Polymer Bulletin (2022) 79:9199–9219

https://doi.org/10.1007/s00289-021-03956-8

REVIEW PAPER

Role of crosslinkers for synthesizing biocompatible,

biodegradable and mechanically strong hydrogels
with desired release profile

Saman Zafar1 · Muhammad Hanif1 · Muhammad Azeem1,2 ·

Khalid Mahmood3 · Sonia Ashfaq Gondal4

Received: 14 June 2021 / Revised: 25 September 2021 / Accepted: 24 October 2021 /

Published online: 30 October 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Over the time, hydrogels have emerged as one of the most potential candidates for
drug delivery and tissue engineering systems due to their swellable and porous
nature. Fabrication process of hydrogel requires addition of crosslinkers. Vari-
ous chemical (e.g., crosslinking by chemical reaction of complementary groups,
polymer–polymer crosslinking, high energy irradiation and enzyme incorporation)
and physical (e.g., charge interactions, crystallization and stereocomplex forma-
tion) approaches have been employed for crosslinking hydrogels. Majority of the
conventionally employed crosslinkers are toxic in nature and unfavorable for use.
Moreover, they have poor water solubility and low biodegradation rate. Various nat-
ural (e.g., vanillin, citric acid, gallic acid, ferulic acid and genipin) and synthetic
(e.g., polymerizable polyphosphate, 1,2,3,4-butanetetracarboxylic dianhydride and
2-chloro-1-methylpyrinium iodide) novel crosslinking agents have been developed
to overcome these limitations and to produce hydrogels with good mechanical prop-
erties. Furthermore, novel non-toxic crosslinkers are being introduced for modulat-
ing release characteristics and attaining controlled drug release profile of hydrogels
made up of highly soluble and erodible polymers. Considering the drawbacks of
conventional crosslinkers, there is a need to search for more biocompatible and bio-
degradable novel polymers to attain safe and efficient hydrogel formulations.

Keywords Hydrogels · Crosslinkers · Crosslinking mechanisms · Physically

crosslinked · Chemically crosslinked · Biodegradable · Biocompatible ·
Mechanically strong · Non-toxic · Controlled drug release

* Muhammad Hanif
muhammad.hanif@bzu.edu.pk
Extended author information available on the last page of the article

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9200 Polymer Bulletin (2022) 79:9199–9219

Introduction

Hydrogels are there-dimensional water-absorbable polymeric networks, com-

monly employed in the medical and pharmaceutical field due to their resem-
blance with the extracellular matrix constituents [1, 2]. Moreover, its non-toxic,
inert and biocompatible nature ensures that it does not influence the metabolic
processes of living organisms; hence, can serve as a potential candidate for drug
delivery systems [3, 4]. Formulation of hydrogels mostly requires the addition of
crosslinkers to prevent the solubilization of polymeric chains before use. Over the
time, more defined roles of crosslinkers have been investigated. More recently,
several promising crosslinkers with improved biocompatibility, biodegradabil-
ity, toxicity and aqueous solubility have been investigated. Moreover, crosslink-
ing agents have been incorporated in the hydrogel formulations for enhancing
the tensile strength and attaining desired drug release characteristics. Hydrogels
exhibit viscoelastic to elastic behavior due to the incorporation of crosslinker
between the polymeric chains.
On the basis of crosslinking, hydrogels are broadly classified into two cat-
egories including physically and chemically crosslinked hydrogels. Exam-
ples of physically crosslinked polymers include polymethacrylic acid-poly-
ethylene glycol, poly(2-ethyl-2-oxazoline)-polycaprolactone, polyethylene
glycol-polyisobutylene and polyethylene glycol-poly (c-benzyl L-glutamate).
Several polymeric hydrogels such as chitosan-polyvinyl alcohol, tetrahy-
droxy polyethylene glycol-lysine, polyvinyl alcohol-glutaraldehyde and gela-
tin-N, N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDAC) have been
crosslinked chemically. The characteristics of both types of gels are described in
Table 1 [5–8]. In some cases, hydrogels may also comprise of a double network
i.e., both physical and chemical interactions may exist (Fig. 1). Cellulose-cin-
namoyl-modified gelatin, acrylamide and gelatin hydrogels are examples of dual
physical and chemical crosslinked hydrogels [9–12].

Table 1  Characteristics of physically and chemically crosslinked hydrogels [5–8]

Physically cross-linked hydrogels Chemically cross-linked hydrogels

Physical/temporary/reversible hydrogels are Chemical/permanent/irreversible hydrogels are

formed by molecular entanglements, hydropho-
bic forces, ionic or hydrogen bonding
They are homogeneous and formulated without
employing crosslinking entities or chemical
modification
They are less stable against degradation
They exhibit poor mechanical properties owing to
physical interactions
They possess both hydrophilic and hydrophobic
regions in their network
formed by covalent bonding
They are non-homogenous and formulated by
incorporating crosslinking entities or chemical
modification
They are relatively more stable against degradation
They exhibit relatively better mechanical properties
They exhibit domains of high crosslink density than
the physical counterparts

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Polymer Bulletin (2022) 79:9199–9219 9201

Fig. 1  Schematic illustration of physical, chemical and double network crosslinking strategies in hydro-
gels [5–8]

Hydrogel crosslinking methods

Chemical and physical crosslinking of hydrogels can be governed by various

approaches (Fig. 2) [4, 7, 8, 13–16].

Fig. 2  Commonly used physical and chemical crosslinking methods of hydrogels [4, 7]

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9202 Polymer Bulletin (2022) 79:9199–9219

Chemically crosslinked hydrogels

Covalent bonding is the main mechanism involved in forming permanent inter-

actions in chemically crosslinked hydrogels. Chemically crosslinked hydrogels
can be produced by various approaches such as chemical reaction of complemen-
tary groups, polymer–polymer crosslinking, high energy irradiation and enzyme
incorporation.

Crosslinking by chemical reaction of complementary groups

In case of aqueous-soluble polymers, several functional groups such as -OH,

-COOH and -NH2 are responsible for forming hydrogels. Covalent linkages between
polymeric chains can be developed by Schiff base formation or reaction of com-
plementary groups, for example, amine-carboxylic acid and an isocyanate-OH/NH2
reaction [14].

Crosslinking by aldehydes Aqueous polymers comprising hydroxyl groups, for

example, polyvinyl alcohol, have been crosslinked using glutaraldehyde under dras-
tic conditions such as additional of methanol as quencher, low pH and high temper-
ature. In case of amine-based polymers, crosslinking (using same crosslinker) can
be carried out under mild conditions resulting in formation of Schiff bases (Fig. 3)
[14, 17, 18]. Several proteins including albumin and gelatin have been success-
fully crosslinked using this approach [14, 19]. In a study, hyaluronic acid hydrogel
films were crosslinked using a macromolecular crosslinker (poly(ethylene glycol)-
propiondialdehyde). Prepared films were degraded by hyaluronidase and regarded
as potential candidates for delivering antibacterial and antiinflammatory drugs in
a sustained release manner at the wound site [20]. Gelatin was crosslinked using
polyaldehydes obtained by partial oxidation of dextran. Formulated gels were
loaded with epidermal growth factor to promote wound healing. The release of
growth factor decreased with an increase in storage time possibly due to ongoing

OH OH

H
2 ROH OR X OR
O O H H

NR NR
2 RNH2
H X H

O H X H
H H
R N N R
NH2
N N
2R N
H
O O
H X H

Fig. 3  Aldehyde-mediated crosslinking of polymers comprising alcohol, amine, hydrazide groups (R

means polymeric chains, X is any spacer, for example, ­(CH2)3 in case of glutaraldehyde) [14]

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Polymer Bulletin (2022) 79:9199–9219 9203

chemical crosslinking and physical structuring of prepared hydrogel formulation.

Moreover, after prolonged storage, very less quantity of protein was released due
to Schiff base formation between ε-lysine and aldehyde groups of protein and oxi-
dized dextran, respectively [21].

Crosslinking by addition reaction Hydrogels comprising water soluble polymers

have been crosslinked by bis or higher functional crosslinking agents. An addition
reaction between these agents and functional groups of aqueous-soluble polymers
leads to crosslinking. Example of reagents used for this purpose include 1,6-hexa-
methylenediisocyanate, divinylsulfone and 1,6-hexanedibromide. The reaction is
mostly carried out in organic solvents to avoid the risk of reaction of water with
the crosslinking reagent. The gels are required to be extracted to remove the traces
of unreacted reagents because majority crosslinking reagents are toxic [14]. In
a study, a reaction between polyethylene glycol-dithiol and polyethylene glycol-
acrylates resulted in the formation of crosslinks in a degradable hydrogel. Solid
particles of albumin were loaded in the prepared formulation by blending tech-
nique. Under physiological conditions, hydrolysis of ester bonds resulted in the
degradation of hydrogel. Release of loaded protein was observed for 5–12 days,
and the functionality of the polyethylene glycol-acrylate regulated the degradation
time (from 5 to 25 days) of formulation [22].

Crosslinking by condensation reaction Condensation reactions have been widely

employed for crosslinking polyester/polyamide-based polymers. Condensation
reaction of several groups, for example, hydroxyl (–OH) or amino (–NH2) with
carboxylic acids and derivatives resulted in the formation of polyesters and poly-
amides chains containing polymers. N, N-(3 dimethylaminopropyl)-N-ethyl car-
bodiimide (EDAC) is an efficient crosslinking reagent which has been employed
for crosslinking aqueous-soluble polymers via forming amide bonds. De Nooy
et al. and Ichi et al. [23, 24] used EDAC as a crosslinker for synthesizing gelatin
hydrogels. N-hydroxy-succinimide was incorporated during this reaction in order
to prevent side reactions and attain relatively higher control over the crosslinking
density of hydrogels [14]. This reagent was utilized by Tan et al. to formulate algi-
nate and polyethylene glycol (PEG)-diamines-based hydrogels exhibiting supe-
rior mechanical properties than ionically crosslinked counterparts. PEG molecu-
lar weight and PEG-diamine concentration in hydrogel significantly influenced
the mechanical properties of hydrogels [25]. Research work conducted by Lee
et al. and Elbert et al. involved preparation of polysaccharide-based hydrogels by
employing Passerini and Ugi condensation reactions (Fig. 4) [26, 27].

Polymer–polymer crosslinking

Activated reactive groups of pre-functionalized polymeric chains have been uti-

lized for preparing hydrogels. This crosslinking approach can be employed for
in situ formulation of chemically crosslinked hydrogels. Polymer–polymer

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9204 Polymer Bulletin (2022) 79:9199–9219

Fig. 4  (Top) Passerini and (bot- O R2

R1-COOH
tom) Ugi condensation reactions
H
[14] R2-CHO N
R1 O R3
R3-NC

R1-COOH O R2

R2-CHO H
N
R3-NC R1 N R3

R4-NH2
R4 O

crosslinking approach has been used for preparing chitosan and hyaluronic acid
polymers-based in situ hydrogels by Schiff bases formed at physiological pH.
Prepared hydrogel remained stable for up to ~ 4 weeks [28]. Michael addition
reactions have been used to produced polymer–polymer crosslinked chitosan
hydrogels [29]. Michael addition between a nucleophile, for example, an amine
or a thiol and a vinyl group has been used for in situ crosslinking [8]. An exam-
ple includes crosslinking of vinyl sulfone-functionalized dextrans with thiolated
polyethylene glycol. This approach offers relative biological inertness and rapid
formation of gel and flexibility in forming multiple types of bonds [29]. Another
example includes formation of a hydrazone bond between an aldehyde and a
hydrazide allowing efficient crosslinking. Hydrazone bond crosslinked hyaluronic
acid delivered tissue plasminogen activator and budesonide to the peritoneum in
a controlled release manner [30, 31]. Though, this crosslinking method eliminates
the requirement of using small molecular weight crosslinkers; multi-step synthe-
sis and purification processes are its limitations [4].

Chemical crosslinking via high energy irradiation

High intensity and energy radiations such as gamma, electron beams, ultraviolet and
microwave have been used to produce permanent gels. Irradiation-mediated chemi-
cal crosslinking process permits formation of gels under mild pH and temperature
without using toxic crosslinking agents and eliminates the need for removing unre-
acted species from final product [4]. In irradiation-induced crosslinking, free rad-
icals are produced which attack the hydrophilic polymeric chains involved in the
polymerization process resulting in production of macroradicals. Covalent bonds
between polymeric chains are synthesized by produced macroradicals with subse-
quent generation of crosslinked networks. Examples of polymers crosslinked by this
crosslinking approach include polyacrylic acid (PAA), polyethylene glycol (PEG)
and polyvinyl alcohol (PVA) [14]. Polymethyl vinyl ether aqueous solutions were
irradiated above phase transition temperature to obtain a thermosensitive hydrogel.
Irradiation dose and polymer concentration influenced swelling ratio and permeabil-
ity of resultant hydrogel [32, 33].

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Polymer Bulletin (2022) 79:9199–9219 9205

Single step gamma-ray irradiation with dose 25–75 kGy has been used for man-
ufacturing metronidazole loaded polyacrylic acid hydrogels. Prepared hydrogels
showed antibacterial activity against several pathogens including Escherichia coli,
Staphylococcus aureus, Streptococcus mutans [34]. Jeong et al. crosslinked poly-
vinyl pyrollidone, para-toluene sulfonate and pyrrole-based conducting hydrogels
using gamma-rays irradiation (25 kGy). The conductivity of prepared hydrogel
was around 13 mS/cm. A good cell viability was observed during CCK-8 assay,
regardless of para-toluene sulfonate and pyrrole concentrations, and it was con-
cluded that prepared hydrogels can be used for biomedical applications [35]. Mitsui
and Hosoi successfully crosslinked polyethylene by gamma radiations at a dose of
1.1 × ­105 rad/h [36]. In a study, glutaraldehyde and gamma irradiation crosslinked
methylcellulose hydrogels were prepared. Glutaraldehyde crosslinked formulations
were less polar and displayed more uniform and homogenous gel structure as com-
pared to the gamma irradiation crosslinked counterparts [37].
Tyramine conjugated gum tragacanth-based chemically crosslinked hydrogels
have been successfully prepared by using electron beam irradiation (dose 5–90 kGy)
[38]. In another study, tetracycline loaded 5-hydroxytryptophan conjugated pectin-
based antibacterial hydrogels were produced by using 10–50 kGy electron beam
radiations [39]. A hydrogel formulation comprising chitosan, polyvinyl pyrrolidone,
polyethylene glycol, polyacrylic acid was loaded with ibuprofen and crosslinked by
electron beam irradiation, at varying doses (ranging from 15 to 25 kGy). During
release study, almost 25% drug was released within 120 min, and maximum release
was observed after 480 min. It was concluded that prepared wet dressing hydrogels
can serve as promising candidates for relieving pain and rapid healing of infected
cutaneous wounds [40]. In a study, methylcellulose wound dressing hydrogel was
prepared by using electron beams (dose 10–40 kGy). The gel fraction, tensile
strength increased and swelling ratio decreased with an increasing dose of irradia-
tion [41].
Azide group (-N3) incorporated chitosan was exposed to ultraviolet light result-
ing in conversion of azide group to nitrene group (R-N:) which bound free amino
groups of chitosan. This led to the in situ production of hydrogel [42]. In a study,
hyperbranched glycidyl methacrylate-co-poly(ethylene glycol) diacrylate polymers-
based formulation was exposed to ultraviolet radiations. Resultant hydrogel showed
a significantly high modulus (over 65 kPa) as compared to unexposed counterpart
(25 kPa) [43]. A formulation comprising polyethylene/ethylene vinyl acetate melt
mixture, trimethylolpropane trimethacrylate (TMPTMA, as crosslinking agent)
and benzophenone (as photoinitiator) was exposed to full ultraviolet radiations of
a 2-kW ultraviolet lamp for 100 s. The elongation at break of prepared gel formula-
tion significantly dropped with an increasing degree of crosslinking [44]. In another
study, aqueous polyethylene oxide solutions were crosslinked by ultraviolet radia-
tions in the presence or absence of oxygen. The ultraviolet irradiation time was var-
ied between 30 and 960 min. The properties of prepared hydrogel were impacted by
presence of oxygen and time of irradiation [45].
Microwave irradiation using temperatures of 100–150 °C produced polyvinyl
alcohol-polyacrylic acid and polyvinyl alcohol- poly(methylvinylether-alt-maleic
anhydride) hydrogels [46]. Larraneta et al. prepared hydrogel forming microneedles

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9206 Polymer Bulletin (2022) 79:9199–9219

comprising poly(methyl vinyl ether-alt-maleic acid) and polyethlene glycol using

microwave assisted crosslinking process at 80 °C. For comparison, hydrogel form-
ing microneedles were also produced using convection oven. The hydrogel-based
formulations produced using microwave radiations displayed equivalent properties
to those prepared by convection oven, however, the production rate of microwave
method was 30 times faster [47]. In a study, chitosan hydrogels doped with tita-
nium dioxide nanoparticles were produced, by microwave irradiation at 180 °C for
30 min, for tissue engineering/skin regeneration applications [48].

Crosslinking using enzymes

During recent years, enzymes have been employed for preparing permanent hydro-
gels, PEG-based hydrogels, being a common example. Several enzymes (for exam-
ple, transglutaminases, peroxidases, tyrosinase, phosphopantetheinyl transferase,
plasma amine oxidase and phosphatases) have been employed for preparing perma-
nent hydrogels [8]. In a study, tetrahydroxy PEG was altered by using glutaminyl
groups (PEG-Q). PEG networks were produced by adding transglutaminase in the
aqueous solutions of PEG-Q and polylysine-co-phenylalanine. A catalytic reaction
was induced between carboxamide and amine group of lysine by transglutaminase
leading to the formation of amide linkage between the polymers [4, 49]. Microbial
transglutaminase enzyme was used to prepare HEK293 cell incorporated thermally
stable and biocompatible gelatin hydrogels. Loaded cells were released in a con-
trolled manner [50]. Horseradish peroxidase catalyzed tyramine modified hyaluronic
acid hydrogel was prepared in two phases. Initially, amide bond was formed between
carboxyl and amine group of hyaluronic acid and tyramine, respectively, resulting
in formation of hyaluronic acid-tyramine conjugate. In the second phase, hyaluronic
acid-tyramine hydrogels were formulated by radical crosslinking reaction involving
Horseradish peroxidase and hydrogen peroxide. The formulated hydrogel success-
fully delivered dexamethasone intra-articularly for managing rheumatoid arthritis
[51].

Physically crosslinked hydrogels

During recent years, physically crosslinked gels have attracted a lot of interest due to
the avoidance of toxic crosslinking agents. Several methods for synthesizing physi-
cally crosslinked hydrogels comprise charge interactions, crystallization and stereo-
complex formation.

Crosslinking by charge interactions

The phenomena of charge interactions involve an interaction between two or mul-

tiple oppositely charged polymers or a polymer and a small molecule resulting in
formation of temporary hydrogels. This approach has been extensively explored for
formulating in situ hydrogel systems. An example of gels processed by this approach
include elastin like polypeptides which were crosslinked by electrostatic interactions

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Polymer Bulletin (2022) 79:9199–9219 9207

between cationic lysine residues and anionic organophosphorus crosslinker [4]. In

a study, dextran microspheres were coated with oppositely charged polymers. The
resulted microsphere showed spontaneous gelation due to formation of ionic com-
plex [52]. Alginate has been crosslinked using calcium ions and used as a matrix
for encapsulating protein and living cells [53–55]. A chelating agent can be used
to extract calcium ions leading to destabilization of gel. The release of protein
from crosslinked alginate microparticles can be regulated by coating the micro-
particles with cationic polymers, for example, chitosan and polylysine [56, 57].
Poly-di(carboxylatophenoxy)phosphazene-based hydrogel formulation has been
crosslinked using calcium ions. For this purpose, an aqueous solution of poly-
di(carboxylatophenoxy)phosphazene was sprayed into an aqueous solution of cal-
cium chloride [58]. Polycations have been crosslinked with anions. Chitosan-based,
obtained by deacetylation of chitin, hydrogels were crosslinked with glycerol-phos-
phate disodium salt. Below room temperature, chitosan solutions remained in the
liquid state in the presence of this salt but rapidly gelated upon heated. The tempera-
ture at which a sol–gel transition took place was found to decrease with an increas-
ing degree of deacetylation [59]. Carrageenan formed a hydrogel with potassium
ions. Carrageenan is capable of forming gel under salt-free conditions as well. How-
ever, hydrogels produced under salt-free conditions are weaker than those prepared
in the presence of metallic ions [60]. Hydrogels have also been prepared by com-
plexation of polyanions and polylysine. Doxorubicin encapsulated nanoparticles
of chitosan hydrogels were prepared by complexation between chitosan and poly-
anions, for example, dextran sulfate or polyphosphoric acid [61].

Crosslinking by crystallization

An example of physically crosslinked hydrogels prepared using crystallization

approach include PVA hydrogels. Formation of PVA hydrogel via crystalliza-
tion method is termed as hydrogen bonding interactions. Aqueous solution of PVA
formed gel with poor mechanical strength at room temperature; however, after
freeze–thaw treatment, a strong and highly elastic hydrogel was obtained. PVA
hydrogel was produced due to formation of crystallites which served as physical
crosslinking sites in the polymeric hydrogel network. PVA molecular weight, con-
centration, temperature and time duration as well as number of freeze–thaw cycles
influenced gel properties. Prepared gel remained stable at 37 °C for ~ 6 months [62].
Aqueous solution of dextran 6000, upon incubation at room temperature, immedi-
ately formed a hydrogel. Microparticles were obtained by stirring the aqueous solu-
tion. The crystallization took place in dextran 6000 solution due to an association of
chains through hydrogen bonding resulting in gel formation [63]. In a study, a physi-
cal double network hydrogel comprising a physically crosslinked polyvinyl alcohol
and a hydrophobically associated polyacrylamide was prepared by in situ polymeri-
zation followed by freeze thaw cycling. The strong crystallization of polyvinyl alco-
hol and the presence of hydrogen bonds among polyvinyl alcohol and polyacryla-
mide chains resulted in the formation of a hydrogel with an improved mechanical
strength [64].

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Crosslinking by stereocomplex formation

Stereocomplex formation approach involves synergistic interactions between poly-

meric chains or low molecular weight compounds with different stereochemistry but
same chemical composition. Homopolymers of poly-L-lactic acid and poly-d-lactic
acid) are an example of polymers crosslinked by stereocomplex formation approach.
Natural polymers can be crosslinked by stereocomplex grafting approach. Grafting
of dextran precursors into the l, d-lactide oligomers leads to spontaneous gelation
in water. In the blends of triblock copolymers of poly-l-lactic acid-polyethylene
glycol- poly-l-lactic acid and poly-d-lactic acid-polyethylene glycol-poly-d-lactic
acid, stereocomplex formation was observed. Bovine serum albumin release from
microspheres prepared using these triblock copolymers was studied, and results
were compared with microspheres based on one enantiomeric form of the triblock
copolymer and poly-l-lactic acid microspheres. A slightly greater burst release was
observed in case of stereocomplex triblock copolymeric microspheres as compared
to poly-l-lactic acid microspheres, most probably, due to higher water-uptake ability
of sterocomplex microspheres owing to the presence of polyethylene glycol. How-
ever, drug release from optically pure homopolymer microspheres was comparable
with that of stereocomplex microspheres [65].
Highly biodegradable and biocompatible hydrogels can be obtained by this
approach without using denaturing agents such as organic solvents and crosslinking
agents. However, this process cannot be used for all types of of polymers [4, 66].
A hydrogel system based on stereocomplex formation was developed by grafting
enantiomeric oligo(lactic acid) side chains onto poly-2-hydroxyethyl methacrylate
(poly-2-hydroxyethyl methacrylate-g-oligolactate). The formulation was obtained
by casting a film from poly-2-hydroxyethyl methacrylate-g-oligo(l)lactate and poly-
2-hydroxyethyl methacrylate-g-oligo(d)lactate, both solubilized in chloroform. The
degradation profile of prepared film formulation was compared with that casted
from the solution of single enantiomer of the graft copolymer. Formulation pre-
pared using blend of l- and d-forms displayed relatively slower degradation than
that obtained from single enantiomer [67]. In a study, crosslinking was established
among lactic acid oligomers of opposite chirality. The l- and d-lactic acid oligom-
ers were coupled with dextran through their terminal hydroxyl group to resulting
in the formation of dex-(l)lactate and dex-(d)lactate, respectively. A hydrogel was
obtained at room temperature by forming aqueous solution of each product sepa-
rately. The hydrogel formation was favored when oligomers were coupled via dif-
ferent functional group, for example, hydroxyl group of one oligomer coupled with
the carboxyl group of other oligomer (i.e., of opposite chirality). This phenomena is
attributed to the parallel packing of oligomers in stereocomplexes [68–70].

Complications associated with crosslinking agents

Several crosslinkers used in preparation of hydrogels are represented in Table 2.

Glutaraldehyde, epichlorhydrin, glyoxal and formaldehyde are the most commonly
employed crosslinkers for preparing hydrogels. Common problems associated with

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 Table 2  Example of crosslinkers commonly used in the preparation of hydrogel formulations
Crosslinkers (nature) Advantages of crosslink- Disadvantages of Crosslinker amounts Hydrogel base materials Applications References
ers crosslinkers

Glutaraldehyde (syn- Inexpensive, water Toxic, difficulty in 0.1% Chitosan Potential for controlled [72]
thetic) miscible reproducing the drug release and tissue
results and scaling up engineering
In terms of molar ratios Collagen Mechanically strong [73]
of CHO/NH2; in ratio hydrogels
0/9
1% Carboxymethyl cellu- pH and thermosensi- [74]
Polymer Bulletin (2022) 79:9199–9219

lose, poloxamer com- tive controlled drug

prising polyethylene release
oxide-polypropylene
oxide-polyethylene
oxide
1.25% Chitosan Potential for controlled [75]
drug release, tissue
engineering
Epichlorhydrin (syn- Inexpensive Toxic 20% w.r.t. dry weight Hydroxyethyl cellulose, Potential for controlled [76]
thetic) of hydrogel base soy protein drug release, tissue
materials engineering
5–15% w.r.t total solu- Cellulose Improved mechanical [77]
tion mass properties
40–80 wt% Dextran Improved water absor- [78]
bency/swelling ability
Polymer/crosslinker Carboxymethyl cel- Controlled drug release [79]
ratio, wt/wt, 1.7–3.1 lulose, gelatin
Formaldehyde (syn- Inexpensive, water Toxic 5 mol/L Chitosan Improved water absor- [80]
thetic) soluble bency
9209

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 Table 2  (continued)
9210

Crosslinkers (nature) Advantages of crosslink- Disadvantages of Crosslinker amounts Hydrogel base materials Applications References
ers crosslinkers

13
Genipin (natural) Non-toxic, water soluble Expensive 3.75 and 7.5% w.r.t. Chitosan Biocompatible, bone [81]
chitosan dry weight tissue engineering

1–3% w.r.t. chitoan dry Chitosan Biocompatible, wound [82]

weight dressing
Glyoxal (synthetic) Relatively less toxic Irritant 0.0025–0.02% Chitosan Potential for controlled [83]
drug release, tissue
engineering

Tripolyphosphate (syn- Non-toxic, water solu- Relatively less strong 0.1% Chitosan Biocompatible, [84]
thetic) ble, inexpensive crosslinker improved swelling
ability

Tripolyphosphate plus Mentioned earlier Mentioned earlier 0.1 M genipin, 0.1 M Chitosan Biocompatible, potential [85]
genipin tripolyphosphate for biomedical appli-
cations
Ferulic acid (natural) Non-toxic, inexpensive Relatively less strong 0.5% w/v Methylcellulose, Biocompatible [86]
crosslinker chitosan
Polymer Bulletin (2022) 79:9199–9219
Polymer Bulletin (2022) 79:9199–9219 9211

these conventional crosslinkers include high toxicity, low biodegradability, biocom-

patibility and aqueous solubility. Hence, several novel crosslinking agents have been
introduced with improved biodegradability, biocompatibility and water solubility.
Various natural compounds such as citric acid, gallic acid, ferulic acid, vanillin, gen-
ipin, flavonoids in foods (catechins, proanthocyanidins) and desmosine analogs have
also gained attention, during recent years, to form biocompatible biomaterials [71].
Moreover, crosslinkers have been incorporated in the formulations to achieve desir-
able release profile and mechanical strength. Various studies on novel crosslinkers
have been conducted for achieving desired characteristics which are summarized
below.

Crosslinkers for improving biodegradability, biocompatibility and water

solubility

Ring opening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with

2-(2-oxo-1,3,2-dioxaphosphoroyl-oxyethylmethacrylate) was performed in order to
produce a novel biodegradable crosslinker polymerizable polyphosphate (PIOP).
PIOP degradation was observed in aqueous medium. The degradation of PIOP was
directly correlated with pH of medium. Complete degradation of PIOP was observed
within 6 days at pH 11. PIOP was employed as a crosslinking agent for preparing
poly [2 methacryloyloxyethyl phosphorylcholine] hydrogel via radical polymeriza-
tion. Prepared hydrogel and crosslinker did not show any cytotoxicity when evalu-
ated using v79 cells [87].
In one study, two polyphenols including gallic acid and ferulic acid were
employed as natural crosslinking agents for synthesizing methylcellulose-chitosan
biocomposites in concentration 1% w/v and 0.5% w/v, respectively. FT-IR (Fourier
transform infrared spectroscopy), DSC (differential scanning calorimetry) and SEM
(scanning electron microscopy) techniques were used to investigate the formation
of the biocomposites. Among the two polyphenols, only ferulic acid was found to
interact with methylcellulose and chitosan. Addition of ferulic acid to methylcellu-
lose and chitosan clear solutions resulted in turbidity. Methylcellulose—ferulic acid
composites exhibited relatively smaller particle size (500–600 nm) as compared to
the chitosan—ferulic acid biocomposites (6–8 μm) [86].
Four crosslinking agents including hexamethylene diisocyanate, PEG diglycidyl
ether (epoxy), glutaraldehyde and genipin were used in concentration 0.5 mol/v to
prepare porous gelatin scaffolds by in situ gas foaming. All crosslinkers were capa-
ble of stabilizing the scaffolds; however, crosslinker type had a profound effect on
physical and mechanical properties of prepared gelatin scaffolds. Epoxy-based for-
mulations showed a relatively higher water absorption capacity and uniform micro-
structure as compared to other formulations. While genipin and GTA-based formula-
tions demonstrated better mechanical properties. Naturally occurring genipin-based
scaffolds showed superior biocompatibility as compared to other formulations par-
ticularly glutaraldehyde-based scaffolds during cytotoxicity analysis [88].
In another study, a “zero-length” crosslinker was utilized for crosslinking hya-
luronic acid molecules. The “zero-length” crosslinker is a coupling reagent i.e.,

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2-chloro-1-methylpyrinium iodide (CMPI) or 1-ethyl-(3,3 dimethylaminopropyl)

carbodiimide hydrochloride (EDC) and facilitates the intra and intermolecular ester
bond formation between carboxyl and hydroxyl groups of hyaluronic acid chains.
CPMI crosslinked hyaluronic acid films exhibited better resistance against hydro-
lytic degradation and higher crosslinking density as compared to EDC crosslinked
films. Furthermore, incorporation of CPMI crosslinker inhibited the inclusion of any
foreign bond into the crosslinked formulation while use of EDC led to the introduc-
tion of N-acylurea groups. Prepared crosslinked films did not show any toxicity risk
and were highly biocompatible and water soluble [89].
Chitosan/PVA hydrogels were prepared by using two crosslinking agents includ-
ing genipin amd glutaraldehyde in concentration 0.05% w/v. The genipin- and glu-
taraldehyde-based hydrogels showed similar morphological features, crystallinity,
thermal characteristics and elongation ratio. However, elastic modulus of genipin-
based formulation was lower (2.08—0:11 MPa) than that processed by glutaralde-
hyde (2.82—0:33 MPa). Though, glutaraldehyde-based hydrogels showed relatively
better mechanical properties, glutaraldehyde is five to ten thousand times more cyto-
toxic as compared to genipin. Hence, genipin can serve as a promising crosslinking
agent for preparing biocompatible hydrogels [90].
Cellulose-based hydrogels were manufactured by esterification crosslinking using
1,2,3,4-butanetetracarboxylic dianhydride (BTCA) as a crosslinking agent in con-
centration 15.5 mmol. Unreacted carboxyl groups were transformed into sodium
carboxylates by incorporating sodium hydroxide resulting in an enhanced aqueous
affinity and absorbency (almost 720 times of dry weight) of hydrogel. Prepared
hydrogel showed good biodegradability; almost 95% degradation was observed
within 7 days in the presence of cellulase [91].

Crosslinkers for improved mechanical strength

Erikci et al. used small charged molecules such as ammonium chloride (NH4+),
glucosamine (GluA+) and a hyaluronic acid disaccharide unit (dHA+) to produce
physically crosslinked hydrogel of thiol modified hyaluronic acid. The degrada-
bility and mechanical properties of hybrid hydrogels were controlled by adjusting
crosslinker type and concentration. Produced hydrogel exhibited good mechanical
properties [92].
In a study, potential of several pluronic samples (in concentration 0.690 mmol/L)
as collagen crosslinking agents was evaluated. Preparation of collagen matrices
involved blending of type I bovine collagen with either pluronics or carbon nano-
tubes dispersed in an aqueous pluronic solution with subsequent crosslinking by
employing carbodiimide chemistry. Resultant collagen hydrogel exhibited good
mechanical strength and differential scanning calorimetry analysis indicated a
change in the denaturation temperature of hydrogels upon addition of crosslinkers.
The collagen hydrogels prepared using pluronics as crosslinkers demonstrated 3–9
folds higher Young’s modulus as compared to non-crosslinked gels. However, non‐
covalent incorporation of carbon nanotubes did not affect the Young’s modulus of
prepared hydrogels [93].

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Polymer Bulletin (2022) 79:9199–9219 9213

Ethylene glycol dimethacrylate, triethylene glycol dimethacrylate and N, N-meth-

ylene bisacrylamide are commonly used crosslinkers for methacrylate network
formation; however, their poorly aqueous-soluble nature limits their potential as
crosslinking agents. Recently, two novel zwitterionic sulfobetaine dimethacrylate
crosslinkers including N, N-bis(methacryloxyethyl)-N-methyl- N-(3-sulfopropyl)
ammonium and N, N-bis(methacryloxyethyl)-N-methyl-N-(4-sulfobutyl) ammo-
nium betaines were prepared. These novel crosslinkers were used to crosslink zwit-
terionic hydrogels comprising N-(methacryloxyethyl)-N, N-dimethyl-N-(3-sul-
fopropyl) ammonium betaine via redox-initiated free-radical polymerization and
mechanical strength of resultant hydrogel was evaluated. Hydrogels prepared using
novel crosslinkers showed relatively higher mechanical strength and crosslink den-
sity than the hydrogels crosslinked using N, N-methylene bisacrylamide and ethyl-
ene glycol dimethacrylate [94].
Enzymatic (e.g., microbial transglutaminase) and ionic (e.g., calcium ions)
crosslinkers were employed for producing hydrogels comprising of gelatin and algi-
nate. Both the polymers were immersed in microbial transglutaminase (20 U / ml)
and calcium ion (1%) solutions. Gelatin-alginate hydrogels crosslinked via enzy-
matic and ionic crosslinking agents exhibited significantly higher tension and elon-
gation (i.e., 62 cN and 739%) as compared to alginate hydrogels crosslinked by cal-
cium ions (i.e., 18 cN and 150%) [95].
Double network hydrogels were formulated by micellar copolymerization of
acrylamide and a rosin-based crosslinker (FPA–PEG200–AC). The crosslinker
was incorporated in concentration 1.5–3.5% w/v. Prepared hydrogel formulations
showed significantly high compressive strain (above 95%) and elongation ratio at
break of 500–1500% [96].
Ionically (calcium ions) crosslinked sodium alginate and kappa-carrageenan dou-
ble network hydrogels good mechanical properties (i.e., stress and elastic modulus
of 1.07 MPa and 1.3 MPa, respectively) [97]. In another study, zinc ions were used
to crosslink polyvinyl alcohol, polyvinyl pyrollidone and acrylic acid triple network
hydrogels. Prepared hydrogels showed fracture stress, strain rate and compressive
stress of 1.87 MPa, 175% and 3.5 MPa, respectively; thus, indicating good mechani-
cal strength [98].

Crosslinkers for desired release characteristics

Highly soluble polymers are unable to provide controlled drug release profile. Addi-
tion of a crosslinker into a polymeric formulation can offer controlled drug delivery.
In a study, solubility and swellability of highly soluble polymer gelatin were modi-
fied by using isophorone diisocyanate as a crosslinking agent (in concentration 1
−30% w.r.t dry weight of gelatin) in order to attain modified drug release proper-
ties. Prepared crosslinked gelatin did not solubilize and was swellable in biofluids. A
reduction in biodegradability, solubility and an increase in amorphous character of
prepared gelatin was observed. Introduction of urea linkage led to a slight reduction
in thermal stability of gelatin. Drug (i.e., 5-Fluorouracil) release from crosslinked
gelatin took place via an anomalous transport mechanism with mild degradative

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diffusion. Results showed that prepared crosslinked gelatin can serve as a promising
carrier for attaining controlled drug release profile [99].
Double network pH responsive alginate and kappa-carrageenan hydrogel for-
mulations were prepared at various calcium chloride and potassium chloride ratios.
Bovine serum albumin was loaded in prepared hydrogel formulations, and in-vitro
release was evaluated in simulated gastric and intestinal fluid. Less than 10% drug
release was observed in simulated gastric fluid while almost 70% drug was released
within 4–5 h in simulated intestinal fluid suggesting controlled release of protein
[100].
Thermosensitive chitosan–gelatin hydrogels were crosslinked by genipin ranging
in concentration from 0 to 100 ug/mL. The release of loaded drug (sodium fluo-
rescein) was inversely correlated with the genipin concentration. within 1 h, almost
16% drug was released from prepared hydrogels. Drug release was 93%, 89%, 84%,
81% and 74% from hydrogels containing 0, 10, 25, 50 and 100 ug/mL of genipin,
respectively [101].
k-carrageenan crosslinked chitosan/hydroxyapatite hydrogels containing cipro-
floxacin. Introduction of hydroxyapatite resulted in sustained release of ciprofloxa-
cin. Chitosan and k-carrageenan complex exhibited 98% drug release; in compari-
son, almost 52 and 66% drug release was observed from hydrogels comprising high
(0.46 g sodium phosphate and 0.3 g calcium chloride) and low (0.23 g sodium phos-
phate and 0.15 g calcium chloride) amount of hydroxyapatite within 120 h suggest-
ing sustained release of drug [102].

Conclusion

Hydrogels are prepared by using several physical and chemical crosslinking

approaches. Various issues (such as toxicity, poorly solubility, biodegradability and
biocompatibility) are associated with majority of the crosslinkers currently being
used for preparing hydrogels. Efforts are being made to develop crosslinking agents
with an improved safety profile and aqueous solubility. During recent years, several
crosslinking agents have been obtained from natural sources in order to overcome
above-mentioned limitations. Moreover, the potential of crosslinkers for improving
the mechanical strength of hydrogels and modulating the release of loaded thera-
peutic agent has also been explored. Future developments may include preparing
hybrid hydrogel formulations by using safe crosslinking and modern biotechnologi-
cal approaches for efficient drug delivery and tissue engineering applications.

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maps and institutional affiliations.

Authors and Affiliations

Saman Zafar1 · Muhammad Hanif1 · Muhammad Azeem1,2 ·

Khalid Mahmood3 · Sonia Ashfaq Gondal4
1
Department of Pharmaceutics, Faculty of Pharmacy, Bahauddin Zakariya University, Multan,
Pakistan
2
Hamdard Institute of Pharmaceutical Sciences, Hamdard University Islamabad Campus,
Islamabad, Pakistan
3
Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan
4
School of Pharmacy, Hajvery University, Lahore, Pakistan

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