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Carbohydrate Polymers 198 (2018) 385–400

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Carrageenan based hydrogels for drug delivery, tissue engineering and T


wound healing
Ramanathan Yegappan, Vignesh Selvaprithiviraj, Sivashanmugam Amirthalingam,

R. Jayakumar
Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682041, India

A R T I C LE I N FO A B S T R A C T

Keywords: Carrageenan is a class of naturally occurring sulphated polysaccharides, which is currently a promising candi-
Carrageenan hydrogels date in tissue engineering and regenerative medicine as it resemblances native glycosaminoglycans. From
Tissue engineering pharmaceutical drug formulations to tissue engineered scaffolds, carrageenan has broad range of applications.
3D bioprinting Here we provide an overview of developing various forms of carrageenan based hydrogels. We focus on how
Cell delivery
these fabrication processes has an effect on physiochemical properties of the hydrogel. We outline the appli-
Drug delivery
cation of these hydrogels not only pertaining to sustained drug release but also their application in bone and
Wound healing
cartilage tissue engineering as well as in wound healing and antimicrobial formulations. Administration of these
hydrogels through various routes for drug delivery applications has been critically reviewed. Finally, we con-
clude by summarizing the current and future outlook that promotes the seaweed-derived polysaccharide as
versatile, promising biomaterial for a variety of bioengineering applications.

1. Introduction abundance (Lee & Mooney, 2012; Varoni et al., 2012; Vignesh,
Sivashanmugam et al., 2018). These biopolymers have known to pos-
Hydrogels are 3D networks of hydrophilic polymeric chains that sess a facile and efficient extraction procedure that makes them ideal to
have 90–99% water content and facilitate efficient oxygen and mass be produced in large scale.
transfer (Seliktar, 2012). Over the past few decades, these hydrophilic In the present scenario, where scientists are in a quest for novel
networks have garnered unprecedented application in tissue en- renewable polymers, the underexploited carrageenan (CRG) seaweeds
gineering owing to their high biocompatibility, low immunogenicity belonging to marine red algae family are an interesting source of
and cytotoxicity, ease of functionalization and tuneable physicochem- polysaccharides with distinctive structure and functional properties.
ical properties (Hoffman, 2012; Saul & Williams, 2013). These poly- Derived from Rhodophyceae, CRG is an anionic, sulphated polygalacton
meric systems are excellent substrates for cell transplantation and dif- consisting of alternating long linear chains of α-1, 3 D-galactose and β-
ferentiation, endogenous regeneration, sustained drug delivery, bio 1, 4 3, 6-anhydro-galactose (3, 6-AG) with ester sulphates (15–40%)
prosthetics and wound healing (Saul & Williams, 2013; Hoffman, 2012; similar to naturally occurring glycosaminoglycans (Campo, Kawano, da
Seliktar, 2012; Anitha et al., 2014). The three dimensional network Silva, & Carvalho, 2009). This seaweed based polysaccharide can be
system of hydrogels mimics the microarchitecture of native tissue ex- conventionally categorized into six basic forms depending on their
tracellular matrix (ECM) and thus provide in vivo niche like conditions sulphate content, source of extraction and solubility as Kappa (κ)-, Iota
for cell survival (Geckil, Xu, Zhang, Moon, & Demirci, 2010; Tibbitt & (ɩ)-, Lambda (λ)-, Mu (μ)-, Nu (ν)- and Theta (θ)-CRG (Fig. 1). Of these
Anseth, 2009). Although a gamut of biopolymers have been employed κ, ɩ and λ are of commercial importance due to their viscoelastic and
in hydrogel biofabrication for cell encapsulation and targeted drug gelling properties (Cunha & Grenha, 2016). CRG type, molecular
delivery, polysaccharide based biomaterials are extensively being stu- weight, concentration and temperature determine the viscosity of CRG
died due to their enhanced biological activity, biocompatibility and gels. Viscosity increases with increase in concentration of the CRG, as
biodegradability, mechanical stability as well as scope for chemical there is more interaction between the macromolecular chains and in the
modification. Recently, hydrogels based on marine derived poly- presence of cations the electrostatic repulsion between the sulphate
saccharides such as alginate, agarose, chitosan, etc. have been widely groups are reduced. κ- and ɩ- CRGs form gels of increased apparent
employed in tissue engineering applications as they are available in viscosity at low temperature and small salt concentrations. The


Corresponding author.
E-mail address: rjayakumar@aims.amrita.edu (R. Jayakumar).

https://doi.org/10.1016/j.carbpol.2018.06.086
Received 23 May 2018; Received in revised form 18 June 2018; Accepted 20 June 2018
Available online 23 June 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

Fig. 1. Chemical structures of different types of carrageenan.


Adapted from ref. (Cunha & Grenha, 2016).

viscosity of CRG gels decreases with increase in temperature (Anderson, application of CRG as promising biomaterial. We provide an overview
Dolan, Lawson, Penman, & Rees, 1968). CRG is used far more widely of the various forms of CRG based hydrogels for drug delivery, tissue
than agar as emulsifier, gelling, thickening and stabilizing agent in engineering and wound healing. Administration of these hydrogels
pharmaceutical and industrial applications (Liu, Zhan, Wan, Wang, & through various routes for drug delivery applications has been critically
Wang, 2015). Apart from its inflammatory and immunomodulatory evaluated. Finally, we have provided our perspectives that promote this
properties, these polysaccharides are used as an anticancer (G. Zhou seaweed-derived polysaccharide as versatile and promising biomaterial
et al., 2004), antihyperlipidemic agents (Panlasigui, Baello, for tissue engineering applications.
Dimatangal, & Dumelod, 2003) and also as herpes (Carlucci, Scolaro, &
Damonte, 1999) and human papillomavirus inhibitors (Buck et al., 2. Different forms of carrageenan based hydrogels
2006).
Among the commercially available CRGs, κ- and ɩ- CRG form three CRG hydrogels are generally formed through thermoreversible ge-
dimensional network of double helix via crosslinking of adjacent sul- lation, ionic crosslinking as well as modification of CRG backbone with
phate groups, while in λ-CRGs the sulphate groups do not undergo photocrosslinking methacrylate moieties (Fig. 2). These modifications
crosslinking and thus do not form gels (Campo et al., 2009). Upon are further explored to fabricate different forms of CRG hydrogels with
cooling and in the presence of appropriate cation (K+, Ca2+), CRGs, interesting features.
kappa in particular undergo, coil to helix transition and helical ag-
gregation to form thermotropic and ionotropic hydrogels. Thermo- 2.1. Photocrosslinking hydrogels
gelation property of κ-CRG hydrogels has been used to develop novel
injectable hydrogels systems. Generally, CRG based hydrogels formed Conventionally, CRG hydrogels are crosslinked via ionic interac-
are brittle in nature with high swelling ratios and poor mechanical tions in presence of K+ and Ca2+ ions resulting in brittle hydrogels
stability under physiological conditions (Thakur et al., 2016). Chemical (Chronakis, Doublier, & Piculell, 2000; Mangione et al., 2005). To cir-
modification of the polymeric backbone can be used to overcome this cumvent this drawback, photocrosslinking hydrogels are made by in-
drawback. CRG is known to possess multifarious functional groups such corporating photocrosslinking moieties such as methacrylate groups
as hydroxyl/sulphate groups which make them an ideal choice for di- into κ-CRG backbone followed by UV crosslinking in presence of che-
verse chemical modifications (Campo et al., 2009). They have been mical photoinitiator (Irgacure 2959) (Mihaila et al., 2013). Varying
oxidized (Zhu et al., 2017), oversulphated (Yuan et al., 2005), car- degree of methacrylation was achieved by replacing the hydroxyl group
boxmethylated (Aparna et al., 2017), acetylated (Yuan et al., 2005), of carrageenan with methacrylate moieties to develop Mκ-CRG. The
methacrylated (Mihaila et al., 2013), as well as phosphorylated (Yuan presence of methacrylate groups limited the interaction between the
et al., 2005). These modifications have not only proven that CRG is a side chains, leading to Mκ-CRG with viscosity much lower than the
robust polymer but have also endowed them with the improved physio- unmodified κ-CRG. Dual crosslinking was also performed, as the an-
chemical properties, new specific functionalities and features. There are ionic character of Mκ-CRG remained unaffected. Swelling ratio of
few excellent reviews that widely explored the basic properties of CRG physically crosslinked Mκ-CRG was lower than chemically crosslinked
such as structure, physiochemical properties, biological activities and hydrogel, as dissolution of ions happened overtime resulting in poor
chemical modifications (Campo et al., 2009; Liu et al., 2015). Further, water retention capabilities. On the other hand, chemically crosslinked
few reviews have also focussed on CRG based drug delivery applica- hydrogels had higher water retention capacity resulting in more flexible
tions using blends and nanocomposites (Cunha & Grenha, 2016; Li, Ni, hydrogel network. Higher degree of methacrylation resulted in lower
Shao, & Mao, 2014; Zia et al., 2017) swelling ratio and lower pore size distribution whereas lower degree of
In this review, we specifically focus on the emerging trends in methacrylation resulted in interconnected pores. Presence of

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R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

Fig. 2. Different crosslinking mechanisms em-


ployed for developing carrageenan based hy-
drogels. Thermoreversible gelation, ionic
crosslinking (in presence of monovalent or di-
valent ions), UV crosslinking (modification of
carrageenan backbone with methacrylate
groups), or dual crosslinking (both UV and
ionic crosslinking).

photoinitiator, methacrylate groups, and UV exposure had no effect on 2.3. Floating hydrogels
cell viability (Mihaila et al., 2013). These results demonstrate that dual
crosslinking of Mκ-CRG rendered greater stability than physical cross- Floating drug delivery system has gained attention among re-
linking thereby preventing the ion exchange in dissolution media. searchers in recent times due to its enhanced gastroretensive property
to stay afloat in stomach and release the drug in a controlled manner.
This extended gastroretention can be attained by utilizing the swelling
2.2. Gradient hydrogels property of the material that absorbs fluid from the surrounding en-
vironment in a controlled manner, making it float above the gastric
Gradient hydrogels exhibit gradual change in one of the physical contents and remain unaffected by the gastric emptying time (Gaikwad,
properties of the material such as viscoelasticity, stiffness, porosity that Yadav, & Gaikwad, 2014; Mayavanshi & Gajjar, 2008). Various studies
could be tailored to mimic the native tissue interface (Genzer & Bhat, have explored sodium bicarbonates (NaHCO3) and calcium carbonates
2008). These hydrogel offers various advantages over conventional (CaCO3) potent as a pore forming agents (Baumgartner, Kristl, Vrečer,
layered hydrogels, providing smooth transition between different tissue Vodopivec, & Zorko, 2000; Chen & Park, 2000; Kumar & Nerella, 2010;
regions similar to the in vivo tissue environment (Khanarian, Haney, Pahwa, Bhagwan, Kumar, & Kohli, 2010). Floating systems using κ-CRG
Burga, & Lu, 2012). Gradient hydrogels are developed by using fabri- hydrogels have been developed by incorporating NaHCO3 and CaCO3 as
cation methods such as electrospinning, microfluidics, gradient makers, effective pore forming agents. Fourier transform infrared spectroscopy
etc. (Seidi, Ramalingam, Elloumi-Hannachi, Ostrovidov, & (FTIR) characterization revealed the presence of carbonates peak in
Khademhosseini, 2011). Instead of these complex techniques, poly- both the groups and a drastic shift in ester sulfatepeak of carrageenan
meric capillary flow can be used to produce two or more layered gra- was observed in NaHCO3 treated group. Sodium carbox-
dient hydrogels (Piraino, Camci-Unal, Hancock, Rasponi, & ymethylcellulose (NaCMC) was blended with κ-CRG hydrogel to en-
Khademhosseini, 2012). Hydrogels of gelatin and CRG modified with hance the swelling behaviour. Porosity increased with increasing con-
methacrylate groups (GelMA, Mκ-CRG) were fabricated by simple re- centration of pore forming agents though no significant difference was
producible technique that utilizes the natural flow property of the observed between CaCO3 and NaHCO3 treated groups. In contrast,
material (Fig. 3). Incorporation of nanosilicates into κ-CRG and gelatin mechanical testing revealed that the gel strength decreased with in-
hydrogels increased the strength and compressive moduli and imparted crease in concentration of pore forming agents, as it made the gel more
a shear thinning property without affecting the gelation time. Micro- porous and fragile. In addition, drug entrapment efficiency decreased
structure analysis using SEM revealed a change in pore structure across when concentration of CaCO3 and NaHCO3 increased from 0.25 to 1%
GelMA towards Mκ-CRG region with the addition of nanosilicates, since due to less dense hydrogel network structure when compared to the
the concentration of nanosilicates played an important role in the dis- control group without pore forming agent. Despite poor entrapment
tribution of pore area. Human mesenchymal stem cells (hMSCs) en- efficiency at increasing concentrations, controlled drug release was
capsulated into GelMA region of gradient hydrogel displayed elongated observed. Comparison of the drug release pattern revealed that, CaCO3
morphology, a characteristic feature of osteoblasts, while cells in Mκ- incorporated hydrogel exhibited more controlled release than NaHCO3,
CRG region had rounded morphology, characteristic to chondrocytes. due to the presence of small pores that led to a sustained drug release
The interface region contained cells of both morphologies (Cross, Shah, profile. Presence of 0.5–1% CaCO3 and NaHCO3 resulted in better
Palani, Peak, & Gaharwar, 2017). These results suggest that change in floating property than control hydrogel (Selvakumaran, Muhamad, &
cell morphology within the gradient hydrogel could be an indicator of Abd Razak, 2016). Similarly, the effect of various concentration of
the hydrogel’s potential to modulate the cell fate. genipin as a crosslinker was studied for developing floating κ-CRG
hydrogel. Increasing the crosslinker concentration resulted in increased

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R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

Fig. 3. Fabrication of gradient hydrogel. 2D nanosilicates have been incorporated into MκCA-GelMA pre-polymer solution, followed by cell encapsulation and
gradient formation. This gradient pre-polymer solution was crosslinked by illuminating UV light to produce gradient hydrogel.
Reproduced from (Cross et al., 2017) with permission from Elsevier, Copyright© 2017.

gel strength, controlled drug release pattern, and low thermal stability 2015). The degradation study revealed that the micropatterned hy-
(Selvakumaran & Muhamad, 2015). Microstructure analysis revealed drogels were stable upto 14 days. Owing to their biodegradability, cell
that the genipin crosslinked hydrogel possessed rough surface mor- encapsulated κCRG hydrogels might have the potential for stem cell
phology when compared to non crosslinked hydrogel. Thus, CRG hy- transplantation and differentiation. Furthermore, hiPSC colonies seeded
drogel could be tailored as a floating hydrogel systems using various on k-CRG hydrogels with circular grid micropatterns, adhered to the
pore forming agents and crosslinkers to enhance its gastroretensive hydrogel surface and were found to proliferate within the micro-
property. patterns. Similarly, low swelling behaviour of methacrylated k-CRG
It may be concluded that these various approaches for fabricating hydrogels enabled generation of reproducible micropatterns using
CRG as gradient hydrogels, photocrosslinkable hydrogels, and floating PDMS (Mihaila et al., 2013). The micromoulding technique allowed
hydrogels highlights scope for application of CRG in various tissue spatial control of microenvironment geometry as well as cell distribu-
engineering approaches. tion, thus generating a cell-biomaterial platform ideal for tissue en-
gineering applications. Taken together, these results show that k-CRG
2.4. Micropatterned hydrogels hydrogels can be micropatterned and used as a platform to study the
effect of physical cues of the microenvironment on stem cell fate.
Micropatterning techniques have been used to design micro-scale
patterns for in vitro cell culture substrate to control the cellular mi- 2.5. Hydrogel scaffolds
croenvironment (Griffin, 2015; Guilak et al., 2009; Lee et al., 2009).
Most of the micropatterning techniques used are tedious, expensive and Various physical and chemical methods have been employed to
often restricted to a narrow range of biopolymers (Mukhopadhyay, develop scaffolds that can mimic the in vivo native extracellular matrix
2007). Many a times, patterns on soft hydrogels are damaged during (ECM) (Bose, Roy, & Bandyopadhyay, 2012; Lu, Li, & Chen, 2013).
curing and demoulding processes. In a recent study, micropatterned Composite scaffold made of κ-CRG and silk fibroin (SF) was fabricated
simple wax moulds were used as templates to micropattern k-CRG hy- by blending different concentrations of κ-CRG into SF solutions. De-
drogels (Vignesh et al., 2018). Using wax mould patterns as prototypes, crease in the concentration of CRG resulted in small and low pore size
hydrogel surfaces were micropatterned with various shapes and sizes distribution, whereas increase in CRG concentration resulted in scaf-
ranging from 620 μm square projections to 1500 μm circular grooves folds with large pore size due to the repulsive forces between SF and
and 600 μm to 1360 μm criss-cross hillocks (Fig. 4). The hydrogels were sulfate groups in CRG. Similarly, swelling ratio was greater in scaffolds
found to be biocompatible upon hMSC encapsulation. Micropatterned with higher CRG concentration in SF solution that directly correlated
hydrogels had a gel strength of ∼40 kPa, which is comparable to that of with larger pore size distribution. In contrast, higher concentration CRG
stiffer gels that favours chondrogenesis and also correlates with other correlated inversely with compressive property of the scaffold; highly
studies that have shown k-CRG to be chondrogenic in nature (Discher, porous scaffolds had low compressive moduli. When immersed in si-
Mooney, & Zandstra, 2009; Popa, Caridade, Mano, Reis, & Gomes, mulated body fluid (SBF), sulfate groups in scaffold attracted Ca2+ ions

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R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

Fig. 4. Micropatterned hydrogels. Hydrogels with varying size of micropatterns created using wax moulds.
Reproduced from (Vignesh, Gopalkrishnan et al., 2018) with permission from Elsevier, Copyright© 2018.

from SBF leading to apatite layer formation at the surface of scaffold polyelectrolyte complex (PEC) such as chitosan and κ-CRG have been
(Nourmohammadi, Roshanfar, Farokhi, & Haghbin Nazarpak, 2017). ɩ- developed by addition of chitosan solution into κ-CRG, followed by
CRG blended with different ratios of poly(vinyl alcohol) (PVA) have increasing the pH to alkaline condition and subsequent drying in hot air
proven to be efficient scaffolds for cryopreservation of cells (Chopra oven (Araujo, Davidenko, Danner, Cameron, & Best, 2014). Chitosan-κ-
et al., 2016). Varying the concentration of ɩ-CRG to PVA ratio produced CRG scaffolds were stable at pH of around 4.5–7.4 beyond which PEC
varying pore size distribution with interconnected pores. Hemo- underwent dissolution due to the repulsive force between sulfate
compatibility studies revealed that the increase in ɩ-CRG concentration groups, and hydrolytic degradation of carrageenan. Lower the chitosan-
led to more RBC lysis in samples where ratio of ɩ-CRG to PVA is greater κ-CRG ratio, higher was the electrostatic repulsion. These results de-
than one. Cells were more viable in PVA-ɩ-CRG ratio of 7:3 and 8:2 due monstrate that water uptake into the scaffold not only depends on the
to the evenly distributed interconnected pores. ɩ-CRG blended with PVA hydrophilicity of the polymer, but also depends on the pore size and
scaffolds have been developed by unidirectionally oriented freeze ability of pores to retain the fluid.
drying method (Zhang et al., 2016). The blends were casted into tube,
rested vertically on top of liquid nitrogen and subjected to uniaxial
2.6. Interpenetrating polymer networks
temperature gradient. Weight loss of the oriented scaffold was much
slower than the non-oriented scaffolds fabricated by traditional freeze
Interpenetrating polymeric network (IPN) hydrogel are having
drying method. Though cells were viable in both the scaffolds, cells
properties ranging from enhanced mechanical behaviour to rapid
were evenly distributed in oriented scaffold than the non-oriented
swelling and deswelling ratio offer several advantages over the con-
scaffold due to their enhanced permeability.
ventional hydrogels. Based on the application and desired properties,
CRG based scaffold for osteogenic differentiation was developed by
IPNs can be fabricated by various methods to produce semi-IPNs, homo-
adding ɩ-CRG solution into chitosan/gelatin blend (CG), followed by
IPNs or fully IPNs (Dragan, 2014; Sperling, 1994). Since CRG’s thixo-
glutaraldehyde crosslinking and subsequent freeze drying (Li et al.,
tropic behaviour fairly mimics the natural mechanics of nucleus pul-
2015). Differential scanning calorimetry (DSC) characterization showed
posus region of the intervertebral disc, it was engineered for application
increase in glass transition temperature with the addition of ɩ-CRG in
in intervertebral disc regeneration (Chan, Boughton, Ruys, & Oyen,
CG scaffold (CCG) due to the strong intermolecular interaction in CCG
2017). κ-CRG gel was infused into sucrose incorporated PCL scaffold to
than CG scaffold. Swelling behaviour and mechanical strength followed
form an IPN and the resulting scaffold exhibited higher mechanical
a similar pattern; increase in ɩ-CRG ratio led to increased swelling and
strength while viscoelastic property remained unaffected. Concentra-
decreased strength. Protein adsorption in CCG scaffold decreased with
tion of κ-CRG in the IPN influenced the tensile moduli. Higher amount
increase in ɩ-CRG ratio owing to the formation of strong hydration layer
of CRG gel greatly reduced the moduli, thus the optimum ratio of gel
(Yu, Zhang, Wang, Brash, & Chen, 2011). Scaffolds made of
infusion into the scaffold resembled the tensile moduli of nucleus

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R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

pulposus.CRG gels promoted adhesion, proliferation, and migration of blended with sodium alginate, added dropwise into ionic crosslinker
3T3 fibroblast cells. Likewise, IPN beads made of κ-CRG and CMC were solution and then into glutaraldehyde for covalent crosslinking. Beads
fabricated using double emulsion (water-in-water) process for the ap- produced from PAAm-g-CRG alone had poor mechanical strength; as a
plication in controlled drug delivery systems (Lohani, Singh, result, it exhibited poor morphology. Whereas IPN formed by mixing
Bhattacharya, Rama Hegde, & Verma, 2016). Various parameters such with sodium alginate possessed higher mechanical strength and hence
as polymer ratio, gelation time, and amount of crosslinker have shown spherical morphology. As discussed earlier, increasing the crosslinker
to influence the formation of IPNs and drug encapsulation efficiency. concentration resulted in smaller bead size with rigid structure and high
Ibuprofen as a model drug was incorporated into IPN beads crosslinked drug entrapment efficiency with enhanced sustained release.
with AlCl3. Owing to the formation of rigid IPN matrix, differential Thermosensitive poly(diethylacrylamide) (PDEA) based κ-CRG
scanning calorimetry (DSC) analysis revealed that drug loaded IPN semi-IPN hydrogels were made by crosslinking only PDEA with me-
beads showed increased melting temperature compared to the control. thylene bisacrylamide, leaving CRG in a linear form (Chen, Liu, Jin, &
In addition, absence of exothermic transition revealed the greater Liu, 2008). Although the surface morphology showed dense structure of
thermal stability of IPN beads than native polymers. Amount of cross- PDEA hydrogel, upon semi-IPN formation using CRG, the hydrogel
linker and gelation time influenced the bead morphology, size, shape obtained a porous structure. Increasing the CRG content increased the
and drug encapsulation efficiency. Higher gelation time and prolonged equilibrium swelling ratio, upon increasing the lower critical solution
exposure to crosslinker solution produced smaller particles with more temperature (LCST), hydrogels underwent temperature dependent
rigid matrix and higher encapsulation efficiency. deswelling due to the destruction of hydrogen bonds. Similarly, pH
IPNs have also been made via cytocompatabile enzymatic and ionic sensitive poly(methacrylic acid) was incorporated into temperature
crosslinking of natural polymers for tissue engineering applications. sensitive PDEA-κ-CRG semi IPN hydrogel (Chen, Liu, & Chen, 2009).
Microbial transglutaminase (mTG) crosslinked gelatin followed by ionic Increasing the pH from acidic to alkaline condition increased the
crosslinked κ-CRG were blended together to form IPN (Wen, Lu, & Li, equilibrium swelling ratio as carboxylic group got deprotonated at al-
2014) (Fig. 5). Use of two different crosslinking mechanisms had a kaline pH, making network chains more flexible. Nanocomposite semi-
significant effect on swelling ratio of the IPN. Crosslinking with mTG IPN hydrogels based on carrageenan and acrylamide were synthesized
alone (Semi-IPN) resulted in increased swelling of gelatin gels making it via solution polymerization (Mahdavinia, Marandi, Pourjavadi, & Kiani,
more flexible, whereas ionic crosslinking reduced the flexibility of κ- 2010). Sodium montmorillonite nano-clay was incorporated into κ-
CRG gels resulting in lower water retention. Thus by dual crosslinking CRG-acrylamide mixture and acrylamide was polymerized in situ by
mechanism using two polymeric systems, IPN with tailorable swelling adding methylene bisacrylamide as crosslinker. Effect of nano-clay on
ratios and degradation can be achieved. CRG has been developed as swelling ratio of the hydrogel at different salt solutions was evaluated.
IPNs in combination with other polysaccharides such as agar, gelatin, Lower concentration led to ionization of sodium montmorillonite
and their drug release profile has been extensively evaluated (Liu, Lin, causing drastic increase in osmotic pressure of the hydrogel and greater
Li, & Liu, 2005). κ-CRG-PVA IPN hydrogels were used for in situ swelling. Therefore, increasing the amount of nano-clay decreases the
synthesis of Fe3O4 nanoparticles in an inert gas free environment osmotic pressure, thus achieving controlled swelling. Semi-IPN hydro-
(Mahdavinia & Etemadi, 2014). κ-CRG and PVA solutions were mixed gels using κ-CRG and poly(N-isopropylacrylamide) (PNIPAAm) were
with the precursors for Fe3O4 nanoparticle synthesis. Upon completion, developed using electron beam radiation technique (Mohanan,
mixture was freeze dried multiple times and subsequently immersed in Vishalakshi, & Ganesh, 2011). In this approach, in addition to pH and
KCl for crosslinking. κ-CRG – PVA hydrogel possessed smooth surface temperature, dose rate and the amount of precursors determined the
topography but in the presence of Fe3O4 NPs, slight change in surface effect of crosslinking and swelling ratios. Mixture of κ-CRG, PNIPAAm,
roughness was apparent. In the presence of magnetic NPs, controlled and methylene bisacrylamide were made into aqueous solution fol-
swelling ratio was observed. Poly(acrylamide) grafted κ-CRG (PAAm-g- lowed by irradiating with electron beam with low dose rate (0.5kGy/
CRG) and sodium alginate was dual crosslinked to form an IPN beads min). Dose less than 20kGy exhibited maximum swelling ratio due to
for controlled drug delivery to intestine (Kulkarni, Boppana, Krishna the lack of complete polymerization, whereas above 20kGy resulted in
Mohan, Mutalik, & Kalyane, 2012). Polyacrylamide was grafted onto κ- rigid gels with least swelling. The highly customizable properties and
CRG via free radical polymerization. The polymeric solution was tuneable features of design and development makes CRG based IPN a

Fig. 5. Schematic representation of enzymatic and ionic crosslinking of gelatin/carrageenan IPNs.


Reproduced from (Wen et al., 2014), with permission from Elsevier, Copyright© 2014.

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R. Yegappan et al. Carbohydrate Polymers 198 (2018) 385–400

Fig. 6. 3D bioprinting of κ-CRG-GelMA hydrogel.


Reproduced from (Li et al., 2018), with permission from American Chemical Society, Copyright © 2018.

suitable polymeric system for various tissue engineering applications. 2.8. Carrageenan as bioink for 3D bioprinting

3D bioprinting of hydrogels with an appreciation of the native tissue


2.7. Nanogels architecture holds promise for generation of engineered tissues.
Thermoresponsive κ-CRG has the ability to form fibrillary networks that
Nanogels have emerged as promising delivery system as they could allows formation of gels with different shapes and tuneable mechanics,
be designed to encapsulate diverse class of biomolecules and could making it an excellent choice for bioink material. Although 3D bio-
provide stability for encapsulated biomolecules (Neamtu, Rusu, printing has emerged as an excellent option for creating biomaterial
Diaconu, Nita, & Chiriac, 2017; Soni, Desale, & Bronich, 2016). LDL, an scaffolds with intricate architecture, the brittle nature of the κ-CRG
integral part of egg yolk is similar to micellar nanostructure that can be hydrogel often stands as a roadblock in their successful applications. A
exploited for variety of nutrient delivery applications (Anton et al., lot of research is now focused on strengthening the hydrogels by re-
2003). CRG have been complexed with low density lipoproteins (LDL), inforcement using nanocomposites, fibres as well as ionic covalent en-
and made as nanogel via spray drying technique (Zhou, Hu, Wang, Xue, largement (ICE) networks. ICE gels are formed by interpenetrating
& Luo, 2016). Curcumin as a model compound was encapsulated into κ- networks of two polymers, one crosslinked with metal ion while the
CRG/LDL complex. Effect of concentration dependent loading was other is with covalent bonds. Tough ICE hydrogels for bioprinting were
evaluated, where, increase in curcumin to LDL ratio increased the generated using κ-CRG and poly (oxyalkylene amine) (Jeffamine®)
polydispersity index (PDI) and size, since more curcumin was en- (Bakarich, Balding, Gorkin, Spinks, & in het Panhuis, 2014). Thermo-
trapped within the nanogel. As the size increases, κ-CRG/LDL nanogels responsive nature of CRG induces its fast gelation, which provides a
precipitated, since it possessed higher negative surface charge that re- structural integrity to the hydrogel system while the epoxy-amine re-
sulted in disruption of lipid bilayers, leading to higher PDI and zeta action provides an ambient temperature for covalent bond formation.
potential. Thermo-sensitive CRG nanogels were also fabricated by re- Similarly, ICE network of GelMA and κ-CRG were reinforced with La-
verse phase microemulsion method (Daniel-da-Silva, Ferreira, Gil, & ponite XLG nSi to form nanoengineered ionic covalent enlargement
Trindade, 2011). κ-CRG as aqueous phase was added into organic phase (NICE) bioink (Chimene et al., 2018). The NICE bioink formulation had
containing n-heptane, cetyltrimethylammonium bromide (CTAB), and enhanced mechanical strength, elasticity and flow characteristics that
butanol as surfactant and co-surfactant, respectively, followed by so- allowed printing of freestanding complex structures with high aspect
nication to produce nanogels. Presence of CTAB had a negative effect ratios. Bioprinted cells were viable and demonstrated attachment and
on size of nanogels produced. The electrostatic interaction between spreading for 120 days of culture. The hybrid system formed by poly-
positively charged surfactant and sulfate group of CRG caused electrolyte complexation, supported C2C12 mouse myoblasts, opening
shrinkage of nanogels when higher percentage of carrageenan was up the possibilities for bioprinting of layered tissue constructs with
used, whereas nanogels with lower polymeric content remained un- interfacial bonding. Likewise, κ-CRG was reinforced with nanosilicates
affected. No significant difference in thermal behaviour was observed (nSi) to bestow it with shear thinning characteristics to form κ-CRG-nSi
for different percentages of CRG while increased CRG content promoted bioink, which is envisioned to bioprint cell laden structures with
higher swelling ratio. Methylene blue (MB), a model drug loaded into tuneable mechanical and shape retention properties (Wilson, Cross,
4% κ-CRG nanogels showed slow burst release and rapid release over- Peak, & Gaharwar, 2017). The high elastic modulus and thermogelation
time, whereas at 1% κ-CRG nanogels displayed burst release initially of the bioink facilitated printing of stable multi-layered tissue con-
and slow release overtime (Daniel-da-Silva et al., 2011). Therefore, size, structs that could be easily crosslinked by ionic interactions. Further-
swelling ratio, thermal behaviour, and release kinetics can be optimized more, the shear thinning nature of the bioink allowed efficient mixing
by varying the percentage of CRG, thereby making nanogels a suitable and printing of mouse preosteoblasts without formation of cellular
drug carrier.

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aggregates. Recently, three cationic hydrogels (chitosan, gelatin, of inferior alveolar nerve (Eppley, Snyders, Winkelmann, & Roufa,
GelMA) were bioprinted in combination with three anionic hydrogels 1991; Levi-Montalcini, 1987). To avoid repetitive injection of NGF,
(alginate, xanthan, κ-CRG) among which κ-CRG-GelMA was found to be collagen/hydroxyapatite/κ-CRG nanocomposite hydrogel was devel-
the best combination of oppositely charged hydrogels for 3D bio- oped with superior bone like native microstructure. CRG conferred
printing (Li, Tan, Liu, & Li, 2018) (Fig. 6). These printable reinforced protective mechanism for NGF, by preventing its degradation during
bioinks are excellent tools for designing large human tissue constructs the period of sustained release (Wang et al., 2010, 2009). Overall, these
in tissue engineering and regenerative medicine. results emphasize their potential use as constructs for bone tissue en-
gineering.
3. Biomedical applications of carrageenan based hydrogels
3.1.2. Cartilage
3.1. Tissue engineering Owing to the presence of sulfate groups in its backbone, CRG mi-
mics sulfated glycosaminoglycans (sGAG) naturally occurring in the
3.1.1. Bone extracellular matrix of cartilage (Bhattacharyya et al., 2010; Popa et al.,
Development of scaffolds and bone substitutes that provides struc- 2015), making CRG based biomaterials a potential candidates in car-
tural and functional support in treating the bone defect remains the tilage tissue engineering. κ-CRG hydrogel incorporated with iron oxide
primary focus in the area of bone tissue engineering. CRG’s ability to magnetic nanoparticles (MNPs) furnished tailorable physical properties
allow apatite layer formation when incorporated with functional for chondrocyte differentiation. Presence of MNPs improved the stabi-
bioactive cues is well established (Daniel-Da-Silva, Lopes, Gil, & lity of hydrogel and affected the cellular response. In response to
Correia, 2007; Kim et al., 2011). κ-CRG when incorporated into col- magnetic field, there was an increase in gene expression of chondro-
lagen-hydroxyapatite composite gel, increased its compressive strength genic markers (Popa, Santo, Rodrigues, & Gomes, 2016; Popa, Reis, &
(Feng et al., 2017). This improvement in mechanical property justifies Gomes, 2012). When implanted in vivo, the fast dissolution rate of io-
CRG as an efficient bone repair material. Bone substitutes based on nically crosslinked κ-CRG prevented accumulation of toxic by-products
nano-hydroxyapatite (n-HAP) have been widely used for their effective thereby readily resorbing and resolving inflammation (Popa et al.,
ion exchange, bioactivity and biocompatibility (Morais et al., 2013). 2014). Encapsulation of human adipose stem cells (hASCs) in κ-CRG
CRG hydrogel blended with different ratios of rod shaped nHAP dis- hydrogel supported proliferation, chondrogenic differentiation in pre-
played promising characteristics for delivery of injectable substitutes sence of chondrogenic growth factors and was found to be non-cyto-
(González & Ossa, 2017). Rod shaped morphology of nHAP influenced toxic (DeLise, Fischer, & Tuan, 2000; Popa et al., 2015). Mechanical
the extent to which it mimics the native apatite crystals in bone, property of the hydrogel was similar to that of native cartilage tissue.
thereby enhancing the cell-cell interaction and cell adhesion (Swain & CRG possesses excellent freeze-thaw stability and maintains structural
Sarkar, 2011). Surface topography of the electrospun nanofiber dictates integrity during cryopreservation-thawing process facilitating easy
the cell proliferation, attachment and migration (Flemming, Murphy, manipulation and efficient use as a storage encapsulation systems (Popa
Abrams, Goodman, & Nealey, 1999). Mechanical property and de- et al., 2013). Combining hydrogel with growth factor and stem cells
gradation behaviour of poly(hydroxybutyrate) (PHB) and poly(hydro- proves to be a promising strategy for cartilage regeneration. TGF-β1 is a
xybutyrate valerate) (PHBV) nanofibers tailored by surface coating of κ- growth factor responsible for chondrocyte differentiation, proliferation,
CRG hydrogel, resulted in enhanced osteoblast differentiation and synthesisof proteoglycans and cell-cell interaction in cartilage matrix
biomineralization. When compared to the control polyester fibers, CRG (DeLise et al., 2000; Holland & Mikos, 2003). TGF-β1 along with
coated fiber exhibited micron-to-nanoscale sized apatite crystals. Upon hADSCs incorporated into κ-CRG hydrogel enhanced the chondrogenic
culturing with osteosarcoma cells, κ-CRG coated fibers exhibited im- differentiation and chondrocyte related gene expression (Rocha, Santo,
proved biomineralization with osteogenic differentiation potential Gomes, Reis, & Mano, 2011). Human articular chondrocyte (HAC) en-
(Goonoo et al., 2017). Though use of selenium related to bone micro- capsulated in CRG/fibrin/hyaluronic acid (HA) hydrogel exhibited
structure is poorly understood (Martiniaková et al., 2013), the trace better elasticity and stiffness when compared to fibrin-HA gel and also
element is thought to possess variety of biological effects such as im- induced cartilage ECM deposition (Pereira et al., 2009). Chondrocytes
proved metabolic cell activity (Chung, Ercan, Roy, & Webster, 2016) were encapsulated within combination of alginate and κ-CRG which
and anti-cancer property (Stolzoff & Webster, 2016), for which it is now were then made into beads and fibers (Popa, Gomes, & Reis, 2011).
widely explored in biomedical applications. Three different solutions Polymer ratio played an important role in cell viability and prolifera-
with κ, ɩ, and λ-CRGs were prepared and sodium selenite was added to tion in 3D hydrogel construct. Hydrogels with high alginate content had
produce selenium nanoparticle (SeNPs). SeNPs produced had poor high metabolically active cells and with intact cell morphology. Che-
colloidal stability, but in presence of −OH, −COOH and sulfate groups mical modification of κ-CRG hydrogels with methacrylate groups upon
found in CRG, they achieved excellent stability. Cell viability of λ-CRG/ UV irradiation formed covalently crosslinked networks. Fabrication of
SeNPs hydrogel when cultured with osteoblast D1 cells showed little to nanosilicates incorporated gradient hydrogel containing GelMA and
no cytotoxic effect. Qualitative analysis of calcium deposits on D1 cells Mκ-CRG possessed multifunctional application in tissue engineering.
using alizarin red staining resulted in good biomineralization (Kim, Lee, Cells seeded at the Mκ-CRG and GelMA region displayed rounded and
& Lee, 2016). Composite scaffolds fabricated using ɩ-CRG, chitosan, and spreaded morphology, a characteristic appearance of chondrocyte and
gelatin showed osteogenic differentiation and survival of adipose de- osteoblast cells, respectively (Fuss, Ehlers, Russlies, Rohwedel, &
rived mesenchymal stem cells (AdMSCs). Presence of various functional Behrens, 2000). Thus, gradient hydrogel could be employed for the
groups like −OH, −COOH, -NH2, and -SO3H in ɩ-CRG/chitosan/gelatin regeneration of bone-cartilage interfacial tissues (Cross et al., 2017).
scaffolds favoured survival and osteogenic differentiation of AdMSCs Furthermore Mκ-CRG with shear thinning behaviour reinforced with 2D
(Li et al., 2015). Wet spinning of CRG to produce fibers, subsequent nanosilicates had improved physiological stability and mechanical
coating with chitosan enhanced the stability of crosslinked CRG fibers. property (Thakur et al., 2016). Human mesenchymal stem cells
Encapsulation of microvascular like endothelial cells within the chit- (hMSCs) encapsulated shear thinning κ-CRG hydrogels showed no toxic
osan coated κ-CRG hydrogel were not only viable for longer time, but effects and maintained the morphology of cell, making it an attractive
also preserved its functionality and phenotype (Mihaila, Popa, Reis, nanoengineered system for regeneration of soft tissues.
Marques, & Gomes, 2014). Nerve growth factor (NGF) is proven to be
crucial for regeneration, maintenance and development of nerves, 3.2. Wound healing
though recurring local injection of NGF to rabbit model with man-
dibular distraction osteogenesis is necessary to accelerate the recovery A plethora of wound dressing materials such as traditional gauzes,

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bandages, cotton wools have been studied upon, with the primary negative than the gram positive bacteria (Prabhu, Venkateswara Rao,
purpose to keep the wounds dry. However, it has been proven that Sesha Sai, & Pavani, 2017).
healing occurs faster in wet/moist environment than in dry environ-
ment (Boateng, Matthews, Stevens, & Eccleston, 2008; Winter, 1962). 3.3. Drug delivery
Hydrogels have been extensively studied as a wound dressing material
because of their high water holding capacity and biocompatibility that Thermoreversible gelation, biocompatibility, tunable viscoelastic
promotes cell migration and re-epithelialization (Kamoun, Kenawy, & properties and simple gelation mechanism makes CRG an ideal polymer
Chen, 2017). Cyclic β- (1–3) (1–6) glucan (CBG) incorporated in ɩ-CRG for controlled drug delivery applications (Keppeler, Ellis, & Jacquier,
hydrogel facilitated accelerated wound healing in vitro and in vivo (Nair, 2009; Santo et al., 2009). Epichlorohydrine crosslinked L-carnosine
Raman, & Doble, 2016). Due to their immunomodulatory properties, incorporated κ-CRG/hyaluronic acid (κ-CRG-HA) was fabricated as
CBG/ɩ-CRG hydrogel displayed superior fibroblast cell migration hydrogel membrane and their drug release kinetics were evaluated (El-
mediated wound healing. Ciprofloxacin incorporated CBG/ɩ-CRG hy- Aassar, El Fawal, Kamoun, & Fouda, 2015). Increased use of crosslinker
drogel was evaluated for antibacterial activity against S. aureus. CBG to exhibited decreased swelling of the hydrogel membrane and rapid
CRG ratio influenced the drug release profile and fibroblast cell mi- mechanical deterioration of hydrogel network. As CRG content de-
gration. Synthetic polyox CRG blends loaded with antibacterial and creases, L-carnosine incorporated κ-CRG-HA membrane showed zero-
anti-inflammatory drug showed promising effect on the healing of order release kinetics due to the improved hydrophilicity of the mem-
chronic wounds (Boateng, Pawar, & Tetteh, 2013). Streptomycin in- brane surface. Quercetin, a flavonoid is shown to possess anti-in-
corporation into the polyox/κ-CRG composite film prevented the flammatory and antioxidant property in vivo and in vitro in various
wound infection in vitro and diclofenac relieved the pain and in- mammalian cell types (Pérez-Mateos, Bravo, Goya, Gómez-Guillén, &
flammation. Gamma irradiated poly(vinylpyrrolidone) (PVP)/κ-CRG Montero, 2005). Different ratios of gelatin-ɩ-CRG (GC) composite hy-
based hydrogel have been extensively explored as a wound dressing drogel were developed and effect of porosity and pore size distribution
material owing to simultaneous sterilization and hydrogel formation in in release of quercetin was studied. As the ratio of CRG was increased,
single step with no use of initiator or crosslinker (Şen & Avci, 2005; De hydrogel became more porous. GC with equal ratio showed release
Silva, Hettiarachchi, Nayanajith, Milani, & Motha, 2011). To overcome pattern similar to gelatin alone, but when CRG ratio was increased,
the drawback of poor mechanical strength, silk was incorporated into higher amounts of quercetin was released, thereby directly relating to
gamma irradiated PVA/PVP/κ-CRG hydrogel (Wu, Bao, Yoshii, & its porosity (Varghese, Chellappa, & Fathima, 2014). κ-CRG beads
Makuuchi, 2001). Long shelf life PVP/κ-CRG/poly(ethylene glycol) crosslinked with KCl were synthesized for sustained delivery of angio-
hydrogel dressing are more efficient when compared to commercially genic growth factor that enhance vascularization in the defect regions
available wound dressings as they can be easily removed without (Santo et al., 2009). Encapsulation of platelet derived growth factor
breaking into pieces and have high tensile strength, thus increasing (PDGF) in κ-CRG hydrogel bead showed sustained release profile by
patient compliance (De Silva et al., 2011). κ-CRG reinforced 2D nano- varying the hardening time. Longer the hardening time, slower the
silicates have been developed by simple mixing of two components at release, thus, makes it an appropriate carrier system for delivery of
different ratios. The incorporation of nanosilicates not only enhanced bioactive molecules.
the mechanical property, injectability and stability of the hydrogel, but Hydrogels incorporated with gold nanoparticles (AuNPs) have been
also provided room for the delivery of therapeutics in a sustained used in a variety of applications ranging from light responsive glucose
manner, possessed better protein adsorption, cell adhesion, migration sensing (Lim, Lee, & Park, 2010) to light initiated bioadhesives
and platelet binding, thereby acting as both hemostatic and wound (Matteini et al., 2010) and drug delivery nanocarriers (Choi et al.,
healing (Lokhande et al., 2018). 2011). Incorporation of AuNPs increased the elastic modulus of κ-CRG
Hydrogels act as physical barriers to microbial attack and accel- hydrogel because of the negative surface charge of AuNPs that attracts
erates the wound healing process. Nano-silver based hydrogels have K+ ions on to the surface, resulting in increased counterions sur-
proved to be very effective in terms of managing the wound infections rounding the nanocarrier. Strong interaction of methylene blue (MB)
(Azizi, Mohamad, Abdul Rahim, Mohammadinejad, & Bin Ariff, 2017; with AuNPs due to surface chemisorption resulted in controlled release
Jayaramudu et al., 2013; Singh, Singh, & Singh, 2015). Seed extract of MB from AuNPs incorporated in the κ-CRG hydrogel (Salgueiro,
from Citrullus colocynthis were used to synthesise silver nanoparticle Daniel-Da-Silva, Fateixa, & Trindade, 2013). There is an emerging trend
(AgNPs). Incorporation of these AgNPs into κ-CRG hydrogel exhibited in the use of carbon nanotubes (CNTs) in the field of nanomedicine and
toxicity towards both gram positive and gram negative bacteria (Azizi biotechnology in past few years (Kostarelos, Bianco, & Prato, 2009;
et al., 2017). Simple and eco-friendly method of green synthesis for Vashist et al., 2011). Near infrared light (NIR) and temperature re-
generation of AgNPs from C.colocynthis paves way for the new possi- sponsive multiwalled CNTs reinforced κ-CRG hydrogel composites have
bilities in AgNPs formulation. The seed extract of C.colocynthis contains been developed for controlled drug release. A concentration dependent
several phytochemicals such as flavonoids, alkaloids, and glycosides enhancement in the mechanical property of CRG hydrogel was ob-
that facilitates the antimicrobial activity of the CRG hydrogel (Dallak served. Upon NIR exposure, faster release of methylene blue was ob-
et al., 2009; Hediat, 2012). Radiation induced synthesis of AgNPs served due to gel-to-sol transition of MWCNTs-CRG hydrogel. At phy-
within PVP/κ-CRG matrices prevented the non-homogeneous distribu- siological temperature without NIR exposure, drug release was slow
tion of nanoparticles and also demonstrated the microbicidal activity of and sustained when compared to control hydrogel without MWCNTs
hydrogel against wound pathogens (Singh et al., 2015). Similarly, green (Estrada, Daniel-da-Silva, & Trindade, 2013).
synthesized AgNPs from leaf extract of Azadirachta indica, when re- κ-CRG blended with CMC were fabricated into beads by conven-
leased from biodegradable CRG hydrogel, attached to the negatively tional dripping method (Hezaveh, Muhamad, Noshadi, Shu Fen, &
charged bacterial cell wall thereby inactivating and killing the bacteria Ngadi, 2012). β-carotene loaded beads crosslinked with genipin, re-
(Jayaramudu et al., 2013). Other metallic nanoparticles, like zinc oxide sulted in slower release of the compound than the native beads. It was
(ZnO-NPs) and Copper oxide (CuO-NPs) have demonstrated promising observed that minimal use of crosslinker resulted in reduced density of
antibacterial activity (Kanmani & Rhim, 2014; Shankar & Rhim, 2014; polymer network, thereby causing more drug to be released into the
Shankar, Teng, Li, & Rhim, 2015). κ-CRG hydrogel with multifunctional surrounding medium. Chitosan dispersed in lactic acid possess en-
properties have been developed with the use of ZnO-NPs and CuO-NPs hanced mucoadhesion and permeation property (Kok-Khiang, Khan, &
as nanofillers (Oun & Rhim, 2017). Due to structural variation in the Ch’ng, 1999). Ionotropic gelation method was adopted to fabricate
bacterial cell wall, CRG hydrogel with cationic surface charged metallic composite hydrogel system by dropwise addition of chitosan dispersion
nanoparticles had more effective antibacterial activity with gram into ɩ-CRG solution. Rheological characterization of this polyelectrolyte

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complex exhibited adhesive property, good spreadability, thixotropic completely clear off the infection whereas CM-ɩ-CRG-Amp B-GNP
behaviour and cohesiveness, making it an appropriate system for treated cells were more effective in eradicating the infection (Aparna
transdermal application. Tramadol hydrochloride entrapped within et al., 2017). Overall, these results suggest that the CRG can be used in
PEC showed delayed release due to strong ionic interaction between targeted drug delivery systems.
chitosan and ɩ-CRG because of the presence of sulfate groups (Kamel &
Abbas, 2013). Curcumin loaded chitosan nanoparticles were synthe- 3.3.2. Oral
sized by ionotropic gelation method to enhance the stability and aqu- Though oral route of administration is most commonly used and has
eous solubility of curcumin. Then alginate-κ-CRG microparticles were high patient compliance, it is necessary to formulate the delivery
prepared by simple blending of alginate with CRG in the aqueous system with utmost care to ensure its stability at extreme pH conditions
mixture containing calcium and potassium ions. Curcumin loaded across the gastrointestinal tract. CRG is widely used in various food
chitosan nanoparticles were then mixed with alginate-κ-CRG micro- applications as a stabilizer, food additives and for microencapsulation
particles and sustained release of curcumin from nano-microparticulate of probiotics (Cheow & Hadinoto, 2013; Cohen & Ito, 2002; Karbowiak
system was achieved (Guzman-Villanueva, El-Sherbiny, Herrera-Ruiz, & et al., 2006). Lactobacillus plantarum, a probiotic gram positive bac-
Smyth, 2013). terium was encapsulated into carboxymethyl cellulose-κ-CRG (CMC/κ-
Stimuli responsive hydrogels are attractive candidate for drug de- CRG) blends crosslinked with calcium and potassium ions, respectively.
livery applications. Alginate and κ-CRG coated iron oxide nanoparticles Being extremely sensitive to acidic pH, encapsulation of L. plantarum in
were used as beads for targeted delivery of riboflavin to colon CMC/κ-CRG blends improved the survival in simulated gastrointestinal
(Mahdavinia, Rahmani, Karami, & Pourjavadi, 2014). Release obtained fluid (SIF). The κ-CRG content in blends made the network denser and
at acidic pH of 1.2 was very much lower than that of release in phy- thus prevented the diffusion of acid inside the matrix. Release of pro-
siological pH 7.4, protecting the majority of drug from degradation in biotic bacteria from CMC/κ-CRG was directly proportional to swelling
acidic environment. This was in direct correlation with swelling ratio and erosion of matrix at SIF and more rapid release was observed at pH
where at acidic pH swelling was minimum compared to that in phy- 7.4. Hence this system may be used as an oral delivery vehicle for colon
siological pH, concluding that drug release is due to diffusion and (Dafe, Etemadi, Zarredar, & Mahdavinia, 2017). Other probiotic gram
polymer relaxation mechanism. Similarly, temperature, magnetic and positive bacteria, Lactobacillus rhamnosus that exists as biofilm in the
pH responsive beads made of carboxymethyl chitosan and κ-CRG gut lining was encapsulated into κ-CRG microspheres. Due to the in-
loaded with diclofenac sodium and anti-cancer drug methotrexate, teraction of L. rhamnosus biofilm with κ-CRG, enhanced thermo-
showed extended release profile (Mahdavinia, Etemadi, & Soleymani, tolerance was achieved and owing to the cryoprotective nature of CRG,
2015; Mahdavinia, Mosallanezhad, Soleymani, & Sabzi, 2017; decreased resistance to freeze drying was observed (Cheow & Hadinoto,
Piyakulawat, Praphairaksit, Chantarasiri, & Muangsin, 2007). A water 2013).
soluble model drug, betamethasone showed swelling dependent loading Use of clay nanoparticles in biomedical application is increasingly
and release from alginate-CRG hydrogel beads (Mohamadnia, gaining attention due to its mechanical and thermal stability, high
Zohuriaan-Mehr, Kabiri, Jamshidi, & Mobedi, 2007). The swelling ef- surface area and drug loading capabilities (Depan, Kumar, & Singh,
fect at basic pH condition was due to repulsive force that exerted be- 2009; Ruiz-Hitzky et al., 2013). Halloysite nanotubes (HNTs) were in-
tween the sulfate and carboxylate groups, whilst it remained deproto- corporated into κ-CRG hydrogel to enhance their mechanical property,
nated at acidic pH. Similarly, during drug release polymer network drug release pattern, thermal stability and biocompatibility. Enhanced
remained contracted at acidic pH and led to minimal drug release while mechanical and thermal stability via interaction between polymer and
relaxation of hydrogel network at physiological pH resulted in max- HNTs was postulated due to the entrapment of CRG products within the
imum drug release. Overall, these results suggest that the electrostatic lumen of HNTs. Swelling was more in HNTs incorporated hydrogel
interaction, and relaxation and contraction of polymeric network con- compared to control hydrogel due to the viscoelastic nature of CRG and
trol the amount of drug release from CRG hydrogels. surface charge of HNTs. Besides swelling, charge of the drug molecule
also influenced the release rate. Cationic dye, rhodamine B (RB) and
3.3.1. Antifungal anionic dye, orange G (OG) were chosen as model drug for en-
Over the past few years, occurrence of fungal infections have dou- capsulation into HNTs. Negatively charged anionic drug, OG was in-
bled owing to numerous factors like chronic use of glucocorticoids, corporated via electrostatic interaction, whereas cationic drug RB ex-
immunosuppressive and antimycotic therapies, and im- perienced electrostatic repulsion with HNTs leading to its faster release
munocompromised patients (Perea & Patterson, 2002). At present, when compared to OG. Thus, addition of HNTs enhanced the drug
fungal infections caused by Candida species accounts for 38% of mor- loading efficiency and release profile influenced by electrostatic re-
tality rate since it evades immune system by residing inside the mac- pulsion (Sharifzadeh, Wahit, Soheilmoghaddam, Whye, & Pasbakhsh,
rophages (Fidel, Vazquez, & Sobel, 1999). Amphotericin B (Amp B), a 2016). Low-density lipoprotein (LDL) complexed κ-CRG was formulated
commonly used antifungal agent employed against Candida infections, as nanogels for oral delivery applications. Curcumin as a model drug
but its use is severely restricted due to nephrotoxicity and renal im- was loaded into LDL/κ-CRG nanogels via hydrophobic interaction and
pairment (Saliba & Dupont, 2008). CRG possesses high affinity for cy- nanogels showed excellent stability in gastric and intestinal environ-
steine rich domain (CysD) in mannose receptor present on the macro- ment. LDL/κ-CRG nanogels showed slower release kinetics and sus-
phages and dendritic cells (Cunha & Grenha, 2016). To target and tained release in both gastric and intestinal condition, making them a
deliver Amp B specific to macrophages, carboxymethylated ɩ-CRG (CM- promising nanodelivery system for oral route of administration (Zhou
ɩ-CRG) conjugated to gelatin nanoparticles (GNP) loaded with Amp B et al., 2016).
was developed (Aparna et al., 2017). Computational docking and mo- Metallic and metal oxide nanoparticles with high surface area to
lecular dynamic simulations were performed to study the receptor-li- volume ratio such as silver and magnesium oxide (MgO) NPs have been
gand interactions. Amp B loaded GNP was conjugated to CM-ɩ-CRG via comprehensively studied in biomedical applications because of their
EDC-NHS chemistry. Due to relaxation of gelatin network, sustained unique physiochemical properties (Hezaveh & Muhamad, 2012a;
release of Amp B was achieved. Further, molecular dynamic simulations Nagireddy et al., 2011; Ohira & Yamamoto, 2012). κ-CRG hydrogels
confirmed that the drug release was due to swelling of gelatin polymer upon attaining the equilibrium swelling ratio were immersed in silver
that resulted in drug diffusion. Rapid uptake of CM-ɩ-CRG conjugated nitrate solution to load silver ions and were reduced with sodium
GNPs than bare GNPs by RAW 264.7 macrophage cells revealed the role borohydride to produce AgNPs. κ-CRG was blended with CMC to en-
of CysD in enhanced cellular uptake of CM-ɩ-CRG conjugation. C. hance its swelling capacity, so as to load maximum number of ions
glabrata infected macrophage cells treated with Amp B-GNP did not inside the hydrogel for in situ synthesis of metallic NPs. Drug release

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profile was studied in simulated gastrointestinal condition with me- 3.3.5. Vaginal
thylene blue (MB) as model drug. Increase in AgNPs in hydrogel Polymeric biomaterials are widely being explored for its use in va-
showed increased release of MB because of the increasing osmotic ginal drug delivery system in recent years (Ghosal, Ranjan, & Bhowmik,
pressure due to the continuous influx of water. Upon crosslinking, 2014; Rohan & Sassi, 2009). Due to the ease of handling, storage, im-
higher concentration of genipin resulted in smaller NPs size and sub- proved drug stability, and enhanced retention time, vaginal films are
sequent release in acidic environment was minimal due to insufficient more advantageous than conventional vaginal dosage forms (Gong
swelling when compared to intestinal condition (Hezaveh & Muhamad, et al., 2017; Ham et al., 2012). PVA films blended with λ-CRG have
2012b). Similarly, metal oxide (MgO) NPs served as reservoir for MB been formulated along with the addition of different plasticizers.
incorporation in κ-CRG-NaCMC hydrogel and displayed higher MB re- Fluorescent PLGA nanoparticles incorporated composite films with
lease than control hydrogel (Hezaveh & Muhamad, 2012a). Thus, in- varying ratios of film formulation showed dynamic changes in me-
corporation of metal and metal oxide NPs in CRG hydrogel could offer chanical properties, and nanoparticle release. It was observed that
controlled drug release profile in gastrointestinal environment. presence of nanoparticle did not alter the property of film, but con-
centration of plasticizer altered the mechanical property. Higher per-
3.3.3. Nasal centage films (i.e. 5%) were less flexible and more rigid when compared
Improved patient compliance, high permeability of nasal epithelium to lower, 2% films. Hence lower percentage film formulations were
and ability to evade first pass metabolism has gained nasal drug de- ideal for use in vaginal drug delivery due to its flexibility, rate of na-
livery system great attention (Touitou & Illum, 2013). Rapid clearance noparticle release and film disintegration (Traore, Fumakia, Gu, & Ho,
of nasal delivered solutions (Guastella et al., 2013), incomplete wetting 2017). Owing to its mucoadhesive nature, chitosan and κ-CRG have
of nasal inserts (Bertram & Bodmeier, 2006), low scalability of micro been used in vaginal drug delivery systems. In situ forming thermo-
and nanoparticles and insufficient drug loading and release (Abdel sensitive hydrogel made of κ-CRG and poloxamer 407 was fabricated
Mouez, Zaki, Mansour, & Geneidi, 2014; Kürti et al., 2013) are major for vaginal delivery. Significant reduction in the release rate of model
setbacks to translation of nasal drug delivery systems. In situ hydrogel drug, acyclovir was observed in a concentration dependant manner due
systems have been developed based on poloxamer-κ-CRG for intranasal to the presence of CRG (Liu, Zhu, Wei, & Lu, 2009). Carbopol, a mu-
delivery where the combination remained as liquid during injection/ coadhesive material beside CRG, prolonged the vaginal residence time
instillation and gelled in situ. Since, poloxamer hydrogel (PH) degrades in mice after being administered with fluorescent-tagged formulation.
faster in aqueous medium, κ-CRG was blended with poloxamer (CPH) to Overall, a good safety profile was observed with no sign of injury,
form in situ gel. Ketorolac tromethamine, an analgesic anti-in- edema or inflammation (Li, Han et al., 2014).
flammatory drug was incorporated by simple dissolving in CPH. Drug CRG has also been used as microbicides to prevent Herpes Simplex
release from CPH was comparatively slower when compared to control Virus (HSV), HIV and papilloma virus infection (Buck et al., 2006;
PH and no nasal ciliotoxicity was observed (Li, Li et al., 2014). Hence, Skoler-Karpoff et al., 2008). Acyclovir, an antiviral drug incorporated
concentration of potassium ions plays a crucial role in erosion of hy- into κ-CRG – hydroxyl-propyl-methyl-cellulose (HPMC) composite hy-
drogel; higher the concentration of K+ ions, slower the erosion rate. drogel displayed prolonged drug release for over a period of 7 days. The
These results demonstrate that CRG provides stability, enhanced drug ratio of κ-CRG to HPMC was crucial for sustained release. Residence
retention and sustained drug release upon blending with poloxamer, time of vaginal mucoadhesive CRG was longer when compared to
and thus, it could be further explored as a new platform for intranasal chitosan and intermediate swelling was observed when combined with
delivery of therapeutics. HPMC, enhancing its patient compliance. This hydrogel system was
biocompatible and might be beneficial in the prevention of genital
3.3.4. Ophthalmic herpes infection (Sánchez-Sánchez et al., 2015). Small interfering RNA
In recent times, stimuli sensitive hydrogels are widely being em- loaded nanoparticles (siRNA-NP) surface coated with anti-HLA den-
ployed in ocular and ophthalmic formulations (Kirchhof, Goepferich, & dritic cell antibody was homogenously incorporated into biodegradable
Brandl, 2015; Lihong, Xin, Yongxue, Yiying, & Gang, 2014). Based on film made of PVA and λ-CRG to target the vaginal immune cells. A
ionic interaction between different biopolymers, in situ gelling system significant reduction in protein expression was observed when SNAP-23
was developed using alginate, κ-CRG and gellan gum at different pro- synaptosome associated 23 kDa protein was targeted with siRNA-NPs
portions (Fernández-Ferreiro et al., 2015). Combination of 20% κ-CRG that resulted in disruption of the SNARE machinery necessary for the
with 80% gellan gum showed increased in vivo corneal retention time. formation of HIV-1 virus particles (Gu, Yang, & Ho, 2015). Vaginal
κ-CRG-gellan gum hydrogel was labelled with fluorine radiotracers suppositories made of κ-CRG were incorporated with tenofovir, an an-
(18F) such as 18F-NaF and 18F-FDG and monitored using positron tiretroviral drug for the prevention of HSV-2 and HIV transmission.
emission tomography/computed tomography (PET/CT), which re- Release study in vaginal simulated fluid revealed that entire drug was
vealed a prolonged corneal contact time when compared to aqueous released via diffusion mechanism into the surrounding environment
solution (Fernández-Ferreiro et al., 2017a). However, quantifying the within 24 h irrespective of the suppository’s size and volume (Zaveri,
clearance of hydrogel from ocular surface was difficult with the use of Hayes, & Ziegler, 2014).
techniques like PET/CT and MRI. The drawback was circumvented by
using scintigraphy, which monitored the physiological pathway of tear 3.3.6. Transdermal
secretion system and drainage (Mansour, Blanksma, Vrakking, & Jager, Transdermal delivery of drugs is another route that sidesteps first
2008). Evaluation was done using technetium pentetate (99mTc-DTPA) pass metabolism and is independent of pH and enzymatic degradation.
radiotracer which showed that the clearance of hydrogel followed ex- Transdermal patch, iontophoresis are few ways in which drugs can be
ponential one-phase decay kinetics (Fernández-Ferreiro et al., 2017b). delivered via transdermal route (Dixit, Bali, Baboota, Ahuja, & Ali,
Thus, using trace amounts of radioactive elements in ophthalmic for- 2007; Schoellhammer, Blankschtein, & Langer, 2014; Wiedersberg &
mulations pose no threat for ocular pharmacokinetic evaluations. Sol- Guy, 2014). Due to lipophilic nature of skin, it is not possible to in-
gel transition of methylcellulose (MC) happens at around 60 °C, which corporate hydrophilic drugs via transdermal patch. Polythiophene
can be further reduced by addition of salts and other additives (PTh), an excellent electroconductive polymer was mixed with κ-CRG
(Bhowmik, Bain, Ghosh, & Chattopadhyay, 2011). The addition of CRG and developed as hydrogel blends. Acetylsalicylic acid (ASA), a model
into MC solution affected the interaction between −OH group of MC, drug was incorporated on to PTh blended κ-CRG hydrogel. Due to
and reduced the sol-gel transition to physiological temperature crosslinking ratio effect, increase in concentration of crosslinking agent
(Bhowmick et al., 2015). Thus, in situ gel forming eye drops based on decreases the hydrogel mesh size. Using smaller ions as crosslinking
CRG could potentially replace the conventional ophthalmic solutions. agent resulted in decreased drug release profile than larger ions

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Table 1
Overview of applications of carrageenan composites in various drug delivery routes with encapsulation of different compounds and various crosslinking mechanisms.
Application Polymer composite Crosslinking method Compounds encapsulated Reference

Anti fungal delivery Carboxymethylated ɩ-CRG/gelatin NPs Ionic Amphotericin B Aparna et al. (2017)
Oral delivery κ-CRG/halloysite nanotube Thermoreversible Rhodamine B, Orange G Sharifzadeh et al. (2016)
LDL/κ-CRG Ionic Curcumin Zhou et al. (2016)
AgNPs/κ-CRG-CMC Chemical Methylene blue Hezaveh and Muhamad (2012b)
MgONPs/κ-CRG-NaCMC Chemical Methylene blue Hezaveh and Muhamad (2012a)
Intranasal delivery Poloxamer-κ-CRG Thermoreversible Ketorolac tromethamine Li, Li et al. (2014)
Ophthalmic delivery κ-CRG-gellan gum Ionic Fluorine radiotracers Fernández-Ferreiro et al. (2017a, 2017b)
CRG-methylcellulose Ionic Pilocarpine hydrochloride Bhowmick et al. (2015)
Vaginal delivery PVA-λ-CRG Thermoreversible Fluorescent PLGA NPs Traore et al. (2017)
κ-CRG-poloxamer Thermoreversible Acyclovir Liu, Zhu, Wei, and Lu, (2009)
κ-CRG – hydroxyl-propyl-methyl-cellulose Ionic Acyclovir Sánchez-Sánchez et al. (2015)
PVA-λ-CRG Thermoreversible SiRNA Gu et al. (2015)
κ-CRG Ionic Tenofovir Zaveri et al. (2014)
Transdermal delivery Polythiophene/κ-CRG Ionic Acetylsalicylic acid Pairatwachapun et al. (2016)
CRG/guar gum/locust bean gum Ionic Tocotrienol rich palm based vitamin E Mei Yee (2016)
Protein delivery Alginate-κ-CRG Ionic Insulin Lim et al. (2017)
κ-CRG-acrylic acid Chemical Insulin Rasool et al. (2010)
Chitosan-κ-CRG Ionic Human recombinant erythropoietin Bulmer et al. (2012)
FeNPs-κ-CRG Thermoreversible Bovine serum albumin Long et al. (2015)

(Ba2+ > Ca2+ > Mg2+). Upon electrical stimulation, a larger ion re- Mashru, Sankalia, & Sutariya, 2006). Human recombinant ery-
laxes the CRG network and facilitates drug diffusion. Electrical stimu- thropoietin (rHU-EPO) was encapsulated into chitosan-κ-CRG (CS/κ-
lation has a drastic effect on CRG microstructure, hence increase in CRG) nanoparticles by simple ionotropic gelation method, for con-
electrical stimulation leads to increased drug release. Thus, presence of trolled release behaviour (Bulmer, Margaritis, & Xenocostas, 2012).
PTh and molecular weight of ions influenced the transdermal delivery Increase in surface charge increased encapsulation efficiency and in-
rate of drugs (Pairatwachapun, Paradee, & Sirivat, 2016). Tocotrienol crease in molecular weight of chitosan in CS/κ-CRG nanoparticles de-
rich palm based vitamin E (TRPE), a hydrophobic compound is widely creased burst release due to multiple bonding interactions between
used in cosmetics owing to its ability to protect skin from harmful UV rHU-EPO and chitosan. Iron oxide nanoparticles (Fe-NPs) prepared by
rays (Budin et al., 2009). TRPE being a hydrophobic compound, semi- reverse phase microemulsion method was surface coated with carbox-
interpenetrating network with hydrophilic polymer was formed in oil- ymethylated CRG via thermally induced gelation. The versatility of
in-water emulsion with the help of surfactants. CRG, guar gum and conjugating IgG antibody via EDC-NHS conjugation chemistry to the
locust bean gum premixed with glycerine was dissolved at high tem- surface functionalized magnetic nanospheres proves to be a convincing
perature to form a uniform gel. TRPE was mixed with the help of so- approach in fabricating nanodelivery systems. In addition, Fe-NPs were
lubiliser, PEG-40 hydrogenated castor oil and preservatives such as surface coated with κ-CRG and used as magnetite carriers for protein
potassium citrate. Increase in concentration of guar gum and potassium delivery application. Since κ-CRG enhanced the viscosity, surface
citrate, increased the gel strength and decreased flexibility of gel, coated nanoparticles possessed weak magnetic property. To overcome
whereas increase in glycerine increased the flexibility. Improved this limitation, chitosan was further coated on to the κ-CRG nano-
emulsification property of PEG-40 hydrogenated castor oil enhanced composite thereby neutralizing the surface charge of free sulfate groups
permeation. Thus, gels were biocompatible, non-irritant and possessed by electrostatic interaction, thus making chitosan coated κ-CRG nano-
good flexibility, strength and improved skin permeation (Mei Yee, composite a promising strategy for protein delivery application with
2016). better magnetic property (Long et al., 2015). Table 1 represents the
overview of applications of carrageenan composites in different routes
of drug delivery.
3.3.7. Protein
Hydrogels as an encapsulation system is a promising strategy to
deliver proteins, growth factors and other therapeutic agents as they 4. Conclusion and future perspective
provide structural support for adhesion, immunoprotection and allows
diffusion of nutrients, oxygen and metabolic wastes (Jen, Wake, & In summary, application of CRG in drug delivery, tissue engineering
Mikos, 1996; Saul & Williams, 2013). Alginate hydrogel bead re- and regenerative medicine is rapidly evolving due to its distinctive
inforced with κ-CRG was used for oral delivery of insulin. The presence gelling mechanism, ample functional groups, strong water absorption
of sulfate groups led to strong ionization degree with low pKa value and favourable physiochemical properties. By adopting variety of fab-
(≈2) facilitating retention of encapsulated insulin within the hydrogel rication strategies to develop CRG based hydrogels, desired therapeutic
under acidic environment (Lim, Ooi, Tey, & Chan, 2017). Once it potential could be achieved. Development of photocrosslinking hydro-
reached the intestine (pH 6.8–7.4), insulin was gradually released fol- gels facilitated to overcome the physiological instability of CRG hy-
lowing zero-order reaction kinetics. Ionic strength, pH and temperature drogels. Floating hydrogel stays afloat and releases drug in a sustained
sensitive hydrogels with different ratios of κ-CRG and acrylic acid with manner thereby it overcomes the limitation faced by conventional oral
vinyltriethoxysilane as crosslinker were fabricated for application in drug delivery systems where drug gets degraded and eliminated due to
insulin delivery. Owing to excellent mucoadhesive property of acrylic rapid gastric emptying time. CRG nanogels possess promising applica-
acid and κ-CRG, increased surface area for protein adsorption and in- tion in drug delivery owing to its high surface area and drug payload.
sulin diffusion across the intestinal wall was achieved (Rasool, Yasin, Use of two different polymers and their ratio played a major role in
Heng, & Akhter, 2010). Surface functionalized magnetic carbox- developing IPNs made of CRG that makes them a suitable biomaterial
ymethylated nanospheres were used for conjugation of bioactive mo- for tissue engineering applications. These CRG hydrogels fabricated in
lecules and antibodies (Daniel-Da-Silva et al., 2009). Protein entrap- different forms are not only excellent excipients for sustained drug re-
ment in ionic crosslinked hydrogel have shown to increase the stability lease but they also find application in protein and growth factor de-
of proteins and reduce the interference with excipients (Sankalia, livery, bone and cartilage tissue regeneration as well as in wound

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healing and antimicrobial formulations. gelation and drug release profiles of methylcellulose-based ophthalmic thermo-re-
Reports on toxicity of CRG are a major limitation that needs to be versible in situ gels. Pharmaceutical Development and Technology, 16(4), 385–391.
Boateng, J. S., Matthews, K. H., Stevens, H. N. E., & Eccleston, G. M. (2008). Wound
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Department of Science and Technology (DST), Government of India
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under the ‘M.Tech’ program (Ref. No. SR/NM/PG-01/2015).A. semi-IPN hydrogel. Materials Chemistry and Physics, 115(1), 339–346.
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Research (CSIR) for the financial support through Senior Research encapsulated in alginate and carrageenan microcapsules exhibiting enhanced ther-
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