinked three-dimensional network structures.1,2 Hydrogels can be defined as natural hydrogels and synthetic hydrogels according to their sources. Natural hydrogels are fabricated from natural polymers include anionic polymers (pectin), cationic polymers (chitosan), and neutral polymers (dextran). In contrast, synthetic hydrogels are fabricated from synthetic polymers including homo-polymers (polyacrylic acid (PAA)), random polymers (PAA-co-poly(isopropyl acrylamide) (PAA-co-PNIPAAm)), and block copolymers (PEG-b-PAA), as shown in Fig. 1. According to the network bonding mode, hydrogels can be divided into physically crosslinked hydrogels (electrostatic interaction, hydrogen bonding, chain entanglement, etc.) and chemically crosslinked hydrogels (chemical bond crosslinking). According to the hydrogel size, they can be divided into macro-hydrogels (according to their shape, they can be further classified into columnar shape, porous sponge shape, fibrous shape, film shape, spherical shape, etc.), injectable hydrogels (amorphous hydrogels), and micro-hydrogels (including nano-hydrogels). According to the response of hydrogels to external stimuli, they can be divided into traditional hydrogels and stimuli-responsive (pH and temperature) hydrogels. Further, according to their application, hydrogels can be divided into water-plugging hydrogels, humidifying hydrogels, biomedical hydrogels, etc. Due to their good characteristics such as hydrophilicity, biocompatibility, non-toxicity, and biodegradability, hydrogels have been widely used in the biomedical field.3
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Fig. 1 Various polymers used for the fabrication of hydrogels.

A variety of natural polymers and synthetic polymers have been used to design hydrogels. Among them, heparin-based hydrogels have aroused extensive interests due to their unique biomedical properties. Heparin is a natural anticoagulant synthesized by the liver, mucous membranes and lungs, and its molecular weight is between 7000–25[thin space (1/6-em)]000 Da. Heparin is a pentosan with a five-carbon sugar ring in its main chain and contains functional groups such as carboxyl, sulfonic acid and hydroxyl groups (Fig. 2). In 2017, Lima et al. reviewed new developments in the use of heparin and related glycosaminoglycans (GAGs) in various fields ranging from thrombosis and neurodegenerative disorders to microbiology and biotechnology.4 Subsequently, Groth et al. gave a brief overview on the structure and biological functions of GAGs, the recent progress in GAG-related biomaterials and their biomedical applications.5 Heparin participates in the interaction of inflammatory mediators, proteases and histamines and is present in the body's mast cell granules. The use of heparin in the construction of hydrogels is proven to be beneficial for the enhancement of biocompatibility and efficacy. Thus, hydrogels can be used as growth factor carriers, cell carriers, and anticancer carriers. The rich functional groups in heparin have potential for conjugation with biomolecules and increase biocompatibility and efficacy, promote cell adhesion, allow cell-mediated protein degradation, and can control the loading and releasing behaviors of growth factors.


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Fig. 2 Structure of heparin.

Growth factors are biological macromolecules based on polypeptides, which can regulate various cellular processes and tissue regeneration, and thus play an important role in the treatment of various diseases.6 However, due to their short half-life, poor stability, complex structure, enzymatic inactivation and other drawbacks, the effective delivery of growth factor is a big challenge. Heparin has a high affinity for a variety of growth factors and is able to isolate them from the extracellular matrix, making it an attractive candidate for binding growth factors.7 Heparin can also stabilize growth factors to prevent them from being degraded by proteases. The interaction between heparin and growth factor occurs mainly through electrostatic attraction between the sulfated residues of heparin and the amino acid residues of growth factor. Also, heparin-based hydrogels play an important role in controlling the release of growth factors.8–10 The heparin-binding growth factors with specific applications are listed in Table 1.

Table 1 The heparin-binding growth factors and their applications
Growth factor Abbreviation Applications
Vascular endothelial growth factor VEGF Angiogenesis,11,12 wound healing6
Fibroblast growth factor FGF Cell proliferation13,14
Insulin-like growth factor IGF Diabetes treatment and adipose regeneration15
Hepatocyte growth factor HGF Tissue engineering,16,17 cancer treatment18
Epidermal growth factor EGF Tissue repair,19 astrocyte proliferation15
Growth and differentiation factor GDF Osseointegration,20,21 ovarian follicle development22
Bone morphogenetic protein BMP Bone regeneration23–25
Transforming growth factor TGF Chondrogenesis26–28


Heparin-containing hydrogels often exhibit excellent properties such as anticoagulant activity, binding to growth factors, and anti-angiogenic and apoptotic effects, giving them broad applications in emerging fields. The presence of sulfonic acid groups and carboxyl groups makes heparin highly negatively charged, which allows it to interact electrostatically with many proteins, such as growth factors, proteases, and chemokines. In most cases, these interactions help stabilize the protein or increase its affinity for cellular receptors. For example, the growth factors fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) can be introduced into a material to design a controlled release platform for tissue engineering. Therefore, the introduction of heparin into hydrogels is an attractive method. The use of heparin as a basic polymer for scaffold fabrication has also been explored, often utilizing the non-covalent binding of heparin with peptides or proteins to promote the self-assembly of hydrogel networks.29

However, heparin has significant supply and safety problems because it is usually derived from animal sources, and has the clinical limitations of bleeding risk and thrombocytopenia. Therefore, analogous heparin-mimicking (also called heparin-inspired) polymers and hydrogels derived from non-animal and/or totally synthetic sources have been widely studied in recent years. Apart from its special sugar ring structure, the heparin molecule also contains functional groups such as carboxyl, sulfonic acid and hydroxyl groups in its main chain. Thus, analogous heparin-mimicking polymers mainly mimic these functional groups and their conformations. In the case of heparin-inspired hydrogels, the strategy is mainly to mimic the rich functional groups. In 2014, we highlighted the progress and potential biomedical applications of surface heparinization and the heparin-inspired modification of polymeric membranes.30 Two years later, Prof. Maynard from the University of California, Los Angeles summarized the major types of various synthetic heparin-mimicking polymers and their applications.31 More recently, Ferro et al. covered the development of heparin mimetics from synthetic polymers, non-carbohydrate small molecules and/or sulfated polysaccharides.32 The research interests in this field are highly evoked due to these reviews. Although some of the research on heparin-inspired hydrogels has been highlighted,33–37 it is strongly desirable to summarize the advances, synthetic strategies and biomedical applications of heparin-inspired hydrogels.

Therefore, in this review, we first focus on the diverse methods to fabricate heparin-based and heparin-inspired hydrogels. Then, we comment on the potential biomedical applications of heparin-based and heparin-inspired hydrogels, including cell culture, drug delivery, and blood contacting. Then finally, the challenges and future directions for the fabrication and industrialization of heparin-based and heparin-inspired hydrogels are discussed.

2. Heparin-based hydrogels

The natural polymer of heparin has been employed to prepare heparin-based hydrogels, which can be classified by their size into macro-hydrogels, injectable hydrogels, and nano-hydrogels. Macro-hydrogels are macroscopic with sizes of one millimeter or more,38 which can be further classified into columnar, spongy, fibrous, filmy, and spherical state according to their shape. Injectable hydrogels belong to the class of amorphous hydrogels, which can be delivered via syringe-needle injection followed by a sol–gel transition to in situ-form hydrogels.39 Micro-hydrogels have a particle size smaller than 5 μm, and nano-hydrogels are a special type of micro-hydrogel with diameters ranging from 10 to 200 nm,40 although some studies reported that hydrogels with a size between 10 and 1000 nm can also be called nano-hydrogels.41

2.1. Heparin-based macro hydrogels

Heparin-based macro hydrogels can be prepared via various methods, and the crosslinking networks of hydrogels can be built by chemical covalent bonding, physical conjugation, and the combination of chemical and physical interactions.
2.1.1 Chemical covalent bonding. Covalent bonding is the most utilized strategy to build heparin-based hydrogels, which includes free radical polymerization of vinyl-containing components, amide bond formation reaction, Michael-type addition reaction, click-chemistry, divinyl sulfone crosslinking, and mussel inspired coating.
2.1.1.1 Free radical polymerization. Since vinyl groups can be utilized to fabricate crosslinking networks, they are frequently introduced onto heparin molecules to prepare heparin-based hydrogels.42–49 For example, Botchwey et al.42,43 fabricated N-desulfated heparin methacrylamide, which was then reacted with poly(ethylene glycol)–diacrylate (PEG–DA, crosslinker) through free radical polymerization to prepare heparin-functionalized PEG–DA hydrogels, as shown in Fig. 3(A). The fabricated hydrogels could load and release the sphingosine analog FTY720 and stromal-derived factor-1α (SDF-1α) with maintained bioactivity, which have the potential to be used for tissue repair with facilitated macrophage transition and vascular network expansion. Besides PEG, other biomacromolecules such as hyaluronan (HA) can also be introduced to heparin-based hydrogel systems via free radical polymerization of glycidyl methacrylated heparin (HPGMA) and glycidyl methacrylated HA (HAGMA) (synthesized by the conjugation of the carboxyl groups of biomacromolecules and the epoxy groups of GMA), as reported by Zhao et al. and shown in Fig. 3(B).50 The fabricated hyaluronan–heparin (HA–HP) hybrid hydrogel was homogeneous and its biomacromolecule content was adjustable, which could be used for the controllable and sustained delivery of bone morphogenetic protein-2 (BMP-2). The free radical polymerization method was also used to prepare multi-functional heparin-based hydrogels. Green's group44 explored the covalent incorporation of bioactive molecules within a conducting hydrogel (CH), which was covalently formed from biosynthetic poly(vinyl alcohol)–heparin, adhesive biomolecules sericin, conductive polymer (CP, poly(3,4-ethylene dioxythiophene)) and gelatin via methacrylate crosslinking, as shown in Fig. 3(C). The electrical properties of the bioactive CH were improved with retained bioactivity of heparin; thus, their hydrogel may be used for nerve growth factor loading and delivering to target cells with outgrowth of neural processes. Degradable heparin-based hydrogels were also fabricated by introducing dithiothreitol, as reported by Temenoff et al.51 They first functionalized heparin derivatives with methacrylamide (MAm) groups by amide bond formation reaction, which were then reacted with poly(ethylene glycol)diacrylate and dithiothreitol to prepare the degradable hydrogels. The hydrogel degradation was tuned by varying the amount of dithiothreitol, and the obtained hydrogels showed controllable binding and release of crystal violet (CV, a positively-charged anti-inflammatory agent) by varying the level of heparin sulfation. Thus, their work may inspire the formulation of promising CV delivery vehicles for a wide range of inflammatory diseases.
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Fig. 3 (A) Hep−N–PEG–DA hydrogels co-release bioactive FTY720 and SDF-1α in vitro. (B) Synthesis pathway for hyaluronan–heparin (HA–HP) hybrid hydrogel. (C) Schematic of biofunctionalized CH, consisting of a conducting polymer component grown through a biosynthetic hydrogel, which comprises an anionic biological dopant, additional covalently bound proteins and mobile diffusible growth factors. (A–C) were reprinted with permissions from ref. 42. Copyright (2018) American Chemical Society, ref. 50. Copyright (2017) De Gruyter, and ref. 44. Copyright (2014) Elsevier, respectively.

2.1.1.2 Amide bond formation reaction. As mentioned above, heparin contains carboxyl groups, which can be used to react with amino groups to introduce vinyl groups for free radical polymerization.42,43,45,46 Furthermore, the amide bond formation reaction can also be utilized for the preparation of heparin-based hydrogels by directly forming crosslinking networks, with more simple fabrication procedures.52–56 For example, Seib's team easily prepared PEG-containing heparin-based hydrogels through amide bond formation reaction, which were loaded with doxorubicin as a focal breast cancer therapy carrier.53 A star-shaped PEG (starPEG)–heparin hydrogel was also prepared via the direct chemical crosslinking (EDC/sulfo-NHS chemistry) of amino-terminated 4-arm PEG (starPEG) and heparin, as reported by Werner's group and shown in Fig. 4(A).54 The obtained scaffold offered mechanical protection and allow the attachment of islets and mesenchymal stromal cells (MSC) accessory cells within its pores, and the MSC could further bio-functionalize the hydrogel scaffold by secreting ECM proteins. Thus, this porous starPEG–heparin hydrogel is a promising vehicle for the housing of pancreatic islets. To combine hemocompatibility and antimicrobial activity, the same group prepared silver-containing PEG–heparin multilayer hydrogel coatings.55 The obtained multi-layered hydrogel coatings performed well for plasmatic coagulation, platelet activation and hemolysis when incubated with human whole blood (Fig. 4(B)), and simultaneously showed long-term antiseptic efficacy against Escherichia coli and Staphylococcus epidermidis strains.
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Fig. 4 (A) Representative SEM image of the hydrogel with a schematic representation of the underlying starPEG–heparin network. (B) Proposed mechanism of multifunctional hydrogel coating-mediated reactions in whole blood. (C) General chemical synthesis scheme for heparin and gelatin-conjugated alginate hydrogel and myogenic differentiation of hSMPCs on hydrogel substrates. (A–C) were reprinted with permissions from ref. 54. Copyright (2016) Elsevier, ref. 55. Copyright (2015) Elsevier, and ref. 57. Copyright (2017) Elsevier, respectively.

Besides directly crosslinked heparin-based hydrogel networks, heparin can also be grafted onto many polymer matrixes by amide bond formation reaction, and further utilized for the preparation of different types of heparin-based hydrogels.57–60 Ljima's group immobilized heparin in collagen (Hep-col) and then gelated at 37 °C to prepare an extracellular matrix (ECM)-modeled hydrogel, which was utilized to mimic the in vivo-like environment and establish an in situ evaluation system for hydrogel-embedded cell responses.58 A quercetin-conjugated heparin hydrogel was developed by Park et al. for the improvement of blood compatibility.59 Also, Zhang's team conjugated heparin and gelatin to modify alginate hydrogel (gelated via a sodium-to-calcium ion exchange method).57 The obtained substrate (Alg-G-H) could enhance the proliferation and differentiation of human skeletal muscle progenitor cells (hSMPCs) together with skeletal muscle ECM, as shown in Fig. 4(C).


2.1.1.3 Michael-type addition reaction. It is convenient to bond different molecules together via Michael-type addition reaction. Among the Michael-type addition reactions, thiol–ene reaction is frequently utilized to fabricate heparin-based hydrogels by grafting ene61–69 or thiol70–86 groups onto heparin molecules. For instance, Kloxin's group reported a degradable heparin-based hydrogel that responds to clinically relevant exogenous stimuli and endogenous stimuli, which was formed with PEG and heparin-based polymer precursors using a Michael-type addition reaction, as shown in Fig. 5(A).63 Recently, a type of matrix metalloprotease (MMP)-responsive starPEG/heparin hydrogel was prepared for cartilage regeneration.61 The carboxyl groups of heparin were reacted with maleimide amine via EDC/sulfo-NHS-activation to generate maleimide group conjugated heparin, then the obtained heparin–maleimide and MMP-responsive-starPEG-conjugates with cysteine termini were crosslinked by Michael-type addition, and the fabricated hydrogel was utilized to embed and culture mesenchymal stromal cells (MSC) and chondrocytes. Levental et al. combined glycosaminoglycan heparin, starPEG, and MMP-cleavable crosslinkers to prepare a biohybrid hydrogel, which was applied to dissect the biophysical and biochemical signals promoting human mammary epithelial cell (MEC) morphogenesis (Fig. 5(B)).65 Their works provided a versatile platform to study mammary epithelial tissue morphogenesis in a chemically defined and precisely tunable 3D in vitro microenvironment; thus, allowing investigation of the biophysical and biochemical aspects of mammary gland biology.
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Fig. 5 (A) Hydrogels were formed by reacting aryl-thiol end-functionalized poly(ethylene glycol) macromolecular precursors (PEG-4-MPA or PEG-4-PD-MPA) with maleimide-end-functionalized precursors (PEG-2-MI or heparin-MI) via Michael-type reaction. (B) Illustration of the study designed to investigate mammary epithelial cell morphogenesis in a hydrogel matrix, which contained star-shaped four-armed PEG (grey), MMP-cleavable peptide sequence (MCP, green), non-cleavable scrambled peptide sequence (scr, red) and glycosaminoglycan heparin (yellow). (C) Schematic illustration of a heparin hydrogel “sandwich” for hepatocyte culture. (A–C) were reprinted with permissions from ref. 63. Copyright (2017) Wiley, ref. 65. Copyright (2017) Elsevier, and ref. 75. Copyright (2015) Elsevier, respectively.

Besides ene group-conjugated heparin precursors, thiol group-conjugated heparin precursors are also commonly introduced to prepare heparin-based hydrogels with various structures via Michael-type addition reaction. For example, a heparin-based hydrogel sandwich was fabricated by Revzin's group via UV light-induced ene–thiol coupling reaction of diacrylated poly(ethylene glycol) (PEGDA) and thiolated heparin (Hep-SH) precursors, which offered a culture system for the maintenance of primary rat hepatocytes (Fig. 5(C)).75 The heparin–PEG hydrogel sandwich exhibited significant flexibility with tunable mechanical properties, adjustable heparin content and photo-promoted gel degradation, which could be utilized as a potential system for maintaining differentiated and polarized primary hepatocytes. Besides the sandwich structure, they also built hydrogel microstructures for the cultivation of primary hepatocytes.76,87 The microwells with both walls and floor composed of heparin hydrogel were constructed via the combination of the micromolding and microcontact printing techniques. Hepatic phenotype expression was enhanced on the heparin gel walls of the microwells, and thus their work indicates that gel microstructures may be a promising hepatic niche for the liver-specific differentiation of stem cells. Heparin-based hydrogels prepared by Michael-type addition reaction can also be utilized for other bio-applications. For example, a hyaluronic acid (HA)-based hydrogel was constructed for Matrix-Assisted Cell Transplantation (MACT), and heparin was introduced to coordinate the presentation of TGF beta 1 and to support the trophic functions of the cardiac progenitor cells (CPCs) via endothelial cell differentiation and vascular-like tubular network formation.84


2.1.1.4 Other covalent bonding approaches. Besides the chemical bonding methods mentioned above, heparin-based hydrogels can also be prepared via other covalent bonding approaches, such as click-chemistry, divinyl sulfone crosslinking, and mussel-inspired. Schaffer's team utilized click-chemistry between dibenzocyclooctyne (DBCO) and azide derivatives to functionalize a hyaluronic acid hydrogel with heparin and RGD, which was then utilized for in vitro maturation and central nervous system (CNS) transplantation of human pluripotent stem cell (hPSC)-derived neural progenitors, as shown in Fig. 6(A).88 The divinyl sulfone (DVS) crosslinking method was also used to synthesize heparin-decorated hyaluronic acid-based hydrogel particles (HGPs) via an inverse emulsion polymerization technique, as seen in Fig. 6(B).89 The covalently immobilized heparin retained its ability to bind bone morphogenetic protein-2 (BMP-2), and the prepared hydrogel particles were utilized as an attractive candidate for the release of BMP-2, and thus applied for cartilage repair and regeneration.
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Fig. 6 (A) Heparin- and RGD-functionalized HA hydrogel crosslinked with PEG-diazide crosslinkers, and confocal images showing the expression of TH and TUJ1 in mDA neuronal clusters cultured within the prepared hydrogels. (B) Crosslinked structures established by DVS in the hybrid hydrogels, and the controlled release of BMP-2 from HP-decorated HA-based hydrogels. (C) Heparin-modified hydrogel tube was obtained by post-functionalization. (A–C) were reprinted with permissions from ref. 88. Copyright (2017) Elsevier, ref. 89. Copyright (2011) Elsevier, and ref. 90. Copyright (2017) Royal Society of Chemistry, respectively.

Since dopamine (DA, a mussel adhesive protein-inspired molecule) can form irreversible covalent bonds to solid surfaces in alkaline aqueous solution, it has drawn intensive interest for surface modification via DA self-polymerization.91–93 Our group anchored dopamine-grafted heparin to an alginate/polyacrylamide double-network hydrogel via a mussel-inspired coating (Fig. 6(C)), and fabricated a heparin post-functionalized and highly stretchable hydrogel tube for potential application as an artificial blood vessel.90 Clotting time, platelet adhesion, hemolysis ratio, and complement activation tests indicated that the heparinized hydrogel tube had excellent hemocompatibility. Furthermore, the endothelial cell culture test showed that the heparinized hydrogel had improved cell adhesion affinity, and thus the obtained hollow hydrogel tube could meet the needs of dynamic functionality of stretchable blood vessels. Other chemicals such as hydrazide-conjugated derivates have also been utilized to fabricate heparin-based hydrogels.52,94 By bonding the N-hydroxysuccinimidyl ester of PEG-bis-butanoic acid (SBA–PEG–SBA) with hydrazide-functionalized heparin (Hep-ADH), Tae's group prepared a crosslinked heparin-based hydrogel, which possessed heparin-binding domains and was utilized as an affinity-based controlled release system for growth factors (VEGF) with an increased density of endothelial cell marker platelet endothelial adhesion molecules (PECAM-1).52

Through covalent bonding, heparin can be introduced to hydrogel matrices to fabricate stable heparin-based hydrogels. However, this chemical strategy has some drawbacks since it may destroy the structure and activity of heparin, and may require many other chemicals with toxicity both humans and the environment. Thus, comparing all the chemical methods mentioned above, mussel-inspired coating may be a promising strategy since it is a green method with a simple procedure.

2.1.2 Physical conjugation. The fabrication of chemical bond crosslinked hydrogels always needs rigorous reaction conditions; whereas, it is relatively easier to form physical conjugated hydrogels via host–guest interaction, electrostatic interaction, hydrogen bonding, hydrophobic interaction, etc.
2.1.2.1 Host–guest interactions. Molecules with functional groups can be nested together through host–guest interactions to form crosslinking networks and prepare heparin-based hydrogels. Zhang's group utilized the host–guest interactions between α-cyclodextrin and amino-terminated poly(ethylene glycol)methyl ether conjugated heparin to prepare a supramolecular hydrogel network with controlled drug release characteristics.95 The prepared hydrogel exhibited good potential as an injectable matrix for encapsulating and releasing model protein bovine serum albumin, and also had a controlled release profile for the conjugated heparin, and thus showed good anticoagulant and blood-compatible properties. Also, an injectable and biodegradable heparin-based hydrogel was designed using the dual physical dynamic bonds of host–guest interaction and electrostatic interaction by Wang's team, as shown in Fig. 7(A).96 They firstly coupled arginine and mono-carboxylic acid-terminated PEG to poly(ethylene aspartate diglyceride) (PEAD) to obtain mPEG-g-PEAD polymer, which was then mixed with heparin and α-cyclodextrin to form a supramolecular hydrogel network via quick gelation, which had shear thinning properties. Their hydrogel showed the sustainable release of FGF2 with a stable rate, which suggested that the fabricated hydrogel system has potential applications in ischemic tissue regeneration and wound healing.
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Fig. 7 (A) Formation of a dually crosslinked heparin incorporated hydrogel based on host–guest interaction and electrostatic interaction, and the steady release of FGF2 from the heparin-based hydrogel. (B) Electrostatic interaction to prepare the CS/Hep/γ-PGA composite hydrogel, and the wound healing and closure rate of the different hydrogels. (C) Schematic of the molecular self-assembly via intra- and inter-molecular hydrogen bonding and ionic interaction. (A–C) were reprinted with permission from ref. 96. Copyright (2016) Royal Society of Chemistry, ref. 97. Copyright (2018) Elsevier, and ref. 99. Copyright (2015) American Chemical Society, respectively.

2.1.2.2 Electrostatic interaction. As mentioned above, electrostatic interaction can also be utilized to form crosslinking networks, and thus can be used to prepare heparin-based hydrogels. Heparin, chitosan (CS) and poly(gamma-glutamic acid) were combined via electrostatic interaction to prepare composite hydrogels, which were further loaded with an antioxidant for the healing of diabetic wounds, as shown in Fig. 7(B).97 The prepared hydrogels showed cytocompatibility to fibroblasts, and could accelerate wound healing by promoting collagen deposition and wound closure after the loading of superoxide dismutase. Wang's group utilized LAPONITE® (a silicate nanoparticle) to bind heparin by electrostatic interaction to prepare a new type of hydrogel. This hydrogel retained heparin's natural affinity towards many proteins and could protect FGF2 from proteolytic degradation, which was released with preserved bioactivity to induce angiogenesis in vitro.98 Additionally, Hartgerink's team combined ionic interaction and hydrogen bonding to prepare a hydrogel using multivalent molecules (heparin, trypan, clodronate, phosphate, and suramin) for drug delivery, as shown in Fig. 7(C).99 Their polymeric delivery vehicles exhibited long-term release behavior, and the drug-loaded crosslinked hydrogels could modulate the cellular phenotype.
2.1.2.3 Hydrogen bonding. Since hydrogen bonding occurs between hydrogen and electro-negative atoms to build crosslinking networks, many researches have reported the use of this method to prepare heparin-based hydrogels.37,99–102 For instance, a heparin-based hydrogel was prepared via the self-assembly of heparin sodium salt and peptide RAD16-I, as shown in Fig. 8(A).37 The prepared hydrogel enhanced the capacity of binding and release of growth factor VEGF165, and thus can be used as a promising system for adipose-derived stem cells (ADSC) survival and chondrogenic commitment. Nam's group applied a high hydrostatic pressure (HHP) method to prepare a heparin-embedded PVA hydrogel crosslinked by inter-molecular hydrogen bonding between the PVA side chains, as shown in Fig. 8(B).100 The heparin–PVA complex hydrogel could prevent clot formation and showed potential as a vascular access, and the HHP method exhibited great promise to prepare specifically-functionalized PVA hydrogels. The hydrogen bonding interactions among peptide CTTHWGFTLC (CTT) was also utilized to prepare an injectable, fast gelation, thermosensitive, and degradable hydrogel for the slow release of the low molecular weight CTT.102
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Fig. 8 (A) Self-assembled heparin-based hydrogel for tissue engineering applications. (B) Heparin-embedded PVA hydrogel fabricated with Dacron mesh. (C) Self-assembled hydrogel/micelle composite constructed by hydrophobic interaction and host–guest interaction. (A–C) were reprinted with permission from ref. 37. Copyright (2015) Elsevier, ref. 100. Copyright (2014) Wiley, and ref. 104. Copyright (2012) Royal Society of Chemistry, respectively.

2.1.2.4 Hydrophobic interaction. Hydrophobic domain-containing polymers exhibit supramolecular self-assembly driven by inter and intra polymeric hydrophobic interactions, and thus can be utilized to fabricate heparin-based hydrogels.69,103–105 GAGs-modified amphiphilic multi-block copolymers were introduced to prepare crosslinker-free hydrogels via self-assembly, which contained triblock poly(ethyleneoxide)–polypropylene oxide copolymers (Pluronics), HP and HA.103 Zhang's team designed a supramolecular hydrogel/micelle composite for the encapsulation and release of granulocyte colony-stimulating factor (G-CSF) and camptothecin (CPT), as shown in Fig. 8(C).104 Heparin-conjugated Pluronic F-127 (Hep-F-127) was initially synthesized as an amphiphilic polymer, which then self-assembled into micelles; meanwhile, G-CSF was loaded to the hydrophilic shell, and CPT was loaded to the hydrophobic core. By further adding α-cyclodextrin (alpha-CD) to the system, a hydrogel/micelle composite was formed, which maintained the biological activity of the encapsulated G-CSF and CPT with prolonged release behavior. Indomethacin (IMC) and basic FGF (bFGF) were entrapped in a poly(ethylene glycol-b-caprolactone-ethylene glycol) (PECL) micelle through hydrophobic interaction and affinity to heparin, which was then utilized as both filler and chemical crosslinker to form chitosan-based hydrogels, and the loaded IMC and bFGF could be released from the prepared hydrogels at a slow rate.105 The hydrophobicity of the fabricated substrate affects the gelation process, which is also an important factor that can affect the degradation of prepared hydrogels.69
2.1.2.5 Other physical interactions. Besides the methods mentioned above, there are many other physical methods that can be used to prepare heparin-based hydrogels. Stimuli-responsive heparin-based hydrogels have been prepared and utilized as candidate materials for the delivery of therapeutic drugs. The temperature-induced sol-to-gel transition of heparin-bearing poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (Hep–PCLA) was used to develop a temperature-responsive injectable hydrogel system for lysozyme delivery.106 The Hep–PCLA conjugates were free-flowing in aqueous solutions (25 °C), and could form a gel under physiological conditions (37 °C), as shown in Fig. 9(A). The model protein lysozyme was loaded into the fabricated hydrogel via hydrophobic and ionic interactions, which was slowly released even during the initial period.
image file: c8tb02671h-f9.tif
Fig. 9 (A) Heparin-based hydrogel prepared by temperature-induced sol-to-gel transition. (B) aFGF- and bFGF-loaded heparin–poloxamer hydrogel for wound healing. (C) Dynamic delivery of IGF-1-loaded heparin nanospheres from a glucose-responsive heparin-based hydrogel via boronate–maltose ester bonds. (A–C) were reprinted with permission from ref. 106. Copyright (2015) Royal Society of Chemistry, ref. 113. Copyright (2016) American Chemical Society, and ref. 116. Copyright (2018) Taylor & Francis Group, respectively.

The triblock copolymer of poloxamer consists of ethylene oxide (EO) and propylene oxide (PO), and its aqueous solution exhibits temperature-induced aggregation phenomena as a result of the hydrophobic nature of the PO block.107 Thus, Zhao's group conjugated poloxamer and heparin to prepare a series of heparin–poloxamer (HP) thermo-sensitive hydrogels for multiple purposes. For example, they utilized the heparin–poloxamer hydrogel for improving vascular anastomosis quality and safety in a rabbit model.107 By combining the advantages of heparin and poloxamer, the HP hydrogel holds high promise for improving vascular anastomosis quality and safety. To treat spinal cord injury (SCI), acidic FGF (aFGF) was mixed with lyophilized HP powder to form a thermo-sensitive aFGF-bridged heparin–poloxamer hydrogel by increasing the temperature to 37 °C, which exhibited the sustained release of aFGF with maintained bioactivity in vitro.108 They also introduced basic fibroblast growth factor (bFGF) and dental pulp stem cells (DPSCs),109,110 Glial cell-derived neurotrophic factor (GDNF)111 and nerve growth factor (NGF)112 in the HP hydrogel for spinal cord injury repair. Different GFs (aFGF and bFGF) were also delivered by the same thermos-sensitive heparin–poloxamer hydrogel for wound healing in vivo, as shown in Fig. 9(B).113 Their work showed superior healing activity to improve wound closure, granulation formation, re-epithelization, and blood vessel density via up-regulation of PCNA proliferation and collagen synthesis. The temperature-sensitive heparin-modified poloxamer (HP) hydrogel could also be used as a support matrix to deliver keratinocyte growth factor (KGF, a potent repair factor for epithelial tissues) and prevent intrauterine adhesion (IUA).114 Epsilon-polylysine (EPL) was further introduced as a functional excipient in the HP hydrogel matrix to fabricate a mucoadhesive EPL–HP hydrogel.115In vitro release of the model drug (KGF) from EPL–HP hydrogel was significantly accelerated by adding EPL in comparison with the HP hydrogel. Thus, the mucoadhesive EPL–HP hydrogel with a suitable KGF release profile may be a more promising than HP hydrogel alone to repair injured endometrium.

To further dynamically deliver an insulin-like growth factor (IGF-1, loaded in heparin nanospheres previously), a heparin-based hydrogel in physiological conditions was developed via the formation of boronate–maltose ester crosslinks between the boronate and maltose groups of heparin derivatives.116 Due to its intrinsic glucose-sensitivity, the exposure of gel scaffold to glucose induced the dissociation of the maltose-functionalized nanospheres from the hydrogel matrix, and the dissociative nanospheres could then dynamically move into the microenvironment, as shown in Fig. 9(C). Thus, the prepared hydrogels were sensitive to blood glucose concentration and could achieve dynamic release when the surrounding glucose concentration increased.

Compared with the covalent bonding method, physical conjugation is easier and simpler for the preparation of heparin-based hydrogels, and the immobilized heparin can maintain its structure and activity. However, physical conjugation also has some shortcomings since the introduced heparin may be eluted from the hydrogel and the amount of heparin in heparin-based hydrogels is limited.

2.1.3 Combined interactions. Heparin-based hydrogel networks can be built by chemical bonding or physical conjugation, while to further construct dual-crosslinked networks, the combination of chemical and physical interactions has also been reported.105,117,118 Park's team combined sol-to-gel transition and photo-initiated ene crosslinking to fabricate a biodegradable heparin/pluronic composite hydrogel for growth factor delivery, as shown in Fig. 10(A).117 Vinyl group-conjugated heparin and diacrylate-terminated Pluronic F127 were firstly mixed together, which underwent a sol-to-gel transition, and then further photo-crosslinked to form the designed hydrogels, and the growth factor bFGF was in situ-loaded during the gelation process. Their work exhibited that the heparin-based hydrogels could load and sustainably release bFGF, and proliferate human umbilical vein endothelial cell (HUVEC) with significant extent of neovascularization, and thus has the potential to be utilized for angiogenesis. Besides physical conjugation followed by chemical bonding, dual-crosslinked heparin-based hydrogels can also be built by firstly chemical bonding and then physical conjugation, as shown in Fig. 10(B). Bettinger et al. introduced biological heparin as a scaffold material and in situ fabricated a hydrogel network with ultra-compliant mechanical property and electronic/ionic conductivity.118 Methacrylic anhydride (MA) was reacted with heparin to prepare Hep–MA through esterification, which was utilized to prepare a photo-crosslinkable heparin scaffold. The scaffold served as a template with a controllable microstructure to dope aniline (electrostatic interaction) for in situ polyaniline polymerization. Their fabricated heparin/polyaniline hydrogel exhibited suitable impedance and conductivity, and also supported the attachment, proliferation, and differentiation of murine myoblasts without any further surface treatments, and thus has potential to be used as a soft tissue material for cell culturing.
image file: c8tb02671h-f10.tif
Fig. 10 (A) Heparin/pluronic composite hydrogel formed by sol-to-gel transition and photo-initiated ene crosslinking. Implantation of bFGF-loaded hydrogel and histological cross-section of underlying tissue. (B) In situ formation of Hep–MA/PANI hydrogel dual network by photo-crosslinking and electrostatic interaction. (A and B) were reprinted with permission from ref. 117. Copyright (2007) Wiley and ref. 118. Copyright (2014) American Chemical Society, respectively.

2.2. Heparin-based injectable hydrogels

Hydrogel materials are applied in the human body mainly via the injection method. Injectable hydrogels are a type of hydrogel with an amorphous form, which can flow under specific conditions (such as shearing, temperature change, and pH change), and can in situ gel to form hydrogels. The preparation of traditional heparin-based macro-hydrogels often requires complex processes with low efficiency, which restrict their application in medical treatment. Thus, recently, the development of rapid and gentle methods to prepare heparin-based hydrogels has become a new hotpot, and the hydrogels prepared via these methods show great potential in the application of injectable materials. Heparin-based injectable hydrogels can be used for tissue engineering,119,120 growth factor combination,72,121 tumor chemotherapy,53,122 and wound healing.96,123 The main methods to prepare heparin-based injectable hydrogels can also be divided into chemical crosslinking and physical crosslinking.

In the case of chemical crosslinking, click chemistry is commonly used for the preparation of heparin-based injectable hydrogels since this method is rapid and efficient. For example, Prestwich's group crosslinked thiol-modified heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol)diacrylate (PEGDA) to create synthetic hydrogel mimics of the extracellular matrix (ECM).72 The covalently bound heparin provided a crosslinkable analog of a heparin sulfate proteoglycan, thus providing a multivalent biomaterial with the controlled release of bFGF. The hydrogels could be injected and crosslinked in situ, and thus constituted highly promising new materials for the controlled release of heparin-binding growth factors in vivo. Another injectable and detachable micro-patterned heparin-based hydrogel was prepared via thiol–ene “click” reaction, which was prepared by manipulating the amount of diacrylated poly(ethylene glycol) (PEG–DA) against a fixed amount of thiolated heparin (Hep-SH).124 The obtained hydrogel was used as an efficient matrix for the hepatic differentiation of human adipose-derived stem cells (hADSCs) and as a cell delivery carrier. This provided a promising total platform from in vitro stem cell differentiation to the enhanced survival and engraftment of cells in vivo upon injection, and may be further applied for the regeneration of damaged liver or other tissues in the future. However, the metabolite of this hydrogel is still a foreign body in the human system.

Considering the abovementioned problem, the use of polysaccharides with reactive groups for crosslinking has become a new idea for the preparation of hydrogels since the applied raw materials are all polysaccharides, which ensure better biocompatibility of hydrogel materials. Wang's team prepared an injectable hydrogel scaffold, which actively facilitated the formation and maturation of blood vessels when implanted in mice.125 Konjac glucomannan (KGM) and heparin (Hep) were modified with tyramine (TA) groups, and the two polysaccharides co-polymerised rapidly to form a hydrogel upon enzyme catalysis. As shown in Fig. 11(A), the co-polymerized hydrogel fully preserved the unique bioactivities of the two polysaccharides by effectively inducing macrophages to secrete pro-angiogenic growth factors and sequestering the macrophage produced growth factors in situ. Abundant and mature blood vessels were found in the hydrogels after 14 days of subcutaneous implantation without the addition of any exogenous pro-angiogenic factors (Fig. 11(B)). Overall, their study demonstrated an innovative and feasible biomaterial-based approach to exploit the power of endogenous growth factors to encourage angiogenesis, which may be applicable for regenerative and therapeutic purposes.


image file: c8tb02671h-f11.tif
Fig. 11 (A) Schematic illustration of the co-polymerised KGM-TA/Hep-TA hydrogel. (B) In vivo sequestration of pro-angiogenic GFs in the KGM-TA/Hep-TA hydrogel. (A and B) were reprinted with permission from ref. 125. Copyright (2017) Elsevier. (C) LMWH–cholesterol conjugates self-assembled into nanoparticles with a core–shell structure due to the hydrophobic interaction between the cholesterol moieties. Reprinted with permission from ref. 126. Copyright (2018) Elsevier.

Physical crosslinking can also be used for the preparation of heparin-based injectable hydrogels, and due to the complexity of the human environment, the hydrophobic interaction is a suitable method for the fabrication of injectable hydrogels. Ci et al.126 prepared low molecular weight heparin (LMWH)-based cholesterol (LHC) conjugates for the intravenous delivery of doxorubicin (DOX), as shown in Fig. 11(C). LHC nanoparticles were prepared with LMWH as the hydrophilic shell and cholesterol as the hydrophobic core. The DOX/LHC nanoparticles (DOX/LHC NPs) had a longer circulation time than that of DOX and exhibited superior anti-metastatic effects in the pulmonary metastasis model. This may be due to the synergistic effect between the cytotoxic drug (DOX) and the drug carrier (LMWH-based nanoparticles), which resulted in anti-metastatic and anti-angiogenic efficiency. Thus, the DOX/LHC nanoparticles are a promising anti-metastatic drug delivery system for postoperative chemotherapy.

The traditional injectable hydrogels face needle clogging issues, which may restrict their biomedical applications. In situ forming hydrogels (specific injectable hydrogels) will not clog the needle, since they undergo a sol–gel transition after being injected inside the human body with stimuli-responsive properties (temperature and pH).127–130 Thus, the introduction of pH and temperature-sensitive functional polymers in the heparin backbone for the preparation of heparin-based injectable hydrogels is attractive. Also, pH and temperature-sensitive heparin-based injectable hydrogels can be applied in various fields ranging from therapeutic delivery to tissue engineering.53,106,124,131,132 For example, Lee et al. developed a temperature-sensitive injectable Hep–PCLA hydrogel system for lysozyme carriage. The Hep–PCLA conjugates underwent sol-to-gel transition upon temperature changes in an aqueous solution, which can freely flow at 25 °C and can form a hydrogel at body temperature (37 °C).106

2.3. Heparin-based nano-hydrogels

Nano-hydrogels (or nanogels) are a particular type of nanoparticle with sizes ranging from 10 to 200 nm and the characteristics of hydrogels. Also, nano-hydrogels are considered a special type of micro-hydrogel since they have a smaller size with high flexibility and versatility. Thus, nano-hydrogels can be considered for application in drug delivery through inhalation, parenteral, and/or topical administration. Due to the size effect, the drug-loaded nanogels are injectable and can be injected into the blood for systemic release or injected to a specific site to achieve intelligent drug release according to environmental changes. However, these functions are difficult to achieve using macro-hydrogels, which are mainly used for pulmonary and oral delivery. Additionally, macro-hydrogels are difficult to apply directly due to the size limitation, but used by transepithelial delivery and placement inside the body. Accordingly, the small size of nano-hydrogels not only makes them needle-injectable but also offers more surface area for drug conjugation, and can lead to facile natural clearance with enhanced penetration through tissue barriers. Heparin-based nano-hydrogels also have crosslinked structures and can be used for drug delivery,133–135 promoting directed differentiation,136 loading RNases,137 and tissue regenerative medicine.138 Also, their preparation methods can be divided into chemical crosslinking, microfluidic, and hydrogen bonding.

For chemical crosslinking, click chemistry is also suitable for the preparation of heparin-based nano-hydrogels, as reported by Park et al.133 Heparin was chemically modified with thiol groups and then crosslinked with disulfide linkages to produce reducible heparin-based nano-hydrogels for the efficient intracellular delivery of free heparin, as shown in Fig. 12(A). Their work provided a new strategy to induce apoptosis in cancer cells using heparin-based nano-hydrogels. The whole synthetic process was convenient, and there were no excess degradation products. Another strategy for preparing heparin-based nano-hydrogels is to initiate the reaction by free radical crosslinking polymerization between double bonds.134 Nano-hydrogels were prepared by derivatizing heparin with vinyl groups, followed by copolymerization with cystamine bisacrylamide in aqueous medium. The prepared nano-hydrogels were used for drug delivery with a low drug release in neutral environment, but fast drug release in a reductive environment. This indicated that the prepared nano-hydrogels were promising for tumor treatment with high efficacy due to their long circulation time, prominent sensitivity to a reductive environment, and high drug accumulation in the tumor site.


image file: c8tb02671h-f12.tif
Fig. 12 (A) Synthetic scheme for disulfide crosslinked heparin nano-hydrogels. Reprinted with permission from ref. 133. Copyright (2008) Elsevier. (B) Photos of the HCT-116 abdominal metastasis tumor after different treatments. (C and D) Tumor weight and nodules in the different treatment groups. (E) Immunohistochemical analysis of Ki-67, TUNEL and hTRAIL expression of tumors in each group. (a) NS; (b) HPR; (c) PEI25K/phTRAIL; (d) HPR/pMCS; and (e) HPR/phTRAIL. (B–E) were reprinted with permission from ref. 139. Copyright (2018) Taylor & Francis Group.

Since heparin contains carboxyl groups, the condensation reaction of carboxyl groups with amino groups is also a convenient way to prepare nano-hydrogels, which does not need heparin to be further modified, and thus maintains its activity. For example, a polyethyleneimine-R8-heparin nano-hydrogel was prepared by covalent binding between the carboxyl group from heparin and the amino group from polyetherimide, which was used for high-efficiency gene delivery.139 The heparin-based nano-hydrogel could condense pDNA compactly and exhibited efficient endolysosomal escape. Meanwhile, the nano-hydrogels presented high transfection efficiency and could deliver plasmid hTRAIL (phTRAIL) into HCT-116 cells to induce significant apoptosis in vitro (Fig. 12(B–D)). Moreover, the heparin/phTRAIL complex exhibited prominent inhibition of tumor growth in a mouse tumor model (Fig. 12(E)). This reduced the toxicity of gene delivery therapy using the shielding effect of heparin, meanwhile, part of the R8 peptide located on the surface of the nano-hydrogel could significantly enhance its cellular uptake. Thus, we believe that this work is very instructive for low-toxic and efficient gene therapy.

Microfluidic technology is a new method in the field of bioengineering, which has also been used to prepare nano-hydrogel materials. The nano-hydrogels prepared via this method are size-controllable, which cannot be achieved via conventional polymerization methods. Revzin's group firstly attempted to prepare heparin-based bioactive nano-hydrogels for the rapid formation and enhanced differentiation of stem cell spheroids via this method.136 As a result, the adsorption of heparin-binding growth factors enhanced the directed differentiation of embryonic stem cells toward the endoderm. Their work focused specifically on endoderm-directed differentiation, and the versatility and simplicity of the fabrication method ensure that this system can be implemented as a scalable platform for general differentiation applications or for the development of tailored cell delivery vehicles.

Physical crosslinking methods are also suitable for the preparation of nano-hydrogel materials. Among them, hydrogen bonding is considered to be a suitable choice due to its changeable bonding force depending on the pH of the environment. Thus, the nano-hydrogels can be disintegrated in the appropriate environment to release drugs. A biopolymer nano-hydrogel was prepared by Tan et al. via hydrogen bonding assembly for vectoring delivery of biopharmaceuticals.138 In the multifunctional nano-hydrogel, the vectoring delivery of bone morphogenetic protein 2 (BMP-2) could be easily controlled. The existence of heparin ensured that the nano-hydrogel had a high loading efficiency of BMP-2, and the duration release of BMP-2 from the nano-hydrogel was low under physiological conditions. The BMP-2-loaded biopolymer nano hydrogels showed high efficiency to promote the viability of MG-63 cells. Thus, multifunctional nano-hydrogels based on biopolymers have great potential in future cartilage and bone tissue regeneration applications.

3. Heparin-inspired hydrogels

As a product derived from animals, the direct utilization of heparin for hydrogel preparation has some drawbacks, e.g. the high cost of heparin inhibits its large-scale use.140,141 Additionally, heparin is a natural polysaccharide, and a dramatic loss in bioactivity and degradation may occur when preparing heparin-based hydrogels via covalent or non-covalent strategies.142 Moreover, when heparin-based hydrogels are exposed to blood or tissue systems, they may covalently or non-covalently interfere with blood components.36 Thus, one of the current research hotspots is developing heparin–mimetic polymers to substitute heparin in the fabrication of hydrogels. Sulfonated polymers and sulfated glycosaminoglycan have been widely recognized as heparin-inspired components since they show similar or some of the bioactivities of heparin, such as anti-clotting and antithrombotic activities, stabilization of growth factors, and promotion of angiogenesis.143–145 Furthermore, these heparin-inspired polymers are usually obtained from facile and scalable chemical synthesis; meanwhile, they have better defined structures and more programmable bioactivity. Moreover, heparin-inspired polymer-modified biomaterials have demonstrated excellent blood and cell compatibility both in vitro and in vivo, which are comparable to that of heparin-immobilized substrates.146–149 Thus, heparin-inspired polymers have great potential in the fabrication of hydrogels for various biomedical applications.

Hence, to better understand the role of heparin-inspired hydrogels and their derivatives in well-designed systems with specific functionalities, a systematic review of this aspect is necessary. This section systematically reviews the various types of heparin-inspired hydrogels (e.g., heparin-inspired macro-hydrogels, surface-attached hydrogel thin films injectable hydrogels, and nano-hydrogels). Additionally, the latest applications (e.g., cell culture, loading of drugs/molecules, blood contacting applications, and other applications) and preparation methods of these heparin-inspired hydrogels will also be highlighted.

3.1. Heparin-inspired macro-hydrogels

Heparin-inspired macro-hydrogels are constructed using heparin–mimetic polymers with three-dimensional network structures. To date, the methods to prepare heparin-inspired macro-hydrogels can also be divided into chemical covalent bonding and physical conjugation. For chemical covalent bonding, besides the ways mentioned above, heparin-inspired macro-hydrogels can also be prepared from the polymerization of monomers containing vinyl and sulfonic acid groups (such as sodium styrene sulfonate (SSNa) and 2-acrylanmido-2-methylpropanesulfonic acid (AMPS), respectively).36 Besides, physical crosslinking methods can also be used to prepare heparin-inspired macro-hydrogels.150,151 The methods for the preparation of heparin-inspired hydrogels are similar to that of heparin-based hydrogels, which have been mentioned in the previous sections. Thus, we will mainly focus on the functions of heparin-inspired hydrogels, which currently are used in the biomedical fields of cell culture, wound healing, drug loading, and blood contacting.
3.1.1 Cell culture. It was reported that heparin-inspired polymers can fix growth factors, and subsequently, the use of heparin-inspired polymers to prepare macro-hydrogels for cell culture became a hotspot since the fabricated hydrogels possess abundant functional groups and porosity. Our group reported a robust, highly elastic and bioactive heparin-inspired hydrogel for cell culture, which was synthesized with doped graphene oxide (GO) as the micro-crosslinker via radical polymerization of acrylic acid (AA) and SSNa, as shown in Fig. 13(A).36 The GO-doped heparin-inspired hydrogel exhibited highly interpenetrating networks with numerous small pores, thin pore walls, and a narrow pore size distribution, and showed reinforced mechanical strength and elastic properties. Furthermore, the heparin-inspired hydrogel exhibited endothelial cell compatibility (Fig. 13(B)). This hydrogel is simple to prepare, but its biocompatibility still needs to be enhanced. To further improve the biocompatibility of heparin-inspired hydrogel materials, Tronci's team investigated the structure–function relationships in chitosan covalent networks.152 Monosodium 5-sulfoisophthalate (PhS) was selected as the growth factor-binding crosslinking segment, whilst 1,4-phenylenediacetic acid (4Ph) and PEG bis(carboxymethyl) ether were employed as sulfonic acid-free diacids of low and high crosslinker length, respectively. The PhS-crosslinked chitosan hydrogels displayed the highest loss (40 ± 6 CFU%) of antibacterial activity upon incubation with Porphyromonas gingivalis, while the cell compatibility of the hydrogel extract was tolerated (no hydrogel-triggered toxic response) in L929 mouse fibroblasts, as shown in Fig. 13(C). The PhS-crosslinked chitosan hydrogels showed great antibacterial property with no obvious cytotoxicity, and thus, their work is inspiring for the design of antibacterial hydrogel materials with cytocompatibility.
image file: c8tb02671h-f13.tif
Fig. 13 (A) Fabrication of GO-doped and interpenetrated heparin-inspired hydrogels, (B) FDA/PI staining and confocal images of the cells cultured with fragmented hydrogels, and the MTT assay to quantify the cell proliferation. (A) and (B) were reprinted with permission from ref. 36. Copyright (2015) Royal Society of Chemistry. (C) Extract cytotoxicity tests were conducted and L929 cell morphology was investigated in either DMEM (a), extract of sample CT-PhS (b), and MTS assay on either extract of sample CT-PhS, DMEM or DMSO (c) and the antibacterial activity displayed by both CT hydrogels and native CT (d). Reprinted with permission from ref. 152. Copyright (2017) Elsevier.
3.1.2 Wound healing. Fibroblast growth factor-2 and vascular endothelial growth factor are vital for the repair of wound injuries. Heparan sulfate proteoglycans of the extracellular matrix also play key roles in blood vessel formation because heparan sulfate chains can interact, accumulate, and promote pro-angiogenic growth factors. Guler and Tekinay's group conducted many studies on the preparation of heparin-inspired materials for wound healing. They reported that a self-assembled heparin–mimetic peptide amphiphile (HM–PA) gel was an effective bioactive wound dressing for the rapid and functional repair of full-thickness excisional wounds in a rat model.150 The HM–PA hydrogel mimicked the activity of heparan sulfates by presenting sulfonate, hydroxyl and carboxylate groups on amino acid side chains. The bioactive gel-treated wounds exhibited increased angiogenesis, re-epithelization, skin appendage formation, and granulation tissue organization compared to the sucrose-treated samples. In the same year, they used the functional and biodegradable peptide hydrogels for burn wounds healing.153 The bioactive hydrogel-treated burn wounds formed well-organized and collagen-rich granulation tissue layers, produced a greater density of newly formed blood vessels, and exhibited increased re-epithelialization and skin appendage development with minimal crust formation, as shown in Fig. 14(A and B). These bioactive hydrogel-formed scaffolds recapitulated the structure and function of the native extracellular matrix through signalling peptide epitopes, which can trigger angiogenesis through their affinity to basic growth factors. Their group also investigated the effect of a heparin-inspired PA nanofiber gel on full-thickness excisional wounds in a db/db diabetic mouse model,154 with emphasis on the ability of the PA nanofiber network to regulate angiogenesis and the expression of pro-inflammatory cytokines. Functionalized PA molecules were synthesized via standard solid phase Fmoc peptide synthesis chemistry. Lauryl-VVAGEGDK(pbs)S-Am (GAG-PA) and Lauryl-VVAGK-Am (K-PA) were synthesized on Rink amide resin. The heparin–mimetic PA gel could support tissue neovascularization, enhance the deposition of collagen and expression of alpha-smooth muscle actin (α-SMA), and eliminate the sustained presence of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the diabetic wound site, as shown in Fig. 14(C–E). Thus, we believe that the heparin-inspired PA nanofiber gel treatment may be a promising candidate for the acceleration of diabetic wound healing by modulating angiogenesis and local immune responses.
image file: c8tb02671h-f14.tif
Fig. 14 (A) Representative images of burn wounds after gel application on different days. (B) Quantification of the wound areas treated with different materials. (A) and (B) were reprinted with permission from ref. 153. Copyright (2017) Elsevier. (C) Bioactive heparin–mimetic peptide nanofiber treatment accelerated the recovery of diabetic wounds. (D) Two wounds (red and blue circle) were created by biopsy punches at the dorsal skin of db/db mice on day 0. (E) Wound area of GAG-PA/K-PA nanofiber-treated animals was smaller than that of the control samples. (C–E) were reprinted with permission from ref. 154. Copyright (2017) Royal Society of Chemistry.
3.1.3 Tissue transplantation. To extend the applications of HM–PA in pancreatic islet transplantation, the same group155 prepared HM–PA hydrogel networks to sustain the viability and functionality of islets during transplantation. The islets cultured with peptide nanofiber gel (containing growth factors) exhibited a similar glucose stimulation index as that of the freshly isolated islets even after 7 days. After transplantation of the islets to streptozocin-induced diabetic rats, 28 day-long monitoring displayed that the islets transplanted in HM–PA nanofiber gels maintained better blood glucose levels compared to the islet-alone transplantation group. In addition, the intraperitoneal glucose tolerance test revealed that the animals implanted with islets within peptide gels showed a similar pattern with the healthy control group. Histological assessment showed that the islets transplanted within peptide nanofiber gels demonstrated better islet integrity with increased blood vessel density. Their work shows that heparin-inspired materials have the potential to be used in the tissue transplantation field.
3.1.4 Loading of drugs. Since heparin-inspired hydrogels have a crosslinking structure and good biocompatibility, they can also be used in drug and protein loading systems. Recently, our group reported hemo- and cyto-compatible GO-hybridized heparin-analogue hydrogels as potential implantable biomaterials for anti-cancer drug and protein delivery, as shown in Fig. 15.156 All the hydrogels exhibited a porous morphology, and their porosity decreased with an increase in the GO concentration (Fig. 15(A–D)). GO was incorporated into the hydrogels to improve their mechanical properties and optimize their drug and protein loading and releasing performances. The DOX-loaded GO/heparin-analogue hydrogels presented efficient anti-cancer cell activity due to their high drug loading and persistent releasing abilities (Fig. 15(E and F)). This work combined graphene oxide materials with heparin–mimetic materials to produce high performance drug-loaded hydrogel materials, and we believe that the synthesized GO doped heparin–mimetic hydrogels have great potential applications in various biomedical fields, such as tissue engineering and implantable drug delivery systems. Subsequently, our group further prepared heparin-inspired hydrogels by introducing chemical crosslinked sulfated polysaccharide.157 Sulfamic acid groups were reacted with konjac glucomannan (KGM) to produce sulfated konjac glucomannan (SKGM), and then heparin-inspired hydrogels were prepared using SKGM, hexamethylene diisocyanate and diols. The hydrogels showed excellent hemocompatibility. Gentamycin sulfate was used as a model drug to be loaded into the hydrogels, and the loading amount increased by ca. 50% after the introduction of SKGM, thus resulting in high bactericidal efficiency. Although these materials have good biocompatibility and sustained drug release, their environmental responsiveness is relatively poor.
image file: c8tb02671h-f15.tif
Fig. 15 (A) Photographs of the synthetic hydrogels. (B) SEM images of the cross-sectional views of the hydrogels in the equilibrium swollen state. Pore-size distributions with various amounts of GO (C) and with various amounts of crosslinker MBA (D). (E) Releasing behaviour of DOX from the drug-loaded hydrogels at pH 7.4 and 5.0. (F) Viability of HeLa cells after exposure to the DOX-loaded hydrogel suspensions. Reprinted with permission from ref. 156. Copyright (2016) Royal Society of Chemistry.

To endow heparin-inspired hydrogels with environmental responsiveness, Park's team reported a heparin-inspired sulfonated reverse thermal hydrogel as a novel protein delivery system, which was prepared via sulfonation of a graft copolymer, poly(serinol hexamethylene urea)-co-poly(N-isopropylacylamide) (PSHU–NIPAAm).158 The sulfonated PSHU–NIPAAm showed a typical temperature-dependent sol–gel phase transition phenomenon, where the polymer solution turned to a physical gel at around 32 °C and could maintain this status at body temperature. In vitro tests revealed that the release of protein from the sulfonated PSHU–NIPAAm was more sustained than that from PSHU–NIPAAm. Furthermore, the sulfonated PSHU–NIPAAm system did not affect the protein structure after a 70 day observation period. Thus, this type of sulfonated PSHU–NIPAAm system can serve as a platform for biomimetic injectable biomaterials for controlled and sustained protein delivery.

When hydrogels are utilized for drug delivery, the mesh size of the hydrogel is a critical control factor. The mesh size is the spacing between the polymer chains of hydrogel crosslinked networks, which allows the diffusion of small solutes and liquids. The mesh size of hydrogels ranges from around 5 nm to 100 nm,159 which is affected by intrinsic factors (basic material characteristics and crosslinker concentration) and outer stimuli (pH and temperature).160,161 When drugs are loaded into hydrogels, the diffusion process is influenced by the mesh size since it controls the steric interaction between the polymer network and the drugs.38 For example, when the mesh size is smaller than the loaded drug, the drug is physically entrapped inside the network and cannot be released directly only by enlarging the mesh size through swelling or degradation.162 When the mesh size approaches the size of the drug, the drug can be released slowly. For hydrogels with a larger mesh size than the immobilized drug, the drug diffusion is almost independent of mesh size and can migrate freely through the network; thus, the drug delivery should be further controlled by the drug–hydrogel interactions (covalent conjugation, electrostatic interactions, hydrophobic associations, etc.).163–165

Thus, to control the release of drugs successfully and efficiently, mesh size is an important factor, as well as the interactions between drugs and hydrogels. Specifically, the designed mesh size should be uniform with a smaller size than that of the target drug, and the hydrogels can be swollen or degraded by external stimuli. Furthermore, the interactions between drugs and hydrogels should also be stimuli-responsive to achieve controlled release. In addition, the hydrogels should be biocompatible and be able to protect the drugs from degradation. Finally, the drug loading amount, hydrogel mechanical property, surface property, size and shape are other factors that should be considered for the design of drug delivery systems.

3.1.5 Blood contacting applications. Heparin-inspired hydrogels can achieve anticoagulant function by adsorbing calcium ions and binding to specific coagulation factors. Moreover, these materials do not trigger hemolysis or decrease blood cells. Therefore, in recent years, a large number of studies have been reported on the use of heparin-inspired hydrogels as blood contact materials. Our group synthesized heparin-inspired chitosan with various carboxymethyl and sulfate groups,166 followed by a crosslinking reaction with glutaraldehyde to prepare heparin-inspired chitosan hydrogels for blood purification, as shown in Fig. 16(A). The activated partial thromboplastin time (APTT) and thrombin time (TT) results indicated that both the heparin-inspired chitosan and corresponding hydrogels exhibited excellent thrombus inhibition property (Fig. 16(B)). Furthermore, the contact activation and complement activation results proved that the hydrogels possessed good blood compatibility, as shown in Fig. 16(C). Our group also prepared GO-based heparin-inspired hydrogels with hemocompatibility via free radical copolymerization.167 The obtained hydrogels exhibited interconnected structures with thin pore walls and high porosity, as shown in Fig. 16(D). The heparin-inspired hydrogels showed superior red blood cell compatibility, anti-platelet adhesion ability and anticoagulant ability, and thus exhibited good hemocompatibility. Therefore, the prepared heparin-inspired hydrogels possessed versatile functionalities and may present great potential to be used in versatile biomedical applications including blood-contact field. However, their poor mechanical properties and obvious swelling behaviors might restrict their application. Thus, we further developed an efficient, dimensionally stable and blood compatible hemoperfusion adsorbent by imitating the structure and functional groups of heparin.168 Carrageenan and PAA crosslinked networks were constructed by the combination of phase inversion of carrageenan and post-crosslinking of AA, as shown in Fig. 16(E). The dual-network structure endowed the beads with improved mechanical properties and restricted swelling ratios. The beads exhibited low protein adsorption, low hemolysis ratio, low cytotoxicity, suppressed complement activation and contact activation, and excellent anticoagulant properties. The beads also showed satisfactory adsorption capacities toward exogenous and endogenous toxins. Thus, the carrageenan-based heparin-inspired gel beads with superior performances showed strong advantages in hemoperfusion.
image file: c8tb02671h-f16.tif
Fig. 16 (A) Proposed synthesis of heparin-like chitosan hydrogel in an acidic medium. (B) Activated partial thromboplastin time and thrombus time for HLCHl and HLCHh. (C) Concentrations of PF4, TAT, C3a, and C5a for the HLCHs. (A–C) were reprinted with permission from ref. 166. Copyright (2016) American Chemical Society. (D) Preparation and SEM images of GO-based heparin-mimicking polymeric hydrogels. Reprinted with permission from ref. 167. Copyright (2015) Royal Society of Chemistry. (E) Preparation of the heparin-mimicking CRG beads. Reprinted with permission from ref. 168. Copyright (2018) American Chemical Society.

3.2. Surface-attached hydrogel thin films

When a material is in contact with blood, its surface blood compatibility is particularly important, since its surface will directly contact with blood. Surface properties such as chemical composition, roughness, and hydrophilicity are essential factors when contacting with external surfaces.169 Thus, the interface design can directly affect the properties and application fields of biomedical materials, where the precise control of surface structures through physical and chemical methods may provide a facile strategy to effectively functionalize materials.170–172 For most biomaterials, conventional surface modification is insufficient since the functional groups are uniformly distributed in the matrices without enrichment on the surface. Considering these drawbacks, many studies have attempted to construct heparin-inspired hydrogel thin films on material surfaces to improve their blood compatibility. Hydrogel thin films are rich in functional groups and show great hydrophilicity; thus, the modification effect is very significant. Surface-attached hydrogel thin films are a special type of macro-hydrogel, and since it can directly affect the property and application of biomaterials we separated this part for individual discussion. For the modification methods, surface chemical crosslinking polymerization, layer-by-layer (LBL) self-assembly, and spin coating methods are mainly used to prepared surface-attached hydrogel thin films.

Using covalent bonding to graft heparin-inspired hydrogel thin films onto a material surface is a stable method. Our group reported a method using heparin-inspired hydrogel thin films to enhance the blood compatibility of polyethersulfone (PES) membranes via covalent bonding.173 Functional groups were firstly introduced onto the PES membrane surface via in situ crosslinking polymerization of hydroxyethyl methacrylate, followed by phase inversion, and then double bonds were introduced via the reaction of acryloyl chloride. Sodium metharylate and sodium ally sulfonate were selected as monomers to form heparin-inspired hydrogel thin films by simultaneous covalent attachment on the functional PES membrane surfaces via surface crosslinking copolymerization. The membranes showed excellent anticoagulant properties with prolonged activated partial thromboplastin time values, and the blood-related complement activation level was suppressed. By using the same strategy, we also developed poly(sulfobetaine methacrylate)/poly(sodium acrylate) antibacterial hydrogel thin films, which were covalently attached onto the PES membrane via surface crosslinking copolymerization.174 Ag ions were adsorbed on the hydrogel layer and reduced to Ag nanoparticles by sodium borohydride, as shown in Fig. 17(A). The designed surfaces not only effectively resisted bacteria attachment, but also killed the surrounding bacteria of E. coli and S. aureus. Additionally, the modified membranes showed excellent hemocompatibility. Also, recently, we further prepared a self-defensive bilayer hydrogel coating, which could switch from a cell adhesion surface to an antibacterial adhesion surface.33 To covalently attach the coating, an antifouling hydrogel thin film was firstly prepared on a thiol-modified substrate as the bottom layer, which was fabricated via thiol–ene click reaction with an ene-functionalized copolymer of poly(sulfobetaine methacrylate acid-2-hydroxyethyl methacrylate). Thereafter, heparin-inspired polymer chains were grafted onto the hydrogel film surface as the upper layer via surface-initiated atom transfer radical polymerization. This modification can be applied to various matrices, such as polymeric, inorganic, and metal substrate implants.


image file: c8tb02671h-f17.tif
Fig. 17 (A) Chemical attachment of hydrogel thin layer onto PES membrane. (B) Fabrication of 3D nano-hydrogel-deposited membranes via surface engineered LBL assembly. (C) Construction of heparin–mimetic hydrogel thin film by LbL interfacial assembly and mussel-inspired oxidative cross-linking. (A–C) were reprinted with permission from ref. 174. Copyright (2017) American Chemical Society, ref. 175. Copyright (2014) Royal Society of Chemistry, and ref. 176. Copyright (2015) American Chemical Society, respectively.

Although grafting the heparin-inspired hydrogel onto the surface of the material via covalent bonding is stable, the synthetic steps are relatively complicated, and the thickness of the hydrogel is difficult to control. In comparison, physical integration is simpler, and using negatively charged heparin-inspired polymers and positively charged polymers to construct hydrogel film on the surface of material through layer-by-layer self-assembly is a common method. Our group fabricated 3D multifunctional layers on biomedical membrane surfaces by employing Ag nanoparticle-embedded nanogels and heparin-inspired polymers through layer-by-layer self-assembly, as shown in Fig. 17(B).175 The approach endowed the membranes with integrated blood compatibility, cell proliferation and antibacterial properties for multiple applications, which may advance the fabrication of biomedical devices. Since the mussel-inspired strategy is commonly substrate-independent, we designed a heparin-inspired hydrogel thin film coating via combined layer-by-layer self-assembly and mussel-inspired post-crosslinking.176 In brief, the substrates were coated with dopamine-grafted heparin-like polymers (DA-g-HepLP) to generate negatively charged surfaces, followed by LbL coating of chitosan and DA-g-HepLP. Finally, the noncovalent multilayers were oxidatively crosslinked by NaIO4 (Fig. 17(C)). The crosslinked hydrogel coatings exhibited excellent long-term stability, low cell toxicity, high promotion ability for cell proliferation and excellent hemocompatibility.

The use of electrostatic self-assembly to build heparin-inspired hydrogel coatings is simple, but lacks stability in a physiological environment. Thus, to solve this problem, our group applied a two-step spin coating method to construct dual-layered polymeric membranes with a top heparin-inspired hydrogel thin film layer.177 Due to the integration of the porous membrane structure, good mechanical strength, excellent hemocompatibility, as well as robust bactericidal capability, the dual-layered membranes have great potential for clinical hemodialysis and many other biomedical therapies.

3.3. Heparin-inspired injectable hydrogels

Heparin-inspired injectable hydrogels can also be applied in many fields such as combining some growth factors,178 loading of drugs179 and tumor chemotherapy.180 Compared with heparin-based injectable hydrogels, the preparation of heparin-inspired injectable hydrogels does not require the participation of heparin, so the monomers used are more flexible and the preparation methods are more diverse. Chemical crosslinking181–184 and physical crosslinking such as hydrogen-bond interaction, electrostatic interaction and hydrophilic interaction185–189 can be used to prepared heparin-inspired injectable hydrogels.

Since chitosan has a similar structure to heparin and is easily modified, using modified chitosan to prepare heparin-inspired injectable hydrogels via chemical crosslinking has become a common method. Lee's group reported an injectable, in situ forming hydrogel system for the co-delivery of human adipose-derived stem cells (hADSC) and platelet-derived growth factor (PDGF).178 In their study, gelatin was modified with tyramine (GTA), while the amino groups of chitosan was functionalized with 4-hydroxyphenyl acetic acid (CHPA), resulting in a chitosan derivative, which was soluble at neutral pH. Also, the hydrogel was obtained as a consequence of the formation of crosslinks between phenolic groups via enzyme-catalyzed oxidative reaction, and also between phenol groups and free amino groups, as shown in Fig. 18(A). Significantly high hADSC viability was observed for the PDGF-BB concentration of 10–20 μg mL−1. This CHPA-GTA system for co-delivering stem cells and growth factor is a promising, minimally invasive approach for tissue regeneration. The support vascularization of irregular defects can be easily adapted for bone tissue engineering or similar applications.


image file: c8tb02671h-f18.tif
Fig. 18 (A) Reaction scheme and dual syringe delivery system of chitosan-4-hydroxylphenylacetamide (CHPA)–gelatin-tyramine (GTA) and CHPA–GTA in situ forming hydrogel. (B) Preparation of 3D crosslinked hydrogels from modified chitosan (Chit–Glu) and PEG–BA crosslinker through Schiff-base linkages. (C) Preparation of self-healing pH-sensitive injectable hydrogels based on cytosine- and guanosine-modified hyaluronic acid. (D) Schematic representation of the crosslinking of CMCh by zinc ions to form supramolecular hydrogels. (A–D) were reprinted with permission from ref. 178. Copyright (2017) IOP Science, ref. 183. Copyright (2018) American Chemical Society, ref. 185. Copyright (2017) Elsevier, and ref. 188. Copyright (2018) Elsevier, respectively.

The time for hydrogel formation based on the traditional crosslinking method is still relatively long since a long reaction time is required to complete the reactions sufficiently, especially for free radical polymerization. In recent years, the preparation of heparin-inspired injectable hydrogel by dynamic covalent bonding has become a new way, and the time for hydrogel formation by this method is shorter. Kellomaki's team prepared a water-soluble derivative of chitosan biopolymer by grafting it with a small anionic amino acid, L-glutamic acid.183 The grafted amino acid greatly suppressed the inter- and intra-molecular interactions between the chitosan chains, thus enhancing the crystallinity and facilitating its water solubility, as shown in Fig. 18(B). The Schiff-base linkages in this hydrogel originating from the amino group of chitosan and aldehyde groups of 4-arm PEG are reversible, making the hydrogel self-healable. Also, the time for hydrogel formation was less than 60 s, and the mechanical properties of these hydrogels could be easily tuned by varying either the crosslinker concentration or the total solid content in the hydrogels. Owing to their injectable, self-healing, and biocompatible nature, these hydrogels are promising candidates for delivery applications in the future.

The preparation of heparin-inspired injectable hydrogels by chemical crosslinking often requires further modification processes, which may limit their applications. In contrast, heparin-inspired injectable hydrogels prepared by physical crosslinking are more flexible, and the different physical crosslinking methods make the prepared heparin-inspired injectable hydrogels more environmentally sensitive. For example, heparin-inspired injectable gels prepared by hydrogen bonding have good pH sensitivity. Self-healing pH-sensitive biodegradable cytosine- and guanosine-modified hyaluronic acid hydrogels (HA-HMDA-C, HA-HMDA-G, and HA-HMDA-C/G) were prepared via hydrogen-bond crosslinking under physiological conditions, as shown in Fig. 18(C).185 The good integrated performances, including pH-sensitivity, self-healing ability, high swelling ratio, biodegradability, effective ability to entrap drug, and ability to sustain release under physiological conditions signified that the modified hyaluronic acid hydrogel can be an attractive candidate as a short- and medium-term polypeptide and protein drug delivery system. Hydrogen bonding can also be used to prepare temperature-sensitive heparin-inspired injectable hydrogels. Wu's group fabricated drug (doxorubicin)-loaded hydrogel precursor solutions, which were injectable and could turn to hydrogels when the temperature increased to body temperature.179 Acid acidic condition (pH 4.0) could trigger the release of the drug, and the cancer cells (HeLa) could adhere to the surface of the hydrogels, thus they are beneficial for the tumor site-specific administration of drugs.

Compared with the preparation of heparin-inspired injectable hydrogels by hydrogen bonding, the preparation of heparin-inspired injectable hydrogels via ion crosslinking is faster and simpler. Heparin-inspired injectable hydrogels can be prepared by mixing polymer substrates such as sodium alginate, chitosan and carrageenan with metal ion solution, and the mechanical properties of the hydrogels can be controlled by changing the concentration of the metal ion solution. For example, an injectable self-healing carboxymethyl chitosan (CMCh) supramolecular hydrogel crosslinked by zinc ions (Zn2+) was reported by Chu et al., as shown in Fig. 18(D).188 The supramolecular hydrogels were obtained by the simple addition of metal ion solution to CMCh solution at an appropriate pH value. The mechanical properties of the hydrogel could be adjusted by varying the concentration of Zn2+. As observed visually and confirmed by rheology, the CMCh–Zn hydrogel with the lowest Zn2+ concentration showed thixotropic property. The CMCh–ZnCMCh–Zn hydrogel also presented injectable properties and Zn2+-dependent antibacterial properties against S. aureus and E. coli. We believe that the prepared supramolecular hydrogels are potential candidates for use in the biomedical field. However, the biggest drawback of using metal ionic solutions to prepare hydrogels is that the introduction of heavy metal ions may have an effect on biocompatibility. For example, the introduction of calcium ions promotes the clotting reaction, and the introduction of zinc ions causes cytotoxicity. Consequently, to date, gentle and simple methods to prepare heparin-inspired injectable hydrogels with low toxicity and rapid gelation process are still a challenge.

3.4. Heparin-inspired nano-hydrogels

Heparin-inspired nano-hydrogels refer to crosslinked heparin-inspired polymer nanoparticles with a three-dimensional network structure, which have become versatile tools in enhancing the biocompatibility of materials, with great miscibility to the material matrix. In our previous study, the antifouling and anticoagulation properties of PES membranes were enhanced by blending them with heparin-inspired nano-hydrogels.141 The heparin-inspired nano hydrogels were synthesized via the typical free radical crosslinked copolymerization of AA, N-vinylpyrrolidone, 2-acrylamido-2-methylpropane sulfonic acid and acrylamide. After incorporation of the heparin-inspired nano-hydrogels, the amount of adsorbed protein decreased by more than 50% and the activated partial thromboplastin time and thrombin time values were prolonged compared with that of the pristine PES membrane. In addition, the platelet adhesion and blood-related complement activation were highly suppressed. Thus, the simple and effective approach to modify polymeric membranes by blending them with heparin-inspired nano-hydrogels may have great potential to be used in blood purification applications.

4. Conclusions and perspectives

In this review, we provided an overview of the heparin and heparin-inspired biomedical hydrogels, which are fabricated by a plethora of fabrication techniques. Firstly, the size effect, fabrication methods and biomedical applications of heparin-based hydrogels were reviewed. Heparin-based macro-hydrogels can be designed via diverse methods, and the crosslinking networks of hydrogels can be built via chemical covalent bonding, physical conjugation, and the combination of chemical and physical interactions. The covalent bonding approaches such as click-chemistry, divinyl sulfone crosslinking and mussel-inspired coating were emphasized. The fabrication of chemical bond crosslinked hydrogels always needs rigorous reaction conditions; whereas, it is relatively easier to form physical conjugated hydrogels via host–guest interaction, electrostatic interaction, hydrogen bonding, and hydrophobic interaction. The applications of heparin-based hydrogels in wound healing, as growth factor carriers, blood vessels, and soft tissue materials for cell culturing were also discussed. Heparin-based injectable hydrogels can be used to mimic the extracellular matrix as promising drug delivery systems for postoperative chemotherapy, cell delivery carrier and the regeneration of damaged liver or other tissues. Heparin-based nano-hydrogels are commonly applied for cancer cell-targeted delivery, as carriers for anti-fibrotic and anti-cancer agents and gene delivery. As an important family of biomedical hydrogels, heparin-based hydrogels may be used as bioactive hydrogels. Since most of the reported heparin-based hydrogels are traditional hydrogels, the fabrication of stimuli-responsive heparin-based hydrogels with smart performances (in situ gelling during hydrogel preparation, swelling or degradation under stimulation for controlled drug delivery) can also be one of the future research interests.

Since heparin has significant safety and supply problems, heparin-inspired hydrogels are one of the current research hotspots to substitute the usage of heparin in the fabrication of hydrogels. Thus, to better understand the role played by heparin-inspired hydrogels and their derivatives in functionality, we reviewed various types of heparin-inspired hydrogels (e.g., heparin-inspired macro-hydrogel, surface thin film hydrogel, injectable hydrogel, and nano-hydrogel). Additionally, the latest applications of these heparin-inspired hydrogels (e.g., cell culture, wound healing, loading of drugs, blood contacting applications, and other applications) were also highlighted. The methods to prepare heparin-inspired macro-hydrogels include in situ polymerization and crosslinking, electrostatic interaction, and hydrophobic interactions. As a new hydrogel form, surface-attached hydrogel thin films can be employed to effectively enhance the membrane biocompatibility. Despite the extensive research in injectable heparin-inspired hydrogels, their applications as a real scaffold can be one of the future directions. Compared with heparin-inspired macro-hydrogels, nano-hydrogels show great miscibility with the membrane matrix.

Although there have been extensive investigations on both heparin-based and heparin-inspired hydrogels, little is known about how the synthetic units of functional polymers work in hydrogels. Thus, more studies on the presentation of functional groups and interactions with proteins and cells are desirable in this regard. Among the fabrication methods, free radical crosslinking polymerization is one of methods suitable for the large-scale fabrication of heparin-based and heparin-inspired hydrogels. However, the mussel-inspired DA strategies endow great benefits due to their facile and green synthesis process. Along with the fast developments in nanomaterials, many novel heparin-based and heparin-inspired nano hydrogels with specific functionalities will be developed to fabricate bioactive hydrogels.

To date, the studies on the industrial and clinical applications of both heparin-based hydrogels and heparin-inspired hydrogels are insufficient. Therefore, increasing attention should be paid to the industrial and clinical applications of heparin-based hydrogels and heparin-inspired hydrogels in future studies. Since both heparin-based hydrogels and heparin-inspired hydrogels have already been intensively prepared with a macro-gel size, filling hydrogels in a column to fabricate anticoagulant columns may be a strategy for non-heparin blood purification. Also, more attention should be paid to the biological response and growth factor binding of hydrogels, which can benefit the functionalization of implants for certain applications in tissue engineering.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially sponsored by the State Key Research Development Programme of China (2016YFC1103000 and 2016YFC1103001), the National Natural Science Foundation of China (No. 51503125, 51673125, 51773127, 51803131, 51803134 and 51873115), Consulting Project of Chinese Academy of Engineering (2017-XZ-08), and the State Key Laboratory of Polymer Materials Engineering (No. sklpme2017-3-07 and sklpme2015-1-03).

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Footnote

These two authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2019