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Colloidal chitin nanogels: A plethora of applications under one shell
Accepted Manuscript Title: Colloidal chitin nanogels: A plethora of applications under one shell Author: M. Vishnu Priya M. Sabitha R. Jayakumar PII: DOI: Reference:
S0144-8617(15)00910-8 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.054 CARP 10356
To appear in: Received date: Revised date: Accepted date:
30-7-2015 16-9-2015 18-9-2015
Please cite this article as: Priya, M. V., Sabitha, M., and Jayakumar, R.,Colloidal chitin nanogels: A plethora of applications under one shell, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights
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Colloidal chitin nanogels are highly versatile nanocarriers Chitin nanogels can be prepared by simple regeneration technique Drugs, quantum dots and metallic nanopaticles can be incorporated into nanogels Chitin nanogels have applications in drug delivery, imaging, sensing and therapy
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*Manuscript
Colloidal chitin nanogels: A plethora of applications under one shell M. Vishnu Priyaa, M. Sabithab, R. Jayakumara* a
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical
Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi-682041,
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India b
Amrita School of Pharmacy, Amrita Institute of Medical Sciences and Research Centre,
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Amrita Vishwa Vidyapeetham University, Kochi-682041, India
___________________________ *Corresponding author: Dr. R. Jayakumar E-mail: [email protected] & [email protected] Tel: +91-484-2801234; Fax:+91-484-2802020 1 Page 2 of 39
Abstract Chitin nanogels (CNGs) are a relatively new class of natural polymeric nanomaterials which have a large potential in the field of drug delivery and nanotherapeutics. These nanogels being very biocompatible are non-toxic when internalised by cells. In this review
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various properties, preparation techniques and applications of CNGs have been described. CNGs because of their nano-size possess certain unique properties which enables them
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to be used in a number of biomedical applications. CNGs are prepared by simple
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regeneration technique without using any cross-linkers. Various polymers, drugs and fluorescent dyes can be blended or incorporated or labelled with the chitin hydrogel
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network. Drugs and molecules encapsulated within CNGs can be used for targeted delivery, in vivo monitoring or even for therapeutic purposes. Here various applications of
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CNGs in the field of drug delivery, imaging, sensing and therapeutics have been discussed.
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Keywords: Chitin nanogels; skin penetration; targeted drug delivery; bioimaging;
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therapeutics.
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List of contents 1. Introduction 2. Synthesis of colloidal chitin nanogels by regeneration chemistry 3. Applications of chitin nanogel
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3.1. DrugDelivery systems 3.1.1. Anti-cancer
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3.1.2. Anti-fungal
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3.1.3. Protein 3.2. Imaging & sensing
3.3.1. Antibacterial
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3.3.2. Radiofrequency assisted cancer
5. Future perspectives
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Acknowledgements
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4. Conclusions
References
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3.3. Therapy
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List of abbreviations
5-Fluorouracil
BSA
Bovine Serum Albumin
CNGs
Chitin nanogels
CCNGs
Curcumin loaded Chitin Nanogels
Cystamine
Cys
DNA
Deoxyribonucleic acid
Dox
Doxorubicin
ECM
Extra Cellular Matrix
EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
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Fluconazole Flu
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Glutathione GSH Hyaluronic acid
HDFs
Human dermal fibroblasts
MeOH
Methanol
NHS
N-hydroxysulfosuccinimide
PCL
poly (caprolactone)
PEG
poly (ethylene glycol)
PEI
poly (ethylenimine)
PLA
poly (lactic acid)
PLGA
poly (lactic-co-glycolic) acid
pNIPAM
poly (N- isopropylacrylamide)
QDs
Quantum Dots
RF
Radiofrequency
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SECM
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HA
RNA
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5- FU
Ribonucleic acid Scanning Electrochemical Microscopy
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1. Introduction In the past few decades, medical field has seen considerable amount of advancements, overcoming various health issues and tackling several diseases. Nevertheless these advancements have not come without their share of drawbacks (Allen & Cullis, 2004). With
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the advent of nanotechnology, researchers are now focussing on using nano-tools to overcome the shortcomings of these conventional methods. Some of the current
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challenges in the conventional systems include lack of proper drug delivery systems with
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efficient bioavailability or drug release capability, lack of efficient imaging and sensing technique, lack of targeted delivery systems, etc. (Utreja, Jain & Tiwary, 2010). As an
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attempt to circumvent these issues, scientists have shifted their focus towards polymeric systems of the nanoscale (Chacko, Ventura, Zhuang & Thayumanavan, 2012; Sahoo,
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Dilnawaz & Krishnakumar, 2008; Sivaram, Rajitha, Maya, Jayakumar & Mangalathillam, 2015). Nanomaterials, with their high surface to volume ratio, possess properties very
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different from their bulk counterparts (Maya et al., 2013). Nanomaterials include nanoparticles, nanogels, nanorods, nanospheres, etc.
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Nanogels are hydrogels having size of few hundred nanometres. Hydrogels with a cross-linked polymeric network are considered to be highly biocompatibility due to their high water retaining capability, low surface tension and three dimensional structure similar
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to the native extracellular matrix (ECM) (Gupta, Vermani & Garg, 2002; Maya et al., 2013; Sivashanmugam, Arunkumar, Priya, Nair S.V.& Jayakumar, 2015). Nanogels have been used for the delivery of drugs, imaging labels, metallic nanoparticles, proteins, Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) (Kim et al., 2011), genes (Xu et al., 2006), growth factors (Kobayashi et al., 2012), etc. These agents interact spontaneously with the polymer matrix via covalent, van-der Waals, electrostatic and/or hydrophobic interactions to form stable nanostructures (Jiang, Chen, Deng, Suuronen & Zhong, 2014;
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Kabanov & Vinogradov, 2009). Because of their ability to swell in aqueous environment, they help in the controlled release of these molecules at the target site (Peppas, Hilt, Khademhosseini& Langer, 2006). Nanogels can further be modified for targeted delivery by chemically conjugating their surface with active moieties for ligand- receptor
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interactions (Garcia, Andrieux, Gil & Couvreur, 2005). Nanogels serve as ideal candidates for the intracellular transport of active moieties as these can be easily taken up by cells
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(Raemdonck, Demeester & De Smedt, 2009). Because of their great affinity towards water
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they are not easily taken up by mononuclear phagocytes and thus can remain in the circulation for a longer time.
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Both synthetic and natural polymers have been used for synthesising nanogels. Polymers like poly (N-isopropylacrylamide) (pNIPAM) (Singh & Lyon, 2007; Zhao et al.,
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2011), poly (ethylene glycol) (PEG) (Lee, Li & Chu, 2006; Mok & Park, 2006), poly(ethylenimine) (PEI) (Ganta et al., 2008; Xu et al., 2006), etc. copolymerised with other
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polymers are being used for fabricating synthetic polymeric nanogels. Natural polymers used for synthesising nanogels include chitosan (Schmitt et al., 2010; Wu, Shen, Banerjee
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& Zhou, 2010), chitin (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011), hyaluronic acid (HA), pullulan (Kobayashi et al., 2012; Kageyama et al., 2008; Morimoto et al., 2012), chondroitin sulphate (Huang et al., 2009; Xi, Zhou & Dai, 2012), etc. Since
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natural polymers degrade into non-toxic products which are naturally present in our body, they are considered to be relatively more biocompatible than synthetic polymers. Chitin is an amino polysaccharide found in the cell walls of fungi, exoskeleton of arthropods such as crustaceans (shrimps, prawns, crabs, etc.) and insects and internal shells of cephalopods such as squid, octopus, etc. (Kumar, 2000; Rinaudo, 2006). The monomeric units of chitin, N-acetyl glucosamine, are same as that of the native ECM. As a result, chitin is biocompatible, biodegradable and non-toxic. Chitin is being processed into
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various forms including hydrogels, nanogels, microparticles, sponges, scaffolds, membranes and nanofibersfor various biomedical applications like tissue engineering and drug delivery (Anitha et al., 2014; Arun Kumar et al., 2015; Pierfrancesco, 2012; Tamura, Furuike, Nair SV & Jayakumar, 2011; Yang, 2011).
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CNGs apart from the above mentioned properties have additional advantages (Fig. 1). CNGs have shown better drug loading capability, controlled release of encapsulated
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drugs, stability of the loaded drug, tuneable design, response to external stimuli, easy
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conjugation with active targeting agents due to large surface area, prolonged circulation time and high stability in aqueous solution (Hamidi, Azadi & Rafiei, 2008; Kabanov &
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Vinogradov, 2009). They can get accumulated at the tumour tissue due to enhanced retention and permeation effect (Maya et al., 2013). Once they enter the cells via endocytic
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pathway, they can release the drugs in the lysosomes or endosomes by pH-controlled hydrolysis (Manchun, Dass & Sriamornsak, 2012) (Fig. 2). Due to these properties, CNGs
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are being used for a wide range of applications (Fig. 3).This review discusses about the
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preparation and application of CNGs in the field of drug delivery, imaging and therapeutics
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Fig. 1. Advantages of colloidal chitin nanogels.
Fig. 2. Chitin nanogels release the drug encapsulated in them in response to external stimuli.
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Fig. 3. Biomedical applications of the prepared colloidal chitin nanogels.
2. Synthesis of colloidal chitin nanogels by regeneration chemistry
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In spite of being highly biocompatible, full potential of chitin has not been explored much mainly due to its insoluble nature. Chitin possesses a high degree of acetylation and
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hydrogen bonding which makes it insoluble in water or in the regular solvents. Harsh solvents like trichloroacetic acid, dichloroacetic acid, lithium thiocyanate, highly polar
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fluorinated solvents like hexafluoroisopropyl alcohol and hexafluoracetonesesquihydrate, inorganic solvents, lithium chloride-tertiary amide solvent systems, etc. have been used in the past to dissolve chitin (Pillai, Paul & Sharma, 2009). In 2006, Tamura et al developed a
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CaCl2-Methanol (MeOH) solvent, which dissolved chitin in a milder environment (Tamura, Nagahama & Tokura, 2006). Calcium chloride dihydrate was dissolved in MeOH to form a supersaturated solution, into which chitin was added to form chitin solution. The solubility of chitin in this solvent depends upon a number of factors such as degree of deacetylation and the molecular weight of chitin, water content of the system and the Ca2+ ion concentration in the CaCl2-MeOH (Tamura, Nagahama & Tokura, 2006). This system serves as a suitable solvent for forming colloidal chitin hydrogel. Chitin is dissolved in
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supersaturated CaCl2-MeOH and is regenerated by adding excess of MeOH which results in the formation of hydrogel. Hydrogel formation occurs as a result of self-assembly of polymeric chains. In this process, hydrophilic polymeric chains capable of hydrophobic or electrostatic interactions aggregate to form a network like structure (Kabanov &
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Vinogradov, 2009). Chitin hydrogel is then washed several times by centrifugation to remove excess solvent. To form gel particles of nano-dimension, hydrogel is dispersed in
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water and subjected to probe sonication at 75% amplitude for 5 minutes after every wash.
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The self-associating property of CNGs has been used extensively to entrap biomolecules in this network, and thus act as excellent drug carriers. CNGs have been
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tagged with dyes such as Rhodamine to monitor their cellular uptake and localization. Such tagged nanogels are prepared by incubating the dye in CNG solution after the final
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wash (Rejinold et al., 2012). Drug loaded CNGs are fabricated either by adding the drug solution to the chitin solution and then regenerating it (Mohammed et al., 2013) or by
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incubating the drug in a CNG solution for a few hours and then subjecting it to a wash to remove the unloaded drug (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013; Jayakumar,
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Nair A., Rejinold, Maya & Nair SV, 2012; Mangalathillam et al., 2013) (Fig. 4). Drugs which are insoluble in aqueous conditions are first dissolved in their suitable solvents and then added drop wise to the chitin solution (Mangalathillam et al., 2012).CNGs have also been
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loaded with proteins; quantum dots (QDs) and metallic nanoparticles. QDs are added directly to a colloidal suspension of CNGs after regeneration, under constant stirring (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011) whereas metallic nanoparticles were added to the chitin solution prior to regeneration (Kumar N.A. et al., 2013; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). Chitin composite nanogels have also been fabricated, wherein chitin is cross-linked or blended with another polymer. Synthetic polymers such as poly (caprolactone) (PCL),
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poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA) were dissolved in their suitable solvents and then added to the chitin solution. This blend was kept under stirring and then regenerated by adding excess of methanol followed by centrifugation and sonication to form nanogels (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013, 2014;
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Rejinold, Biswas, Chellan & Jayakumar, 2014). Polymers such as HA were cross-linked to chitin with the help of EDC and cystamine (Kumar NA., Maya & Jayakumar, 2014). In such
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systems, cystamine is conjugated with HA by EDC/ N-hydroxy sulfosuccinimide (NHS)
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conjugation chemistry. The carboxyl group of HA which is activated by EDC interacts with one of the amine groups of cystamine. The hydroxyl group of chitin interacts with the other
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amine group thus forming a cross-linked network (Kumar NA., Maya & Jayakumar, 2014).
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Fig. 4. Preparation of colloidal chitin nanogel and drug loaded chitin nanogels.
3. Applications of CNGs 3.1. Drug delivery Any system to be used for drug delivery should have good interaction between the system and the molecule which has to be delivered. Nanogel due to its highly porous nature serves as an excellent delivery system. The molecules can be easily encapsulated/ entrapped in these pores. In certain cases, the molecules can even physically adsorb onto 12 Page 13 of 39
the surface of the gel particles. When these nanogel systems are placed in the body, the molecules are slowly released as the gel matrix degrades/disperses. Such systems help in the controlled release of the molecules, thereby reducing the chances of dose-dependent toxicity. CNGs have been used to deliver anti-cancer drugs, anti-fungal drugs and proteins.
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3.1.1. Anti-cancer Conventional anti-cancer drugs are associated with problems like dose-dependent toxicity,
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improper distribution at the tumour site, off-target delivery, toxicity to off-target sites,
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bioaccumulation or quick degradation, etc (Maya et al., 2013). CNGs with their highly versatile nature can be used to overcome most of these issues.
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pH responsive CNGs encapsulated with anti-cancer drugs have been developed. These gels are loaded with various molecules to enable their targeted delivery to the
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specific target site. Colloidal CNGs incorporated with Doxorubicin (Dox) were synthesized with size of around 130-160 nm. They showed a higher drug release at a lower pH of 4.5
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than in neutral pH. These CNGs were taken up by fibroblasts as well as cancer cell lines but showed more toxicity towards cancer cell lines like MCF7, HEPG-2 and A549
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(Jayakumar, Nair A., Rejinold, Maya & Nair, SV., 2012). Similarly Chitin-PLA nanogels loaded with Dox were developed for treating hepatic cell carcinoma. More amount of Dox was found to be adsorbed onto the surface of the nanogel rather than being encapsulated
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within it. Such adsorption seemed to have improved the stability of the colloidal nanogel suspension (Arunraj, Rejinold, Kumar NA & Jayakumar, 2014). This system also showed a better drug release in acidic environment. Dox loaded PLA-CNGs showed better cellular uptake and dose dependent toxicity towards HepG2 cells (Arunraj, Rejinold, Kumar NA & Jayakumar, 2014). Dox loaded PCL-CNGs were synthesized for non-small cell lung cancer which was also pH-responsive and showed dose-dependent toxicity towards A549 cells (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013). The higher drug release in acidic
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pH could be attributed to the higher swelling ratio of CNGs in an acidic environment which was demonstrated (Rejinold et al., 2012). Since cancer cells are known to have an acidic pH, these systems have successfully shown more toxicity towards cancer cell lines. In order to further improve the targeted drug delivery systems, redox responsive
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chitin-HA composite nanogels conjugated using cystamine (HA-Cys-CNGs) were prepared (Kumar NA., Maya & Jayakumar, 2014). These nanogels were loaded with Dox and
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targeted for delivery to colon cancer cells. HA, a ligand for CD-44 receptors which are
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over-expressed in colon cancer cells seemed to facilitate specific internalization of the drug loaded nanogels by HT29 (CD-44 +ve) cells (Fig. 5). These gels showed higher in
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vitro drug release in the presence of glutathione and more toxicity towards CD-44 +ve
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presence of acidic pH and glutathione.
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cells. Such systems can be used for specific targeted delivery to the tumor tissue in the
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Fig. 5. (i) Schematic representation of the possible mechanism by which Dox-HA-Cys-CNGs induce toxicity in CD+ve cells. (ii) Fluorescent images showing the specific uptake of DoxHA-Cys-CNGs by CD+ve and CD-ve cells (B-C: HT29 (CD+ve); D-E: IEC6 (CD-ve)) [A & Dcontrol HT-29 & IEC cells respectively without nangels treatment] (Ashwin et al., 2014).
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Figures reproduced with kind permission from RSC. Apart from Doxorubicin, two other anti-cancer drugs curcumin and 5-fluorouracil (5-
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FU) have been loaded into CNGs for treating skin cancer via transdermal route
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(Mangalathillam et al., 2012). Both chitin and curcumin are highly hydrophobic in nature whereas Curcumin loaded CNGs (CCNGs) showed a good and stable colloidal dispersion
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in water. Cell uptake and cytotoxicity studies were carried out using human dermal fibroblasts (HDFs) and A375 melanoma cells. The melanoma cells showed higher cellular
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uptake of CCNGs compared to HDF cells (Mangalathillam et al., 2012) (Fig. 6(i)). These gels also showed higher toxicity on A 375 melanoma cells compared to human dermal
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fibroblast cells. However the innate toxicity of curcumin on HDF cells, as reported earlier was found to be reduced in the CCNG formulation, due to the antioxidant effect of chitin.
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That means the chitin nanogel formulation was found to retain the cancer specific toxicity of curcumin and at the same time reduced its toxicity on HDF. Ex vivo studies carried out using porcine skin showed higher penetration for curcumin loaded in CNGs (CCNGs) in
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comparison to control curcumin solution. The steady state flux for skin permeation was found to be 4 fold for CCNGs compared to control curcumin solution and retention of CCNGs was found to be more in the deeper layers of epidermis and dermis when compared with control curcumin solution (Fig. 6(ii)). Histopathological evaluations of the skin treated with the samples clearly indicated the loosening as well as fragmentation of stratum corneum in case of the skin treated with CCNGs. 5-FU loaded in CNGs (FCNGs) was also tested for cytotoxicity and uptake on A375 and HDF cells (Mangalathillam et al.,
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2013). Skin permeation studies of FCNGs showed steady state flux similar to 5-FU solution, but 5-FU loaded in CNGs showed 4-5 times higher retention in the deeper layers of the skin than the control 5-FU solution. Curcumin loaded chitin nanogel was found to be more effective for melanoma via the transdermal route because of its specific toxicity and
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enhanced skin penetration, when compared with 5-FU loaded chitin nanogel.
Fig. 6. (i) CCNGs showing enhanced uptake by A375 cells (A); (ii) Fluorescent images
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showing deeper skin penetration by CCNGs (Mangalathillam et al., 2012).Figures reproduced with kind permission from RSC. 3.1.2. Anti-fungal
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Anti- fungal drug delivery to the cornea faces a major issue of poor bioavailability. The presence of various static barriers like different layers of cornea, sclera and retina, dynamic barriers like choroidal and conjunctival blood flow and physiological processes like tear drainage and blinking greatly reduce the residence time of the topically applied drug. This in turn reduces the efficacy of the drug (Urtti, 2006).It is reported that less than 10% of the eye drops permeate and reach the target tissue to achieve effective drug concentration (Diebold & Calonge, 2010). As an attempt to overcome these issues, Fluconazole (Flu) loaded CNGs were developed which showed better deeper tissue 16 Page 17 of 39
penetration for porcine cornea (Mohammed et al., 2013). This system also showed very slow and controlled release of the Flu in neutral pH. Such a release is considered important for anti-fungal treatment, where the drug is required to be present for longer time duration for better efficacy (Mudgil, Gupta, Nagpal & Pawar, 2012). This system showed
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good anti- fungal activity towards Candida tropicalis without causing any toxicity towards HDFs. Hemocompatability studies showed that these nanogels did not interfere with the
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plasma coagulation pathway and further ex vivo studies showed no signs of inflammation
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or destruction to the corneal cells. Cornea treated with Flu loaded CNGs showed loosening and ablation of corneal epithelium, thereby penetrating into deeper layers of
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cornea (Fig. 7). Such a penetration could not be seen in cornea treated with Flu. The cationic charges of Flu loaded CNGs could interact with the corneal glycoproteins, thus
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increasing their permeation. Such an interaction might form a pre-corneal depot, from which the drug can release at a controlled rate for a prolonged time period. Apart from this,
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such systems can also protect the drug from degradation and increase its retention though
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bioadhesion. Thus Flu loaded CNGs can serve as a good antifungal formulation.
Fig. 7. Flu-CNGs showing penetration into deeper layers of cornea (Mohammed et al., 2013). Figures reproduced with kind permission from American Scientific Publishers.
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3.1.3. Protein Besides drugs, CNGs can be used for delivering proteins also. Bovine Serum Albumin (BSA) was used as the model protein to study the protein delivering potential of CNGs (Rejinold, Chennazhi, Tamura, Nair, SV & Jayakumar, 2011). Multifunctional CNGs were
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developed by incorporating BSA in Quantum Dots (QD)-CNGs. BSA was expected to load into CNGs and QD-CNGs through physical adsorption chemistry. Both the systems
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showed high BSA loading. Release study was carried out in acidic as well as neutral pH.
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BSA loaded in CNGs as well as QD-CNGs showed controlled release in neutral pH and higher release in acidic pH. Higher release in acidic pH was attributed to the acidic
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degradation of the nanogel followed by its swelling (Raemdonck, Demeester & De Smedt, 2009). These multifunctional CNGs did not show any haemolytic activity and were non-
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toxic towards L929, VERO, MCF7, KB, PC3 and NIH3T3 cells (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011). These studies show that CNGs can be used for
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3.2. Imaging and sensing
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delivering molecules like drugs, proteins, growth factors, etc. for drug delivery and tissue
Apart from drug delivery systems, CNGs have found application in the field of bioimaging and biosensing as well. Mercaptopropionic acid capped CdTe QDs have been
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incorporated into CNGs via physical adsorption chemistry for imaging purposes (Rejinold, Chennazhi, Tamura, Nair SV. & Rangasamy, 2011). Electrochemical analysis showed that complexing QDs with CNGs did not reduce its electrochemical stability and luminescent properties. These properties are important as they are required for imaging the cells in in vivo experiments. L929, VERO and PC3 cells were stained with QD loaded CNGs and observed under fluorescent microscope. It was seen that the QDs did not undergo complete quenching even after cellular internalization and thus can be used for cell-
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labelling under physiological conditions. The cells did not show any change in their morphology even after 24 hrs of incubation with the QD nanogels. Similarly CdTe/ZnTe-QD-chitin/PLGA composite nanogels and umbelliferone labelled chitin/PLGA-composite-nanogels have been reported to be taken up by microbes
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like Escherichia coli, Staphylococcus aureus and Candida albicans and thus could be used for microbial monitoring (Rejinold, Biswas, Chellan & Jayakumar, 2014). These nanogels
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did not show any toxicity towards HDFs. Although semiconductor QDs has been
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previously used for cellular imaging, they were retained in the cells for very short time duration (~2 hours). But these QD loaded CNGs showed retention up to 24 hrs with no
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toxicity towards the cells (Clift, Brandenberger, Rutishauser, Brown & Stone, 2011). Such systems can thus facilitate simultaneous optical diagnosis and monitor the distribution of
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nano polymeric drug conjugates and drug response (Rejinold, Chennazhi, Tamura, Nair SV. & Jayakumar, 2011).
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3.3. Therapy
CNGs have also been utilized for therapeutic purposes. As these nanogels provide high
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surface area, a large number of molecules can be adsorbed on their surface as well as get entrapped within them. When these nanogels reach a specific physiological environment, they will degrade and release the entrapped molecules. These molecules in turn exhibit
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their specific functions. Metallic nanoparticle loaded CNGs have been used for antibacterial and radiofrequency (RF) assisted cancer therapy (Kumar N.A. et al., 2013; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). QDs loaded composite nanogels have been used to provide hyperthermia by RF application to the cancer cells (Rejinold, Biswas, Chellan & Jayakumar, 2014). These two applications have been discussed here briefly.
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3.3.1. RF assisted Au, QDs, and several metal oxides have been used for RF assisted cancer therapy. However, toxicity by some of these QDs and metal oxides towards the normal cells, has promoted the use of these therapeutic agents by encapsulating them in biocompatible
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nanocarries. Au, Fe3O4, CdTe/ZnTe QDs loaded chitin-PLGA composite nanogels showed good amount of heating when exposed to RF signals (Rejinold, Biswas, Chellan &
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Jayakumar, 2014). These gels were taken up more efficiently by MCF7 than HDFs which
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may be due to difference in their surface membranes.In another similar work Au based chitin-MnO2 ternary composite nanogels were used for similar application (Rejinold,
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Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). In this work chitin solution and MnO2nanorods containing methanol was mixed together and regenerated by adding
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excess of methanol to obtain MnO2-chitin composite nanogel. To this composite nanogel a colloidal dispersion of gold nanoparticles (10nm) was added to form the final ternary
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composite nanogel. This system was further labelled with rhodamine-123 to study their cellular localization. These nanogels showed good conductivity when analysed by
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scanning electrochemical microscopy (SECM), which was attributed to the gold nanoparticles. They did not show any toxicity towards L929, HDF, MG63, T47D and A375 cells even at higher concentrations (1mg/ml). They were easily internalized by the cells
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without disturbing the cellular morphology. However the uptake was more by the cancer cells than HDFs. When the breast cancer cell line (T47D) was exposed to 100watts of RF signals for 2 minutes, ablation of the cells could be seen by live dead assay (Fig. 8). Thus concentration dependent heating efficiency of these nanogels can be used for magnetic hyperthermia for cancer cells (Rejinold, Biswas, Chellan& Jayakumar, 2014; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014).
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Fig. 8. Complete ablation of the breast cancer cells shown by live/dead assay after treating them with ternary CNGs at 100 watts/2 min RF exposure (Rejinold et al, 2014b). Figures
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reproduced with kind permission from RSC. 3.3.2. Anti-bacterial
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Nickel (Ni) nanoparticles with a size of around 50nm were loaded in CNGs to produce Ni-
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CNGs of around 120-150nm (Kumar NA. et al., 2013). These nanogels showed good antibacterial activity against Staphylococcus aureus by serial dilution method. Though
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these nanogels were lethal towards bacteria, they did not show any toxicity towards L949 and A549 cell lines. Studies have reported that nickel nanoparticles with a positive charge can bind to the negatively charged bacterial cell membrane and disrupt it, thereby
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exhibiting an antibacterial activity (Visurraga, Gutierrez, Plessing & Garcia, 2011). These nanoparticles have also shown to generate reactive oxygen species, which damage the bacterial cells (Visurraga, Gutierrez, Plessing & Garcia, 2011). Rhodamine labelled NiCNGs showed good cellular localization in L949 and A549 cell lines. Though Ni-loaded CNGs showed good antibacterial activity, the use of nickel for therapeutic purposes may be banned in some countries due to its occasional allergic responses and other side effects. These issues have to be addressed before this system can be taken for clinical purposes. 21 Page 22 of 39
4. Conclusions This review overviewed the properties and preparation of CNGs along with their application in the field of drug delivery, imaging, sensing and therapeutics. Chitin, apart from being highly biocompatible, can also be easily fabricated into nanogels by simple
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regeneration technique without using any cross-linkers. Due to the inherent properties of nanogels, these can be loaded with a wide range of molecules. Here various techniques of
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developing composite CNGs loaded with various molecules have been described. Further,
therapeutics have also been discussed in detail.
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their applications in the field of targeted drug delivery, bioimaging, biosensing and This review is expected to help
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researchers working in the field of polymeric nanogels and drug delivery systems to have a
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better understanding about the vast potential of CNGs.
5. Future perspectives
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Though a wide range of innovative applications have been found in the field of drug delivery and therapeutics with the advent of nanotechnology, there is still a large void in
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the area of bench to bedside translation. In order to bridge this gap, extensive in vivo studies need to be carried out to confirm the efficacy of CNGs in practical conditions. The functionality of these nanocarriers when scaled up must be thoroughly investigated.
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Various regulatory issues concerning the toxicity related to certain drugs, metallic particles or molecules need to be carefully addressed. Once all these aspects have been looked into these nanogels can be taken for further clinical trials. Acknowledgements The author R. Jayakumar is grateful to Department of Biotechnology (DBT), India, for providing research support. The author M. Sabitha is also grateful to Department of Science
22 Page 23 of 39
and Technology (DST), India, for providing financial support under DST fast track scheme (Ref No: SB/YS/LS-124/2013).
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List of Figure Captions Fig. 1. Advantages of colloidal chitin nanogels. Fig. 2. Chitin nanogels release the drug encapsulated in them in response to external stimuli.
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Fig. 3. Biomedical applications of the prepared colloidal chitin nanogels. Fig. 4. Preparation of colloidal chitin nanogel and drug loaded chitin nanogels.
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Fig. 5. (i) Schematic representation of the possible mechanism by which Dox-HA-Cys-CNGs
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induce toxicity in CD+ve cells. (ii) Fluorescent images showing the specific uptake of DoxHA-Cys-CNGs by CD+ve and CD-ve cells (B-C: HT29 (CD+ve); D-E: IEC6 (CD-ve)) [A & D-
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control HT-29 & IEC cells respectively without nangels treatment] (Ashwin et al., 2014). Figures reproduced with kind permission from RSC.
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Fig. 6. (i) CCNGs showing enhanced uptake by A375 cells(A); (ii) Fluorescent images showing deeper skin penetration by CCNGs (Mangalathillam et al., 2012).Figures
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reproduced with kind permission from RSC.
Fig. 7. Flu-CNGs showing penetration into deeper layers of cornea (Mohammed et al.,
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2013). Figures reproduced with kind permission from American Scientific Publishers. Fig. 8. Complete ablation of the breast cancer cells shown by live/dead assay after treating them with ternary CNGs at 100 watts/2 min RF exposure (Rejinold et al, 2014b). Figures
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reproduced with kind permission from RSC.
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S0144-8617(15)00910-8 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.054 CARP 10356
To appear in: Received date: Revised date: Accepted date:
30-7-2015 16-9-2015 18-9-2015
Please cite this article as: Priya, M. V., Sabitha, M., and Jayakumar, R.,Colloidal chitin nanogels: A plethora of applications under one shell, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights
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Colloidal chitin nanogels are highly versatile nanocarriers Chitin nanogels can be prepared by simple regeneration technique Drugs, quantum dots and metallic nanopaticles can be incorporated into nanogels Chitin nanogels have applications in drug delivery, imaging, sensing and therapy
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*Manuscript
Colloidal chitin nanogels: A plethora of applications under one shell M. Vishnu Priyaa, M. Sabithab, R. Jayakumara* a
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical
Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi-682041,
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India b
Amrita School of Pharmacy, Amrita Institute of Medical Sciences and Research Centre,
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Amrita Vishwa Vidyapeetham University, Kochi-682041, India
___________________________ *Corresponding author: Dr. R. Jayakumar E-mail: [email protected] & [email protected] Tel: +91-484-2801234; Fax:+91-484-2802020 1 Page 2 of 39
Abstract Chitin nanogels (CNGs) are a relatively new class of natural polymeric nanomaterials which have a large potential in the field of drug delivery and nanotherapeutics. These nanogels being very biocompatible are non-toxic when internalised by cells. In this review
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various properties, preparation techniques and applications of CNGs have been described. CNGs because of their nano-size possess certain unique properties which enables them
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to be used in a number of biomedical applications. CNGs are prepared by simple
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regeneration technique without using any cross-linkers. Various polymers, drugs and fluorescent dyes can be blended or incorporated or labelled with the chitin hydrogel
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network. Drugs and molecules encapsulated within CNGs can be used for targeted delivery, in vivo monitoring or even for therapeutic purposes. Here various applications of
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CNGs in the field of drug delivery, imaging, sensing and therapeutics have been discussed.
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Keywords: Chitin nanogels; skin penetration; targeted drug delivery; bioimaging;
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therapeutics.
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List of contents 1. Introduction 2. Synthesis of colloidal chitin nanogels by regeneration chemistry 3. Applications of chitin nanogel
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3.1. DrugDelivery systems 3.1.1. Anti-cancer
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3.1.2. Anti-fungal
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3.1.3. Protein 3.2. Imaging & sensing
3.3.1. Antibacterial
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3.3.2. Radiofrequency assisted cancer
5. Future perspectives
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Acknowledgements
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4. Conclusions
References
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3.3. Therapy
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List of abbreviations
5-Fluorouracil
BSA
Bovine Serum Albumin
CNGs
Chitin nanogels
CCNGs
Curcumin loaded Chitin Nanogels
Cystamine
Cys
DNA
Deoxyribonucleic acid
Dox
Doxorubicin
ECM
Extra Cellular Matrix
EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
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Fluconazole Flu
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Glutathione GSH Hyaluronic acid
HDFs
Human dermal fibroblasts
MeOH
Methanol
NHS
N-hydroxysulfosuccinimide
PCL
poly (caprolactone)
PEG
poly (ethylene glycol)
PEI
poly (ethylenimine)
PLA
poly (lactic acid)
PLGA
poly (lactic-co-glycolic) acid
pNIPAM
poly (N- isopropylacrylamide)
QDs
Quantum Dots
RF
Radiofrequency
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SECM
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HA
RNA
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5- FU
Ribonucleic acid Scanning Electrochemical Microscopy
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1. Introduction In the past few decades, medical field has seen considerable amount of advancements, overcoming various health issues and tackling several diseases. Nevertheless these advancements have not come without their share of drawbacks (Allen & Cullis, 2004). With
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the advent of nanotechnology, researchers are now focussing on using nano-tools to overcome the shortcomings of these conventional methods. Some of the current
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challenges in the conventional systems include lack of proper drug delivery systems with
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efficient bioavailability or drug release capability, lack of efficient imaging and sensing technique, lack of targeted delivery systems, etc. (Utreja, Jain & Tiwary, 2010). As an
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attempt to circumvent these issues, scientists have shifted their focus towards polymeric systems of the nanoscale (Chacko, Ventura, Zhuang & Thayumanavan, 2012; Sahoo,
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Dilnawaz & Krishnakumar, 2008; Sivaram, Rajitha, Maya, Jayakumar & Mangalathillam, 2015). Nanomaterials, with their high surface to volume ratio, possess properties very
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different from their bulk counterparts (Maya et al., 2013). Nanomaterials include nanoparticles, nanogels, nanorods, nanospheres, etc.
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Nanogels are hydrogels having size of few hundred nanometres. Hydrogels with a cross-linked polymeric network are considered to be highly biocompatibility due to their high water retaining capability, low surface tension and three dimensional structure similar
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to the native extracellular matrix (ECM) (Gupta, Vermani & Garg, 2002; Maya et al., 2013; Sivashanmugam, Arunkumar, Priya, Nair S.V.& Jayakumar, 2015). Nanogels have been used for the delivery of drugs, imaging labels, metallic nanoparticles, proteins, Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) (Kim et al., 2011), genes (Xu et al., 2006), growth factors (Kobayashi et al., 2012), etc. These agents interact spontaneously with the polymer matrix via covalent, van-der Waals, electrostatic and/or hydrophobic interactions to form stable nanostructures (Jiang, Chen, Deng, Suuronen & Zhong, 2014;
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Kabanov & Vinogradov, 2009). Because of their ability to swell in aqueous environment, they help in the controlled release of these molecules at the target site (Peppas, Hilt, Khademhosseini& Langer, 2006). Nanogels can further be modified for targeted delivery by chemically conjugating their surface with active moieties for ligand- receptor
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interactions (Garcia, Andrieux, Gil & Couvreur, 2005). Nanogels serve as ideal candidates for the intracellular transport of active moieties as these can be easily taken up by cells
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(Raemdonck, Demeester & De Smedt, 2009). Because of their great affinity towards water
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they are not easily taken up by mononuclear phagocytes and thus can remain in the circulation for a longer time.
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Both synthetic and natural polymers have been used for synthesising nanogels. Polymers like poly (N-isopropylacrylamide) (pNIPAM) (Singh & Lyon, 2007; Zhao et al.,
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2011), poly (ethylene glycol) (PEG) (Lee, Li & Chu, 2006; Mok & Park, 2006), poly(ethylenimine) (PEI) (Ganta et al., 2008; Xu et al., 2006), etc. copolymerised with other
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polymers are being used for fabricating synthetic polymeric nanogels. Natural polymers used for synthesising nanogels include chitosan (Schmitt et al., 2010; Wu, Shen, Banerjee
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& Zhou, 2010), chitin (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011), hyaluronic acid (HA), pullulan (Kobayashi et al., 2012; Kageyama et al., 2008; Morimoto et al., 2012), chondroitin sulphate (Huang et al., 2009; Xi, Zhou & Dai, 2012), etc. Since
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natural polymers degrade into non-toxic products which are naturally present in our body, they are considered to be relatively more biocompatible than synthetic polymers. Chitin is an amino polysaccharide found in the cell walls of fungi, exoskeleton of arthropods such as crustaceans (shrimps, prawns, crabs, etc.) and insects and internal shells of cephalopods such as squid, octopus, etc. (Kumar, 2000; Rinaudo, 2006). The monomeric units of chitin, N-acetyl glucosamine, are same as that of the native ECM. As a result, chitin is biocompatible, biodegradable and non-toxic. Chitin is being processed into
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various forms including hydrogels, nanogels, microparticles, sponges, scaffolds, membranes and nanofibersfor various biomedical applications like tissue engineering and drug delivery (Anitha et al., 2014; Arun Kumar et al., 2015; Pierfrancesco, 2012; Tamura, Furuike, Nair SV & Jayakumar, 2011; Yang, 2011).
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CNGs apart from the above mentioned properties have additional advantages (Fig. 1). CNGs have shown better drug loading capability, controlled release of encapsulated
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drugs, stability of the loaded drug, tuneable design, response to external stimuli, easy
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conjugation with active targeting agents due to large surface area, prolonged circulation time and high stability in aqueous solution (Hamidi, Azadi & Rafiei, 2008; Kabanov &
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Vinogradov, 2009). They can get accumulated at the tumour tissue due to enhanced retention and permeation effect (Maya et al., 2013). Once they enter the cells via endocytic
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pathway, they can release the drugs in the lysosomes or endosomes by pH-controlled hydrolysis (Manchun, Dass & Sriamornsak, 2012) (Fig. 2). Due to these properties, CNGs
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are being used for a wide range of applications (Fig. 3).This review discusses about the
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in detail.
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preparation and application of CNGs in the field of drug delivery, imaging and therapeutics
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Fig. 1. Advantages of colloidal chitin nanogels.
Fig. 2. Chitin nanogels release the drug encapsulated in them in response to external stimuli.
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Fig. 3. Biomedical applications of the prepared colloidal chitin nanogels.
2. Synthesis of colloidal chitin nanogels by regeneration chemistry
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In spite of being highly biocompatible, full potential of chitin has not been explored much mainly due to its insoluble nature. Chitin possesses a high degree of acetylation and
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hydrogen bonding which makes it insoluble in water or in the regular solvents. Harsh solvents like trichloroacetic acid, dichloroacetic acid, lithium thiocyanate, highly polar
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fluorinated solvents like hexafluoroisopropyl alcohol and hexafluoracetonesesquihydrate, inorganic solvents, lithium chloride-tertiary amide solvent systems, etc. have been used in the past to dissolve chitin (Pillai, Paul & Sharma, 2009). In 2006, Tamura et al developed a
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CaCl2-Methanol (MeOH) solvent, which dissolved chitin in a milder environment (Tamura, Nagahama & Tokura, 2006). Calcium chloride dihydrate was dissolved in MeOH to form a supersaturated solution, into which chitin was added to form chitin solution. The solubility of chitin in this solvent depends upon a number of factors such as degree of deacetylation and the molecular weight of chitin, water content of the system and the Ca2+ ion concentration in the CaCl2-MeOH (Tamura, Nagahama & Tokura, 2006). This system serves as a suitable solvent for forming colloidal chitin hydrogel. Chitin is dissolved in
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supersaturated CaCl2-MeOH and is regenerated by adding excess of MeOH which results in the formation of hydrogel. Hydrogel formation occurs as a result of self-assembly of polymeric chains. In this process, hydrophilic polymeric chains capable of hydrophobic or electrostatic interactions aggregate to form a network like structure (Kabanov &
ip t
Vinogradov, 2009). Chitin hydrogel is then washed several times by centrifugation to remove excess solvent. To form gel particles of nano-dimension, hydrogel is dispersed in
cr
water and subjected to probe sonication at 75% amplitude for 5 minutes after every wash.
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The self-associating property of CNGs has been used extensively to entrap biomolecules in this network, and thus act as excellent drug carriers. CNGs have been
an
tagged with dyes such as Rhodamine to monitor their cellular uptake and localization. Such tagged nanogels are prepared by incubating the dye in CNG solution after the final
M
wash (Rejinold et al., 2012). Drug loaded CNGs are fabricated either by adding the drug solution to the chitin solution and then regenerating it (Mohammed et al., 2013) or by
ed
incubating the drug in a CNG solution for a few hours and then subjecting it to a wash to remove the unloaded drug (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013; Jayakumar,
ce pt
Nair A., Rejinold, Maya & Nair SV, 2012; Mangalathillam et al., 2013) (Fig. 4). Drugs which are insoluble in aqueous conditions are first dissolved in their suitable solvents and then added drop wise to the chitin solution (Mangalathillam et al., 2012).CNGs have also been
Ac
loaded with proteins; quantum dots (QDs) and metallic nanoparticles. QDs are added directly to a colloidal suspension of CNGs after regeneration, under constant stirring (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011) whereas metallic nanoparticles were added to the chitin solution prior to regeneration (Kumar N.A. et al., 2013; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). Chitin composite nanogels have also been fabricated, wherein chitin is cross-linked or blended with another polymer. Synthetic polymers such as poly (caprolactone) (PCL),
10 Page 11 of 39
poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA) were dissolved in their suitable solvents and then added to the chitin solution. This blend was kept under stirring and then regenerated by adding excess of methanol followed by centrifugation and sonication to form nanogels (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013, 2014;
ip t
Rejinold, Biswas, Chellan & Jayakumar, 2014). Polymers such as HA were cross-linked to chitin with the help of EDC and cystamine (Kumar NA., Maya & Jayakumar, 2014). In such
cr
systems, cystamine is conjugated with HA by EDC/ N-hydroxy sulfosuccinimide (NHS)
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conjugation chemistry. The carboxyl group of HA which is activated by EDC interacts with one of the amine groups of cystamine. The hydroxyl group of chitin interacts with the other
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ed
M
an
amine group thus forming a cross-linked network (Kumar NA., Maya & Jayakumar, 2014).
11 Page 12 of 39
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Fig. 4. Preparation of colloidal chitin nanogel and drug loaded chitin nanogels.
3. Applications of CNGs 3.1. Drug delivery Any system to be used for drug delivery should have good interaction between the system and the molecule which has to be delivered. Nanogel due to its highly porous nature serves as an excellent delivery system. The molecules can be easily encapsulated/ entrapped in these pores. In certain cases, the molecules can even physically adsorb onto 12 Page 13 of 39
the surface of the gel particles. When these nanogel systems are placed in the body, the molecules are slowly released as the gel matrix degrades/disperses. Such systems help in the controlled release of the molecules, thereby reducing the chances of dose-dependent toxicity. CNGs have been used to deliver anti-cancer drugs, anti-fungal drugs and proteins.
ip t
3.1.1. Anti-cancer Conventional anti-cancer drugs are associated with problems like dose-dependent toxicity,
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improper distribution at the tumour site, off-target delivery, toxicity to off-target sites,
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bioaccumulation or quick degradation, etc (Maya et al., 2013). CNGs with their highly versatile nature can be used to overcome most of these issues.
an
pH responsive CNGs encapsulated with anti-cancer drugs have been developed. These gels are loaded with various molecules to enable their targeted delivery to the
M
specific target site. Colloidal CNGs incorporated with Doxorubicin (Dox) were synthesized with size of around 130-160 nm. They showed a higher drug release at a lower pH of 4.5
ed
than in neutral pH. These CNGs were taken up by fibroblasts as well as cancer cell lines but showed more toxicity towards cancer cell lines like MCF7, HEPG-2 and A549
ce pt
(Jayakumar, Nair A., Rejinold, Maya & Nair, SV., 2012). Similarly Chitin-PLA nanogels loaded with Dox were developed for treating hepatic cell carcinoma. More amount of Dox was found to be adsorbed onto the surface of the nanogel rather than being encapsulated
Ac
within it. Such adsorption seemed to have improved the stability of the colloidal nanogel suspension (Arunraj, Rejinold, Kumar NA & Jayakumar, 2014). This system also showed a better drug release in acidic environment. Dox loaded PLA-CNGs showed better cellular uptake and dose dependent toxicity towards HepG2 cells (Arunraj, Rejinold, Kumar NA & Jayakumar, 2014). Dox loaded PCL-CNGs were synthesized for non-small cell lung cancer which was also pH-responsive and showed dose-dependent toxicity towards A549 cells (Arunraj, Rejinold, Kumar NA & Jayakumar, 2013). The higher drug release in acidic
13 Page 14 of 39
pH could be attributed to the higher swelling ratio of CNGs in an acidic environment which was demonstrated (Rejinold et al., 2012). Since cancer cells are known to have an acidic pH, these systems have successfully shown more toxicity towards cancer cell lines. In order to further improve the targeted drug delivery systems, redox responsive
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chitin-HA composite nanogels conjugated using cystamine (HA-Cys-CNGs) were prepared (Kumar NA., Maya & Jayakumar, 2014). These nanogels were loaded with Dox and
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targeted for delivery to colon cancer cells. HA, a ligand for CD-44 receptors which are
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over-expressed in colon cancer cells seemed to facilitate specific internalization of the drug loaded nanogels by HT29 (CD-44 +ve) cells (Fig. 5). These gels showed higher in
an
vitro drug release in the presence of glutathione and more toxicity towards CD-44 +ve
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presence of acidic pH and glutathione.
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cells. Such systems can be used for specific targeted delivery to the tumor tissue in the
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Fig. 5. (i) Schematic representation of the possible mechanism by which Dox-HA-Cys-CNGs induce toxicity in CD+ve cells. (ii) Fluorescent images showing the specific uptake of DoxHA-Cys-CNGs by CD+ve and CD-ve cells (B-C: HT29 (CD+ve); D-E: IEC6 (CD-ve)) [A & Dcontrol HT-29 & IEC cells respectively without nangels treatment] (Ashwin et al., 2014).
ip t
Figures reproduced with kind permission from RSC. Apart from Doxorubicin, two other anti-cancer drugs curcumin and 5-fluorouracil (5-
cr
FU) have been loaded into CNGs for treating skin cancer via transdermal route
us
(Mangalathillam et al., 2012). Both chitin and curcumin are highly hydrophobic in nature whereas Curcumin loaded CNGs (CCNGs) showed a good and stable colloidal dispersion
an
in water. Cell uptake and cytotoxicity studies were carried out using human dermal fibroblasts (HDFs) and A375 melanoma cells. The melanoma cells showed higher cellular
M
uptake of CCNGs compared to HDF cells (Mangalathillam et al., 2012) (Fig. 6(i)). These gels also showed higher toxicity on A 375 melanoma cells compared to human dermal
ed
fibroblast cells. However the innate toxicity of curcumin on HDF cells, as reported earlier was found to be reduced in the CCNG formulation, due to the antioxidant effect of chitin.
ce pt
That means the chitin nanogel formulation was found to retain the cancer specific toxicity of curcumin and at the same time reduced its toxicity on HDF. Ex vivo studies carried out using porcine skin showed higher penetration for curcumin loaded in CNGs (CCNGs) in
Ac
comparison to control curcumin solution. The steady state flux for skin permeation was found to be 4 fold for CCNGs compared to control curcumin solution and retention of CCNGs was found to be more in the deeper layers of epidermis and dermis when compared with control curcumin solution (Fig. 6(ii)). Histopathological evaluations of the skin treated with the samples clearly indicated the loosening as well as fragmentation of stratum corneum in case of the skin treated with CCNGs. 5-FU loaded in CNGs (FCNGs) was also tested for cytotoxicity and uptake on A375 and HDF cells (Mangalathillam et al.,
15 Page 16 of 39
2013). Skin permeation studies of FCNGs showed steady state flux similar to 5-FU solution, but 5-FU loaded in CNGs showed 4-5 times higher retention in the deeper layers of the skin than the control 5-FU solution. Curcumin loaded chitin nanogel was found to be more effective for melanoma via the transdermal route because of its specific toxicity and
ed
M
an
us
cr
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enhanced skin penetration, when compared with 5-FU loaded chitin nanogel.
Fig. 6. (i) CCNGs showing enhanced uptake by A375 cells (A); (ii) Fluorescent images
ce pt
showing deeper skin penetration by CCNGs (Mangalathillam et al., 2012).Figures reproduced with kind permission from RSC. 3.1.2. Anti-fungal
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Anti- fungal drug delivery to the cornea faces a major issue of poor bioavailability. The presence of various static barriers like different layers of cornea, sclera and retina, dynamic barriers like choroidal and conjunctival blood flow and physiological processes like tear drainage and blinking greatly reduce the residence time of the topically applied drug. This in turn reduces the efficacy of the drug (Urtti, 2006).It is reported that less than 10% of the eye drops permeate and reach the target tissue to achieve effective drug concentration (Diebold & Calonge, 2010). As an attempt to overcome these issues, Fluconazole (Flu) loaded CNGs were developed which showed better deeper tissue 16 Page 17 of 39
penetration for porcine cornea (Mohammed et al., 2013). This system also showed very slow and controlled release of the Flu in neutral pH. Such a release is considered important for anti-fungal treatment, where the drug is required to be present for longer time duration for better efficacy (Mudgil, Gupta, Nagpal & Pawar, 2012). This system showed
ip t
good anti- fungal activity towards Candida tropicalis without causing any toxicity towards HDFs. Hemocompatability studies showed that these nanogels did not interfere with the
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plasma coagulation pathway and further ex vivo studies showed no signs of inflammation
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or destruction to the corneal cells. Cornea treated with Flu loaded CNGs showed loosening and ablation of corneal epithelium, thereby penetrating into deeper layers of
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cornea (Fig. 7). Such a penetration could not be seen in cornea treated with Flu. The cationic charges of Flu loaded CNGs could interact with the corneal glycoproteins, thus
M
increasing their permeation. Such an interaction might form a pre-corneal depot, from which the drug can release at a controlled rate for a prolonged time period. Apart from this,
ed
such systems can also protect the drug from degradation and increase its retention though
Ac
ce pt
bioadhesion. Thus Flu loaded CNGs can serve as a good antifungal formulation.
Fig. 7. Flu-CNGs showing penetration into deeper layers of cornea (Mohammed et al., 2013). Figures reproduced with kind permission from American Scientific Publishers.
17 Page 18 of 39
3.1.3. Protein Besides drugs, CNGs can be used for delivering proteins also. Bovine Serum Albumin (BSA) was used as the model protein to study the protein delivering potential of CNGs (Rejinold, Chennazhi, Tamura, Nair, SV & Jayakumar, 2011). Multifunctional CNGs were
ip t
developed by incorporating BSA in Quantum Dots (QD)-CNGs. BSA was expected to load into CNGs and QD-CNGs through physical adsorption chemistry. Both the systems
cr
showed high BSA loading. Release study was carried out in acidic as well as neutral pH.
us
BSA loaded in CNGs as well as QD-CNGs showed controlled release in neutral pH and higher release in acidic pH. Higher release in acidic pH was attributed to the acidic
an
degradation of the nanogel followed by its swelling (Raemdonck, Demeester & De Smedt, 2009). These multifunctional CNGs did not show any haemolytic activity and were non-
M
toxic towards L929, VERO, MCF7, KB, PC3 and NIH3T3 cells (Rejinold, Chennazhi, Tamura, Nair SV & Jayakumar, 2011). These studies show that CNGs can be used for
engineering purposes.
ce pt
3.2. Imaging and sensing
ed
delivering molecules like drugs, proteins, growth factors, etc. for drug delivery and tissue
Apart from drug delivery systems, CNGs have found application in the field of bioimaging and biosensing as well. Mercaptopropionic acid capped CdTe QDs have been
Ac
incorporated into CNGs via physical adsorption chemistry for imaging purposes (Rejinold, Chennazhi, Tamura, Nair SV. & Rangasamy, 2011). Electrochemical analysis showed that complexing QDs with CNGs did not reduce its electrochemical stability and luminescent properties. These properties are important as they are required for imaging the cells in in vivo experiments. L929, VERO and PC3 cells were stained with QD loaded CNGs and observed under fluorescent microscope. It was seen that the QDs did not undergo complete quenching even after cellular internalization and thus can be used for cell-
18 Page 19 of 39
labelling under physiological conditions. The cells did not show any change in their morphology even after 24 hrs of incubation with the QD nanogels. Similarly CdTe/ZnTe-QD-chitin/PLGA composite nanogels and umbelliferone labelled chitin/PLGA-composite-nanogels have been reported to be taken up by microbes
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like Escherichia coli, Staphylococcus aureus and Candida albicans and thus could be used for microbial monitoring (Rejinold, Biswas, Chellan & Jayakumar, 2014). These nanogels
cr
did not show any toxicity towards HDFs. Although semiconductor QDs has been
us
previously used for cellular imaging, they were retained in the cells for very short time duration (~2 hours). But these QD loaded CNGs showed retention up to 24 hrs with no
an
toxicity towards the cells (Clift, Brandenberger, Rutishauser, Brown & Stone, 2011). Such systems can thus facilitate simultaneous optical diagnosis and monitor the distribution of
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nano polymeric drug conjugates and drug response (Rejinold, Chennazhi, Tamura, Nair SV. & Jayakumar, 2011).
ed
3.3. Therapy
CNGs have also been utilized for therapeutic purposes. As these nanogels provide high
ce pt
surface area, a large number of molecules can be adsorbed on their surface as well as get entrapped within them. When these nanogels reach a specific physiological environment, they will degrade and release the entrapped molecules. These molecules in turn exhibit
Ac
their specific functions. Metallic nanoparticle loaded CNGs have been used for antibacterial and radiofrequency (RF) assisted cancer therapy (Kumar N.A. et al., 2013; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). QDs loaded composite nanogels have been used to provide hyperthermia by RF application to the cancer cells (Rejinold, Biswas, Chellan & Jayakumar, 2014). These two applications have been discussed here briefly.
19 Page 20 of 39
3.3.1. RF assisted Au, QDs, and several metal oxides have been used for RF assisted cancer therapy. However, toxicity by some of these QDs and metal oxides towards the normal cells, has promoted the use of these therapeutic agents by encapsulating them in biocompatible
ip t
nanocarries. Au, Fe3O4, CdTe/ZnTe QDs loaded chitin-PLGA composite nanogels showed good amount of heating when exposed to RF signals (Rejinold, Biswas, Chellan &
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Jayakumar, 2014). These gels were taken up more efficiently by MCF7 than HDFs which
us
may be due to difference in their surface membranes.In another similar work Au based chitin-MnO2 ternary composite nanogels were used for similar application (Rejinold,
an
Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014). In this work chitin solution and MnO2nanorods containing methanol was mixed together and regenerated by adding
M
excess of methanol to obtain MnO2-chitin composite nanogel. To this composite nanogel a colloidal dispersion of gold nanoparticles (10nm) was added to form the final ternary
ed
composite nanogel. This system was further labelled with rhodamine-123 to study their cellular localization. These nanogels showed good conductivity when analysed by
ce pt
scanning electrochemical microscopy (SECM), which was attributed to the gold nanoparticles. They did not show any toxicity towards L929, HDF, MG63, T47D and A375 cells even at higher concentrations (1mg/ml). They were easily internalized by the cells
Ac
without disturbing the cellular morphology. However the uptake was more by the cancer cells than HDFs. When the breast cancer cell line (T47D) was exposed to 100watts of RF signals for 2 minutes, ablation of the cells could be seen by live dead assay (Fig. 8). Thus concentration dependent heating efficiency of these nanogels can be used for magnetic hyperthermia for cancer cells (Rejinold, Biswas, Chellan& Jayakumar, 2014; Rejinold, Ranjusha, Balakrishnan, Mohammed & Jayakumar, 2014).
20 Page 21 of 39
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Fig. 8. Complete ablation of the breast cancer cells shown by live/dead assay after treating them with ternary CNGs at 100 watts/2 min RF exposure (Rejinold et al, 2014b). Figures
an
reproduced with kind permission from RSC. 3.3.2. Anti-bacterial
M
Nickel (Ni) nanoparticles with a size of around 50nm were loaded in CNGs to produce Ni-
ed
CNGs of around 120-150nm (Kumar NA. et al., 2013). These nanogels showed good antibacterial activity against Staphylococcus aureus by serial dilution method. Though
ce pt
these nanogels were lethal towards bacteria, they did not show any toxicity towards L949 and A549 cell lines. Studies have reported that nickel nanoparticles with a positive charge can bind to the negatively charged bacterial cell membrane and disrupt it, thereby
Ac
exhibiting an antibacterial activity (Visurraga, Gutierrez, Plessing & Garcia, 2011). These nanoparticles have also shown to generate reactive oxygen species, which damage the bacterial cells (Visurraga, Gutierrez, Plessing & Garcia, 2011). Rhodamine labelled NiCNGs showed good cellular localization in L949 and A549 cell lines. Though Ni-loaded CNGs showed good antibacterial activity, the use of nickel for therapeutic purposes may be banned in some countries due to its occasional allergic responses and other side effects. These issues have to be addressed before this system can be taken for clinical purposes. 21 Page 22 of 39
4. Conclusions This review overviewed the properties and preparation of CNGs along with their application in the field of drug delivery, imaging, sensing and therapeutics. Chitin, apart from being highly biocompatible, can also be easily fabricated into nanogels by simple
ip t
regeneration technique without using any cross-linkers. Due to the inherent properties of nanogels, these can be loaded with a wide range of molecules. Here various techniques of
cr
developing composite CNGs loaded with various molecules have been described. Further,
therapeutics have also been discussed in detail.
us
their applications in the field of targeted drug delivery, bioimaging, biosensing and This review is expected to help
an
researchers working in the field of polymeric nanogels and drug delivery systems to have a
M
better understanding about the vast potential of CNGs.
5. Future perspectives
ed
Though a wide range of innovative applications have been found in the field of drug delivery and therapeutics with the advent of nanotechnology, there is still a large void in
ce pt
the area of bench to bedside translation. In order to bridge this gap, extensive in vivo studies need to be carried out to confirm the efficacy of CNGs in practical conditions. The functionality of these nanocarriers when scaled up must be thoroughly investigated.
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Various regulatory issues concerning the toxicity related to certain drugs, metallic particles or molecules need to be carefully addressed. Once all these aspects have been looked into these nanogels can be taken for further clinical trials. Acknowledgements The author R. Jayakumar is grateful to Department of Biotechnology (DBT), India, for providing research support. The author M. Sabitha is also grateful to Department of Science
22 Page 23 of 39
and Technology (DST), India, for providing financial support under DST fast track scheme (Ref No: SB/YS/LS-124/2013).
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List of Figure Captions Fig. 1. Advantages of colloidal chitin nanogels. Fig. 2. Chitin nanogels release the drug encapsulated in them in response to external stimuli.
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Fig. 3. Biomedical applications of the prepared colloidal chitin nanogels. Fig. 4. Preparation of colloidal chitin nanogel and drug loaded chitin nanogels.
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Fig. 5. (i) Schematic representation of the possible mechanism by which Dox-HA-Cys-CNGs
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induce toxicity in CD+ve cells. (ii) Fluorescent images showing the specific uptake of DoxHA-Cys-CNGs by CD+ve and CD-ve cells (B-C: HT29 (CD+ve); D-E: IEC6 (CD-ve)) [A & D-
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control HT-29 & IEC cells respectively without nangels treatment] (Ashwin et al., 2014). Figures reproduced with kind permission from RSC.
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Fig. 6. (i) CCNGs showing enhanced uptake by A375 cells(A); (ii) Fluorescent images showing deeper skin penetration by CCNGs (Mangalathillam et al., 2012).Figures
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reproduced with kind permission from RSC.
Fig. 7. Flu-CNGs showing penetration into deeper layers of cornea (Mohammed et al.,
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2013). Figures reproduced with kind permission from American Scientific Publishers. Fig. 8. Complete ablation of the breast cancer cells shown by live/dead assay after treating them with ternary CNGs at 100 watts/2 min RF exposure (Rejinold et al, 2014b). Figures
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reproduced with kind permission from RSC.
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