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Thermoresponsive magnetic nanoparticle – Aminated guar gum hydrogel system for sustained release of doxorubicin hydrochloride
Accepted Manuscript Title: Thermoresponsive magnetic nanoparticle - aminated guar gum hydrogel system for sustained release of doxorubicin hydrochloride Author: Ragothaman Murali Ponraj Vidhya Palanisamy Thanikaivelan PII: DOI: Reference:
S0144-8617(14)00433-0 http://dx.doi.org/doi:10.1016/j.carbpol.2014.04.076 CARP 8837
To appear in: Received date: Revised date: Accepted date:
24-1-2014 24-3-2014 15-4-2014
Please cite this article as: Murali, R., Vidhya, P., & Thanikaivelan, P.,Thermoresponsive magnetic nanoparticle - aminated guar gum hydrogel system for sustained release of doxorubicin hydrochloride, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.076 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.
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Thermoresponsive magnetic nanoparticle - aminated guar gum hydrogel
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system for sustained release of doxorubicin hydrochloride
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Ragothaman Murali, Ponraj Vidhya, Palanisamy Thanikaivelan*
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Advanced Materials Laboratory, Centre for Leather Apparel & Accessories Development,
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Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar,
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Chennai 600020, India
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*Corresponding author. Tel/Fax: +91 44 24910953. E-mail address: [email protected] (P.
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Thanikaivelan) 1 Page 1 of 22
ABSTRACT
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Hydrogel based sustained drug delivery system has evolved as an immense treatment method
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for solid tumors over the past few decades with long term theranostic ability. Here, we
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synthesized an injectable hydrogel system comprising biocompatible aminated guar gum,
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Fe3O4-ZnS core-shell nanoparticles and doxorubicin hydrochloride. We show that amination
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of guar gum resulted in attraction of water molecules thereby forming the hydrogel without
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using toxic crosslinking agents. Hydrogel formation was observed at 37°C and is stable up to
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95°C. The prepared hydrogel is also stable over a wide pH range. The in-vitro studies show
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that the maximum de-gelation and drug release up to 90% can be achieved after 20 days of
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incubation. Studies reveal that the drug and the core-shell nanoparticles can be released
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slowly from the hydrogel to provide the healing and diagnosis of the solid tumor thereby
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avoiding several drug administrations and total excision of organs.
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Keywords: Solid tumor, Biomedical imaging, Aminated guar gum, Core shell nanoparticles,
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Doxorubicin HCl.
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1. Introduction Direct administration of chemotherapeutic agents in cancerous solid tumors has shown
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severe side effects in the system (Kintzel & Dorr, 1995). Injectable hydrogel is an alternative
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and effective system that can deliver the drug to specific tissues thereby reducing the drug
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side effects as well as the injection frequency (Arola et al., 2000; Kim et al., 2012). Currently,
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injectable hydrogel system comprising both therapeutic and diagnostic imaging agents is an
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emerging research area for effective guided therapy (Kim et al., 2012). Guar gum (GG) is one
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of the high-molecular weight, water-soluble, non-ionic biopolymer derived from the
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endosperm of guar beans, which has been widely used in the pharmaceuticals products
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primarily due to its unique ability to alter rheological properties (Burke, Park, Srinivasarao, &
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Khan, 2000). Several studies have been carried out to increase the mechanical, thermal and
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viscosity properties of the GG by chemical modification (Bajpai, Saxena, & Sharma, 2006;
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Huang, Lu, & Xiao, 2007). The chemically modified GG cross-linked with glutaraldehyde,
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phosphate, urea-formaldehyde or borax has been used for various applications such as
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controlled drug release (Soppirnath & Aminabhavi, 2002), colon-specific drug delivery
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(Chourasia & Jain, 2004; Gliko-Kabir, Yagen, Baluom, & Rubinstein, 2000), liquid pesticide
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(Kulkarni, Soppimath, Aminabhavi, Dave, & Mehta, 1999), water retention (Tayal, Pai, &
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Khan, 1999), synthetic cervical mucus (Burruano, Schnaare, & Malamud, 2002) and
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transdermal drug delivery systems (Narasimha Murthy, Hiremath, & Paranjothy, 2004) due to
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its high biocompatibility and biodegradability. However, the preparation of chemically cross-
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linked hydrogel using toxic crosslinkers poses significant limitations in the applications
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(Chang & Zhang, 2011).
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Inclusion of magnetic resonance imaging (MRI) agents in the injectable hydrogel helps in
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depiction of targeted agent and diagnosis of the lesion sites. Iron oxide nanoparticles (IONP)
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are prominently used as MRI agent for biological applications (Corot, Robert, Idee, & Port,
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2006). A suitable chemical modification has been indispensable for the IONP, which has
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intended to reduce the alteration of magnetic properties, agglomeration and rapid degradation
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(Kim et al., 2012; Santra et al., 2001). It has been used clinically for various biomedical
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applications, such as hyperthermia, drug delivery, tissue repair, cell and tissue targeting and
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transfection (Gupta & Gupta, 2005; Gupta, Naregalkar, Vaidya, & Gupta, 2007).
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Doxorubicin hydrochloride (DOX) is one of the most widely-used chemotherapeutic
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anticancer drugs, however, it initiates serious side effects such as cardiotoxicity, bone
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marrow and gastrointestinal toxicity if applied directly (Arola et al., 2000; Von Hoff et al.,
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1979). Several studies reveal that the encapsulated DOX reduces the toxicity and side effects
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of the drug (Gelperina et al., 2002; Yoo, Lee, Oh, & Park, 2000). It has been shown that the
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conjugation of DOX with the dextran and liposomes not only reduces the cytotoxicity but
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also enhances the efficiency of the drug (Mitra, Gaur, Ghosh, & Maitra, 2001; Wong et al.,
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2006). Here, an approach has been made to prepare aminated guar gum (AGG) from GG by
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reacting with ethylene diamine and sodium borohydride (Simi & Abraham, 2010). The
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reaction leads to the substitution of –OH group with –NH2 group at the 2nd, 3rd, and 6th
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position in the monosaccharide units of polysaccharide backbones. We also report a simple
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approach to prepare injectable hydrogel system using the AGG incorporated with iron oxide
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– zinc sulphide core shell nanoparticles (CSNP) and DOX, which could eliminate the
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limitations such as instability and cytotoxicity. The prepared AGG, IONP, CSNP and
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injectable hydrogel systems were investigated for the chemical, structural, magnetic and
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biological properties.
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2. Materials and methods
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2.1. Materials
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Ferric chloride (FeCl3) was purchased from M/s SD Fine-Chem, Mumbai, India. Ferrous
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sulphate (FeSO4.7H2O) and acetone were purchased from Merck, Mumbai, India.
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Ammonium hydroxide (25%) was procured from M/s Sisco Research Lab, Mumbai, India.
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Zinc acetate (Zn(CH3COO)2), sodium borohydride (NaBH4) and guar gum powder were
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procured from Himedia, India. Ethylene diamine (C2H8N2), sodium sulfide (Na2S) and
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doxorubicin HCl were purchased from Sigma-Aldrich Chemicals. All other reagents were of
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analytical grade.
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2.2. Synthesis of aminated guar gum
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The synthesis of AGG was carried out by the addition of C2H8N2 as an aminating agent to
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the GG, which substituted the -OH group of the GG with -NHCH2CH2NH2. Further the
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-NHCH2CH2NH2 group was reduced to -NH2 group by the addition of NaBH4 (Simi &
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Abraham, 2010). 1g (w/v) of GG was dissolved in 250 ml of distilled water and stirred to get
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homogenous solution for 30 min. 25 ml of ethylene diamine was added to the homogenous
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solution of GG and allowed to react with continuous stirring for overnight at room
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temperature. Then, 50 ml of 5% NaBH4 solution was added to the reaction mixture and stirred
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vigorously for 3 h. After the completion of the reaction, the mixture was precipitated and
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washed several times with acetone. The precipitated sample was lyophilized and then
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powdered to uniform particles.
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2.3. Synthesis of core shell nanoparticles
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The synthesis of CSNP was carried out in two steps. In the first step, iron oxide
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nanoparticles were synthesized. In the typical Fe3O4 nanoparticles synthesis, 20 ml of 0.1M
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FeCl3 and 20 ml of 0.05M FeSO4 were mixed and stirred for 30 min at room temperature. To
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this solution, NH4OH was added drop wise to attain pH 10. A black color precipitate was
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formed signifying the formation of Fe3O4 nanoparticles. The precipitate was washed
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repeatedly with acetone and dried at 55°C. In the second step, the Fe3O4 nanoparticles (10 ml,
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0.1% w/v) were sonicated to remove the agglomeration for 30 min at 30±2°C. After
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sonication, Na2S (5 ml, 0.15M) was added and stirred continuously for 30 min to facilitate
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deposition of sulphide ions on the surface of Fe3O4 nanoparticles. Then, 10 ml of 0.3M
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Zn(CH3COO)2 was added to the solution and stirred for 3 h. The precipitated CSNP were
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washed with acetone several times. The collected precipitate was dried at 55°C and then
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ground to make fine powder.
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The amino group in the AGG was determined by 2,4,6-trinitrobenzenesulfonic acid
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(TNBS) assay (Bubnis & Ofner III, 1992). It is known that the TNBS reacts with amines,
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hydrazines and hydrazides groups to form a stable trinitrophenyl complex. Briefly, 5 mg of
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samples from GG and AGG were dissolved in 1 ml of distilled water. To this solution, 1 ml
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of 4% w/v NaHCO3 and 1 ml of freshly prepared 0.5% (v/v) TNBS solution were added and
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kept at 60°C for 4 h. After that, 1 ml of the reacted solution was transferred to a fresh test
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tube and mixed with 3 ml of 6M HCl. The tubes were kept at 40°C for 1.5 h and the
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absorbance
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spectrophotometer (Schimadzu, UV-1800) at 420 nm. The percentage of amino group
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substituted in the GG was calculated using the equation below.
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The GG, AGG, IONP and CSNP were analyzed using Perkin Elmer FT-IR spectrometer.
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The samples were ground with KBr and compressed to form pellets. The pellets were 6 Page 6 of 22
analyzed in a single beam mode at the range of 400 - 4000 cm-1 with an average of 4 scans
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and 2 cm-1 resolution. X-ray diffraction was performed to identify the crystallographic
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structure of IONP and CSNP by using Rigaku Miniflex (II) desktop diffractometer (Ni
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filtered CuKα radiation with λ=0.15418 nm). The samples were analyzed in the 2θ range of 10
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to 80° in a step of 0.02° and with a counting time of 10 sec per step. Elemental composition
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of IONP and CSNP was investigated through energy dispersive X-ray analysis (EDX) using
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Oxford Instruments NanoAnalysis (INCA Energy 250 Microanalysis System). The magnetic
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properties of the synthesized IONP and CSNP were measured using the vibrating sample
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magnetometer (VSM, Lakeshore, 7410 model) at room temperature. The morphology, size
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and structure of the IONP and CSNP nanoparticles were analyzed using high resolution -
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scanning electron microscopy (HRSEM, Quanta 200 series, FEI Company) and high
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resolution - transmission electron microscopy (HRTEM, JEOL 3010).
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2.5. Preparation of injectable hydrogel
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The injectable AGG-CSNP-DOX hydrogel (100/0.125/0.05 wt.%) was prepared by the
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following method as shown in Fig. 1. Initially, 25 ml of 0.01% w/v solution of CSNP was
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sonicated for 30 min at 30±2°C. After the complete dispersion, the AGG (2g) and DOX (1
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ml, 0.1% w/v) were added to the solution. These solutions were uniformly dispersed by
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sonication at 37°C for 15 min to form stable injectable AGG-CSNP-DOX (100/0.125/0.05
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wt.%) hydrogel. Likewise, the AGG (100 wt.%) and AGG-CSNP (100/0.125 wt.%)
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hydrogels were prepared by using the respective dispersed compositions as above.
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Fig. 1. Schematic showing (a) the synthesis of CSNP from IONP, (b) the synthesis of AGG
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from GG and (c) the preparation of AGG-CSNP-DOX injectable hydrogel system.
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2.6. Effect of temperature and pH on the hydrogel
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The effect of different temperature and pH conditions on the as-synthesized AGG, AGG-
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CSNP and AGG-CSNP-DOX hydrogel systems were studied. For studying the effect of
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temperature (0 to 95oC) on the hydrogel systems, temperature controlled heating water bath
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and ice bath were employed. For studying the effect of pH (2 to 12) on the hydrogel systems,
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1M NaOH and 1M HCl solutions were employed.
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2.7. In-vitro de-gelation studies
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The in-vitro de-gelation of the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel
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systems was carried out by incubating a known weight of hydrogel into the 25 ml of
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phosphate buffered saline (PBS) solution (pH 7.4) at 37°C. The weight of in-vitro de-gelated
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hydrogels was measured after removal of surface water using filter paper at different time
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periods. The percentage de-gelation was calculated by using the equation below.
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2.8. In-vitro drug release study The in-vitro drug release tendency was studied by incubating the AGG-CSNP-DOX
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injectable hydrogel into 25 ml PBS (pH 7.4) solution at 37°C. The amount of DOX released
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from the encapsulated hydrogel was determined at different time periods using UV-Visible
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spectrophotometer at 486 nm. The cumulative DOX release from the injectable hydrogel was
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calculated using the equation below.
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3. Results and discussion
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3.1. Degree of substitution
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The AGG was prepared by reacting C2H8N2 and NaBH4 with the GG. It is seen that the
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degree of substitution of amino group in guar gum is up to 61% as analyzed through the
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TNBS method. The results clearly indicate the presence of NH2 group in AGG and confirm
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the modification of GG. The presence of amino group in the GG biopolymer attracts water
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molecules thereby leading to the formation of hydrogel. The amination process expected to
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create ionic imbalances in the biopolymer due to the formation of NH3+ (Simi & Abraham,
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2010). We propose that the cationic charge attracts more water molecules in order to
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neutralize the system by forming AGG-NH3+ - –(δ-(OH--H+))n hydrogel. Each water molecule
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provides a partial negative charge (δ-) such that several water molecules can cumulatively
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satisfy and neutralize one positive charge of the AGG. Therefore, AGG with several cationic
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amino groups would require more number of water molecules to neutralize several positive
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charges thereby forming the hydrogel.
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3.2. FTIR analysis The FTIR spectrum of pure GG, AGG, IONP and CSNP are shown in Fig. 2. The FT-IR
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spectrum of pure GG depicts a broad absorption band at 3352 cm−1, which indicates the
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presence of significant quantity of hydroxyl groups in the structure (Fig. 2a). In the modified
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GG (Fig. 2b), this band became sharper and shifted from 3352 to 3423 cm−1 due to the
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substitution of -NH2 functional group to the -OH group in the structure (Simi & Abraham,
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2010). Further, the AGG shows appearance of a sharp peak around 1030 cm−1, which
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corresponds to C-N stretching vibration of aliphatic amine. Hence, these results are in
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agreement with the amino group determination study of the AGG.
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Fig. 2. FTIR Spectrum of (a) GG, (b) AGG, (c) IONP and (d) CSNP.
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FT-IR spectrum of IONP exhibits major bands at 1740, 1628, 1401, 1123, 1051, 593 and
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460 cm−1 (Fig. 2c). The band at 1628 cm-1 corresponds to hydroxyl group of moisture while
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the bands at 1740, 1401, 1123 and 1051 cm-1 correspond to C-O vibration arising from the
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presence of CO2 from air. The characteristic peaks seen at 593 and 460 cm−1 correspond to
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the Fe-O stretching vibration of the IONP (Maity & Agrawal, 2007). The spectrum of CSNP
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depicts the characteristic peaks at 676, 617 and 499 cm−1 due to the stretching vibration of
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ZnS molecule (Salavati-Niasari, Ranjbar, & Ghanbari, 2012). It is interesting to note that the
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characteristic peaks of IONP are not visible in the spectrum of CSNP (Fig. 2d). This is due to
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the formation of core shell structures with high deposition of ZnS on the surface of IONP.
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XRD pattern of the IONP (Fig. 3a) shows the phases such as (220), (311), (400), (422),
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(511) and (440). The position of the entire indexed diffraction peaks matches well with that
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of Fe3O4 (JCPDS No. 85-1436). Fig. 3b shows the XRD pattern of CSNP, which depicts new
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phases such as (111) and (220), apart from the phases of Fe3O4. These new phases indexed in
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the CSNP indicate the presence of cubic zinc blende structure of ZnS, which matches well
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with the standard (JCPDS No. 05-566). These results indicate that the ZnS is well-
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crystallized and coated around the surface of the IONP (Salavati-Niasari, Ranjbar, &
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Ghanbari, 2012; Weesiong et al., 2010; Yu, Ji, Wang, Sun, & Du, 2010).
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Fig. 4 shows the EDX spectrum of IONP and CSNP. EDX spectrum of IONP show the
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presence of iron and oxygen at 75.2 and 23 wt.%, respectively (Fig. 4a). The presence of
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sulphur and chlorine elements in traces is also noted in the prepared IONP, which may be due
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to the unreacted FeCl3 and FeSO4. On the other hand, the CSNP show the presence of zinc,
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sulphur, iron and oxygen at 37.4, 10.5, 21.2 and 30.9 wt.%, respectively (Fig. 4b). These
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results substantiate the formation of CSNP with ZnS shells around the core of IONP.
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Fig. 4. EDX spectrum of (a) IONP and (b) CSNP; (c) Magnetization curves of IONP and
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CSNP analyzed using VSM. Inset shows the magnetization curve of CSNP showing the
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ferromagnetic behavior.
3.5. VSM analysis
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The magnetization curves of the IONP and CSNP are shown in Fig. 4c. The saturation
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magnetization of IONP is 58.69 emu/g at room temperature with 0 Oe coercivity
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demonstrating the ferromagnetic behavior. However, the saturation magnetization of CSNP is
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only 3.06 emu/g. The reduction in magnetic properties is due to the formation of core-shell
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structure over the surface of the IONP. It is known that ZnS is a diamagnetic II–VI
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semiconductor and hence the coating of ZnS on IONP is expected to reduce the magnetic
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properties.
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3.6. Electron microscopic studies High resolution scanning electron microscopic images showing the surface morphology of
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the synthesized IONP and CSNP are given in Fig. 5. The SEM micrographs of the IONP
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show that the average particle size is 16±1.5 nm and the particles are spherical (Fig. 5a). On
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the other hand, the morphology of CSNP is not clear due to the excessive deposition of ZnS
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on the surface of IONP leading to aggregation (Fig. 5b). High resolution transmission
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electron microscopic image of CSNP shows aggregated core-shell nanoparticles with
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spherical shape (Fig. 5c). The average particle size is estimated as 25±4 nm. The presence of
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dark iron oxide spherical nanoparticles surrounded by lighter ZnS confirms the formation of
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core-shell nanoparticles.
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Fig. 5. High resolution scanning electron microscopic images showing the morphology of (a)
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IONP and (b) CSNP; (c) High resolution transmission electron microscopic image showing
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the morphology of CSNP.
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3.7. Effect of temperature and pH
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The aminated GG attains a strong gel structure by interpenetrating polymeric networking
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without using any toxic crosslinking agents. The digital photographs of AGG, AGG-CSNP
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and AGG-CSNP-DOX hydrogel systems at 27 and 37°C are shown in Fig. 6a and 6b,
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respectively. When the AGG solution is sonicated and heated at 37°C, the gelation occurs 14 Page 14 of 22
and hydrogel forms. However, it does not form hydrogel at 0, 5 and 27°C. It is also observed
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that the formed hydrogel systems show remarkably high stability up to 95°C. Also, the
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hydrogel systems do not show any significant change in their structure at different pH
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conditions (pH 2 to 12). The results revealed that the prepared injectable hydrogel system is
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highly stable when compared to various other hydrogel systems reported based on synthetic
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polymers and toxic cross linking agents (Kim et al., 2012; Tan et al., 2012). Hence, it is
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possible to use the prepared thermo-responsive and thermo-stable hydrogel system for solid
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tumor treatment applications such as hyperthermia.
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3.8. In-vitro hydrogel stability and drug release studies
The injectable biodegradable hydrogel can offer a long-term theranostic and drug release
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to the infection site within the tumor tissues. Recently, it evolved as a direct treatment for the
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tumor tissues by preventing the side effects and providing long term diagnosis to the patients
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(Al-Abd, Hong, Song, & Kuh, 2010; Chun et al., 2009). Hence, the de-gelation studies are
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important to understand the stability of the hydrogel during the tumor treatment process. The
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in-vitro de-gelation studies of the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel
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systems are shown in Fig. 6c. It is seen that the maximum de-gelation for all the AGG, AGG-
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CSNP and AGG-CSNP-DOX hydrogel systems is around 90% after 20 day incubation. It is
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important to note that the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel systems do not
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show any significant difference in the de-gelation rate. This implies that the CSNP and DOX
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materials have evenly dispersed and interlaced in the interpenetrating polymeric network of
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the AGG hydrogel system. The results confirm that the CSNP and DOX do not alter the
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hydrogel system and provide long term stability, drug delivery and theranostic properties for
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the solid tumor treatment.
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Fig. 6. Digital images showing the physical state of AGG, AGG-CSNP and AGG-CSNP-
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DOX systems as a function of temperature at (a) 27°C and (b) 37°C; (c) In-vitro de-gelation
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patterns of AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel systems; (d) In-vitro drug
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release pattern of AGG-CSNP-DOX hydrogel.
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Fig. 6d shows the release of DOX from AGG-CSNP-DOX hydrogel system determined
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using UV-Visible spectrophotometer. A progressive linear release pattern of DOX is seen
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from 0th day up to 21st day of incubation demonstrating the sustained drug delivery. It is seen
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that the mean cumulative amount of DOX released was up to 92% after 21st day of
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incubation. It should be noted that the long term DOX release was primarily due to the strong
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interlacing of DOX within the aminated GG polymeric network chains. It is seen that the de-
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gelation rate is directly proportional to the rate of DOX release from the hydrogel. Hence, the
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results show a sustained CSNP and DOX release from the hydrogel. Hence, the results
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suggest that the prepared hydrogel can be used directly for drug delivery, hyperthermia and
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theranostic applications.
332 4. Conclusions
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A stable injectable magnetic nanoparticle incorporated hydrogel system was prepared as
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an alternative for the long-term drug release and diagnosis to the cancer patients. The
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injectable hydrogel system consisted of AGG as the biodegradable polymer and gelling
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agent, DOX as the anticancer drug and CSNP as the imaging agent without using any toxic
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crosslinkers. Studies reveal that a strong gel was formed by the reaction between NH3+
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groups and water molecules at 37°C and stable over a wide temperature and pH range. The
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in-vitro drug releasing tendency of DOX is seen up to 21st day of incubation demonstrating
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the sustained delivery over long period. Hence, the prepared thermoresponsive injectable
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hydrogel system has promising applications such as sustained drug release, hyperthermia and
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theranostic for the solid tumor treatment.
344 Acknowledgements
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Authors thank SERC, Department of Science and Technology, India for providing
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financial assistance under the Fast Track Scheme for Young Scientists (SERC/ET-
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0359/2011). We also thank SAIF, IIT Madras for providing the VSM and SEM facility.
17 Page 17 of 22
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351
Al-Abd, A. M., Hong, K. Y., Song, S. C., & Kuh, H. J. (2010). Pharmacokinetics of
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Bajpai, S. K., Saxena, S. K., & Sharma, S. (2006). Swelling behavior of barium ions-
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Graphical Abstract
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Highlights • Aminated guar gum and core shell nanoparticles were synthesized via simple method. • Injectable hydrogel system was prepared without using toxic crosslinkers. • Hydrogel forms at body temperature and is stable up to 95oC. • In-vitro drug release shows the sustained release of DOX for more than 21 days. • Inclusion of core shell nanoparticles would help in diagnosis of lesion sites.
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S0144-8617(14)00433-0 http://dx.doi.org/doi:10.1016/j.carbpol.2014.04.076 CARP 8837
To appear in: Received date: Revised date: Accepted date:
24-1-2014 24-3-2014 15-4-2014
Please cite this article as: Murali, R., Vidhya, P., & Thanikaivelan, P.,Thermoresponsive magnetic nanoparticle - aminated guar gum hydrogel system for sustained release of doxorubicin hydrochloride, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.076 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.
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Thermoresponsive magnetic nanoparticle - aminated guar gum hydrogel
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system for sustained release of doxorubicin hydrochloride
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Ragothaman Murali, Ponraj Vidhya, Palanisamy Thanikaivelan*
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Advanced Materials Laboratory, Centre for Leather Apparel & Accessories Development,
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Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar,
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Chennai 600020, India
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*Corresponding author. Tel/Fax: +91 44 24910953. E-mail address: [email protected] (P.
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Thanikaivelan) 1 Page 1 of 22
ABSTRACT
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Hydrogel based sustained drug delivery system has evolved as an immense treatment method
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for solid tumors over the past few decades with long term theranostic ability. Here, we
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synthesized an injectable hydrogel system comprising biocompatible aminated guar gum,
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Fe3O4-ZnS core-shell nanoparticles and doxorubicin hydrochloride. We show that amination
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of guar gum resulted in attraction of water molecules thereby forming the hydrogel without
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using toxic crosslinking agents. Hydrogel formation was observed at 37°C and is stable up to
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95°C. The prepared hydrogel is also stable over a wide pH range. The in-vitro studies show
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that the maximum de-gelation and drug release up to 90% can be achieved after 20 days of
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incubation. Studies reveal that the drug and the core-shell nanoparticles can be released
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slowly from the hydrogel to provide the healing and diagnosis of the solid tumor thereby
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avoiding several drug administrations and total excision of organs.
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Keywords: Solid tumor, Biomedical imaging, Aminated guar gum, Core shell nanoparticles,
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Doxorubicin HCl.
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1. Introduction Direct administration of chemotherapeutic agents in cancerous solid tumors has shown
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severe side effects in the system (Kintzel & Dorr, 1995). Injectable hydrogel is an alternative
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and effective system that can deliver the drug to specific tissues thereby reducing the drug
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side effects as well as the injection frequency (Arola et al., 2000; Kim et al., 2012). Currently,
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injectable hydrogel system comprising both therapeutic and diagnostic imaging agents is an
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emerging research area for effective guided therapy (Kim et al., 2012). Guar gum (GG) is one
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of the high-molecular weight, water-soluble, non-ionic biopolymer derived from the
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endosperm of guar beans, which has been widely used in the pharmaceuticals products
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primarily due to its unique ability to alter rheological properties (Burke, Park, Srinivasarao, &
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Khan, 2000). Several studies have been carried out to increase the mechanical, thermal and
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viscosity properties of the GG by chemical modification (Bajpai, Saxena, & Sharma, 2006;
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Huang, Lu, & Xiao, 2007). The chemically modified GG cross-linked with glutaraldehyde,
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phosphate, urea-formaldehyde or borax has been used for various applications such as
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controlled drug release (Soppirnath & Aminabhavi, 2002), colon-specific drug delivery
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(Chourasia & Jain, 2004; Gliko-Kabir, Yagen, Baluom, & Rubinstein, 2000), liquid pesticide
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(Kulkarni, Soppimath, Aminabhavi, Dave, & Mehta, 1999), water retention (Tayal, Pai, &
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Khan, 1999), synthetic cervical mucus (Burruano, Schnaare, & Malamud, 2002) and
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transdermal drug delivery systems (Narasimha Murthy, Hiremath, & Paranjothy, 2004) due to
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its high biocompatibility and biodegradability. However, the preparation of chemically cross-
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linked hydrogel using toxic crosslinkers poses significant limitations in the applications
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(Chang & Zhang, 2011).
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Inclusion of magnetic resonance imaging (MRI) agents in the injectable hydrogel helps in
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depiction of targeted agent and diagnosis of the lesion sites. Iron oxide nanoparticles (IONP)
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are prominently used as MRI agent for biological applications (Corot, Robert, Idee, & Port,
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2006). A suitable chemical modification has been indispensable for the IONP, which has
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intended to reduce the alteration of magnetic properties, agglomeration and rapid degradation
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(Kim et al., 2012; Santra et al., 2001). It has been used clinically for various biomedical
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applications, such as hyperthermia, drug delivery, tissue repair, cell and tissue targeting and
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transfection (Gupta & Gupta, 2005; Gupta, Naregalkar, Vaidya, & Gupta, 2007).
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Doxorubicin hydrochloride (DOX) is one of the most widely-used chemotherapeutic
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anticancer drugs, however, it initiates serious side effects such as cardiotoxicity, bone
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marrow and gastrointestinal toxicity if applied directly (Arola et al., 2000; Von Hoff et al.,
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1979). Several studies reveal that the encapsulated DOX reduces the toxicity and side effects
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of the drug (Gelperina et al., 2002; Yoo, Lee, Oh, & Park, 2000). It has been shown that the
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conjugation of DOX with the dextran and liposomes not only reduces the cytotoxicity but
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also enhances the efficiency of the drug (Mitra, Gaur, Ghosh, & Maitra, 2001; Wong et al.,
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2006). Here, an approach has been made to prepare aminated guar gum (AGG) from GG by
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reacting with ethylene diamine and sodium borohydride (Simi & Abraham, 2010). The
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reaction leads to the substitution of –OH group with –NH2 group at the 2nd, 3rd, and 6th
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position in the monosaccharide units of polysaccharide backbones. We also report a simple
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approach to prepare injectable hydrogel system using the AGG incorporated with iron oxide
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– zinc sulphide core shell nanoparticles (CSNP) and DOX, which could eliminate the
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limitations such as instability and cytotoxicity. The prepared AGG, IONP, CSNP and
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injectable hydrogel systems were investigated for the chemical, structural, magnetic and
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biological properties.
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2. Materials and methods
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2.1. Materials
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Ferric chloride (FeCl3) was purchased from M/s SD Fine-Chem, Mumbai, India. Ferrous
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sulphate (FeSO4.7H2O) and acetone were purchased from Merck, Mumbai, India.
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Ammonium hydroxide (25%) was procured from M/s Sisco Research Lab, Mumbai, India.
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Zinc acetate (Zn(CH3COO)2), sodium borohydride (NaBH4) and guar gum powder were
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procured from Himedia, India. Ethylene diamine (C2H8N2), sodium sulfide (Na2S) and
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doxorubicin HCl were purchased from Sigma-Aldrich Chemicals. All other reagents were of
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analytical grade.
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2.2. Synthesis of aminated guar gum
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The synthesis of AGG was carried out by the addition of C2H8N2 as an aminating agent to
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the GG, which substituted the -OH group of the GG with -NHCH2CH2NH2. Further the
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-NHCH2CH2NH2 group was reduced to -NH2 group by the addition of NaBH4 (Simi &
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Abraham, 2010). 1g (w/v) of GG was dissolved in 250 ml of distilled water and stirred to get
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homogenous solution for 30 min. 25 ml of ethylene diamine was added to the homogenous
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solution of GG and allowed to react with continuous stirring for overnight at room
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temperature. Then, 50 ml of 5% NaBH4 solution was added to the reaction mixture and stirred
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vigorously for 3 h. After the completion of the reaction, the mixture was precipitated and
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washed several times with acetone. The precipitated sample was lyophilized and then
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powdered to uniform particles.
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2.3. Synthesis of core shell nanoparticles
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The synthesis of CSNP was carried out in two steps. In the first step, iron oxide
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nanoparticles were synthesized. In the typical Fe3O4 nanoparticles synthesis, 20 ml of 0.1M
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FeCl3 and 20 ml of 0.05M FeSO4 were mixed and stirred for 30 min at room temperature. To
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this solution, NH4OH was added drop wise to attain pH 10. A black color precipitate was
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formed signifying the formation of Fe3O4 nanoparticles. The precipitate was washed
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repeatedly with acetone and dried at 55°C. In the second step, the Fe3O4 nanoparticles (10 ml,
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0.1% w/v) were sonicated to remove the agglomeration for 30 min at 30±2°C. After
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sonication, Na2S (5 ml, 0.15M) was added and stirred continuously for 30 min to facilitate
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deposition of sulphide ions on the surface of Fe3O4 nanoparticles. Then, 10 ml of 0.3M
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Zn(CH3COO)2 was added to the solution and stirred for 3 h. The precipitated CSNP were
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washed with acetone several times. The collected precipitate was dried at 55°C and then
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ground to make fine powder.
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The amino group in the AGG was determined by 2,4,6-trinitrobenzenesulfonic acid
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(TNBS) assay (Bubnis & Ofner III, 1992). It is known that the TNBS reacts with amines,
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hydrazines and hydrazides groups to form a stable trinitrophenyl complex. Briefly, 5 mg of
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samples from GG and AGG were dissolved in 1 ml of distilled water. To this solution, 1 ml
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of 4% w/v NaHCO3 and 1 ml of freshly prepared 0.5% (v/v) TNBS solution were added and
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kept at 60°C for 4 h. After that, 1 ml of the reacted solution was transferred to a fresh test
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tube and mixed with 3 ml of 6M HCl. The tubes were kept at 40°C for 1.5 h and the
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absorbance
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spectrophotometer (Schimadzu, UV-1800) at 420 nm. The percentage of amino group
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substituted in the GG was calculated using the equation below.
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using
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The GG, AGG, IONP and CSNP were analyzed using Perkin Elmer FT-IR spectrometer.
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The samples were ground with KBr and compressed to form pellets. The pellets were 6 Page 6 of 22
analyzed in a single beam mode at the range of 400 - 4000 cm-1 with an average of 4 scans
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and 2 cm-1 resolution. X-ray diffraction was performed to identify the crystallographic
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structure of IONP and CSNP by using Rigaku Miniflex (II) desktop diffractometer (Ni
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filtered CuKα radiation with λ=0.15418 nm). The samples were analyzed in the 2θ range of 10
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to 80° in a step of 0.02° and with a counting time of 10 sec per step. Elemental composition
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of IONP and CSNP was investigated through energy dispersive X-ray analysis (EDX) using
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Oxford Instruments NanoAnalysis (INCA Energy 250 Microanalysis System). The magnetic
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properties of the synthesized IONP and CSNP were measured using the vibrating sample
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magnetometer (VSM, Lakeshore, 7410 model) at room temperature. The morphology, size
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and structure of the IONP and CSNP nanoparticles were analyzed using high resolution -
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scanning electron microscopy (HRSEM, Quanta 200 series, FEI Company) and high
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resolution - transmission electron microscopy (HRTEM, JEOL 3010).
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2.5. Preparation of injectable hydrogel
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The injectable AGG-CSNP-DOX hydrogel (100/0.125/0.05 wt.%) was prepared by the
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following method as shown in Fig. 1. Initially, 25 ml of 0.01% w/v solution of CSNP was
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sonicated for 30 min at 30±2°C. After the complete dispersion, the AGG (2g) and DOX (1
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ml, 0.1% w/v) were added to the solution. These solutions were uniformly dispersed by
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sonication at 37°C for 15 min to form stable injectable AGG-CSNP-DOX (100/0.125/0.05
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wt.%) hydrogel. Likewise, the AGG (100 wt.%) and AGG-CSNP (100/0.125 wt.%)
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hydrogels were prepared by using the respective dispersed compositions as above.
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Fig. 1. Schematic showing (a) the synthesis of CSNP from IONP, (b) the synthesis of AGG
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from GG and (c) the preparation of AGG-CSNP-DOX injectable hydrogel system.
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2.6. Effect of temperature and pH on the hydrogel
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The effect of different temperature and pH conditions on the as-synthesized AGG, AGG-
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CSNP and AGG-CSNP-DOX hydrogel systems were studied. For studying the effect of
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temperature (0 to 95oC) on the hydrogel systems, temperature controlled heating water bath
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and ice bath were employed. For studying the effect of pH (2 to 12) on the hydrogel systems,
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1M NaOH and 1M HCl solutions were employed.
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2.7. In-vitro de-gelation studies
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The in-vitro de-gelation of the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel
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systems was carried out by incubating a known weight of hydrogel into the 25 ml of
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phosphate buffered saline (PBS) solution (pH 7.4) at 37°C. The weight of in-vitro de-gelated
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hydrogels was measured after removal of surface water using filter paper at different time
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periods. The percentage de-gelation was calculated by using the equation below.
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2.8. In-vitro drug release study The in-vitro drug release tendency was studied by incubating the AGG-CSNP-DOX
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injectable hydrogel into 25 ml PBS (pH 7.4) solution at 37°C. The amount of DOX released
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from the encapsulated hydrogel was determined at different time periods using UV-Visible
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spectrophotometer at 486 nm. The cumulative DOX release from the injectable hydrogel was
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calculated using the equation below.
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3. Results and discussion
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3.1. Degree of substitution
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The AGG was prepared by reacting C2H8N2 and NaBH4 with the GG. It is seen that the
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degree of substitution of amino group in guar gum is up to 61% as analyzed through the
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TNBS method. The results clearly indicate the presence of NH2 group in AGG and confirm
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the modification of GG. The presence of amino group in the GG biopolymer attracts water
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molecules thereby leading to the formation of hydrogel. The amination process expected to
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create ionic imbalances in the biopolymer due to the formation of NH3+ (Simi & Abraham,
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2010). We propose that the cationic charge attracts more water molecules in order to
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neutralize the system by forming AGG-NH3+ - –(δ-(OH--H+))n hydrogel. Each water molecule
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provides a partial negative charge (δ-) such that several water molecules can cumulatively
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satisfy and neutralize one positive charge of the AGG. Therefore, AGG with several cationic
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amino groups would require more number of water molecules to neutralize several positive
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charges thereby forming the hydrogel.
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3.2. FTIR analysis The FTIR spectrum of pure GG, AGG, IONP and CSNP are shown in Fig. 2. The FT-IR
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spectrum of pure GG depicts a broad absorption band at 3352 cm−1, which indicates the
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presence of significant quantity of hydroxyl groups in the structure (Fig. 2a). In the modified
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GG (Fig. 2b), this band became sharper and shifted from 3352 to 3423 cm−1 due to the
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substitution of -NH2 functional group to the -OH group in the structure (Simi & Abraham,
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2010). Further, the AGG shows appearance of a sharp peak around 1030 cm−1, which
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corresponds to C-N stretching vibration of aliphatic amine. Hence, these results are in
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agreement with the amino group determination study of the AGG.
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Fig. 2. FTIR Spectrum of (a) GG, (b) AGG, (c) IONP and (d) CSNP.
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FT-IR spectrum of IONP exhibits major bands at 1740, 1628, 1401, 1123, 1051, 593 and
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460 cm−1 (Fig. 2c). The band at 1628 cm-1 corresponds to hydroxyl group of moisture while
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the bands at 1740, 1401, 1123 and 1051 cm-1 correspond to C-O vibration arising from the
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presence of CO2 from air. The characteristic peaks seen at 593 and 460 cm−1 correspond to
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the Fe-O stretching vibration of the IONP (Maity & Agrawal, 2007). The spectrum of CSNP
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depicts the characteristic peaks at 676, 617 and 499 cm−1 due to the stretching vibration of
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ZnS molecule (Salavati-Niasari, Ranjbar, & Ghanbari, 2012). It is interesting to note that the
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characteristic peaks of IONP are not visible in the spectrum of CSNP (Fig. 2d). This is due to
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the formation of core shell structures with high deposition of ZnS on the surface of IONP.
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XRD pattern of the IONP (Fig. 3a) shows the phases such as (220), (311), (400), (422),
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(511) and (440). The position of the entire indexed diffraction peaks matches well with that
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of Fe3O4 (JCPDS No. 85-1436). Fig. 3b shows the XRD pattern of CSNP, which depicts new
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phases such as (111) and (220), apart from the phases of Fe3O4. These new phases indexed in
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the CSNP indicate the presence of cubic zinc blende structure of ZnS, which matches well
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with the standard (JCPDS No. 05-566). These results indicate that the ZnS is well-
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crystallized and coated around the surface of the IONP (Salavati-Niasari, Ranjbar, &
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Ghanbari, 2012; Weesiong et al., 2010; Yu, Ji, Wang, Sun, & Du, 2010).
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Fig. 4 shows the EDX spectrum of IONP and CSNP. EDX spectrum of IONP show the
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presence of iron and oxygen at 75.2 and 23 wt.%, respectively (Fig. 4a). The presence of
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sulphur and chlorine elements in traces is also noted in the prepared IONP, which may be due
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to the unreacted FeCl3 and FeSO4. On the other hand, the CSNP show the presence of zinc,
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sulphur, iron and oxygen at 37.4, 10.5, 21.2 and 30.9 wt.%, respectively (Fig. 4b). These
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results substantiate the formation of CSNP with ZnS shells around the core of IONP.
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Fig. 4. EDX spectrum of (a) IONP and (b) CSNP; (c) Magnetization curves of IONP and
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CSNP analyzed using VSM. Inset shows the magnetization curve of CSNP showing the
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ferromagnetic behavior.
3.5. VSM analysis
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The magnetization curves of the IONP and CSNP are shown in Fig. 4c. The saturation
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magnetization of IONP is 58.69 emu/g at room temperature with 0 Oe coercivity
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demonstrating the ferromagnetic behavior. However, the saturation magnetization of CSNP is
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only 3.06 emu/g. The reduction in magnetic properties is due to the formation of core-shell
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structure over the surface of the IONP. It is known that ZnS is a diamagnetic II–VI
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semiconductor and hence the coating of ZnS on IONP is expected to reduce the magnetic
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properties.
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3.6. Electron microscopic studies High resolution scanning electron microscopic images showing the surface morphology of
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the synthesized IONP and CSNP are given in Fig. 5. The SEM micrographs of the IONP
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show that the average particle size is 16±1.5 nm and the particles are spherical (Fig. 5a). On
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the other hand, the morphology of CSNP is not clear due to the excessive deposition of ZnS
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on the surface of IONP leading to aggregation (Fig. 5b). High resolution transmission
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electron microscopic image of CSNP shows aggregated core-shell nanoparticles with
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spherical shape (Fig. 5c). The average particle size is estimated as 25±4 nm. The presence of
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dark iron oxide spherical nanoparticles surrounded by lighter ZnS confirms the formation of
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core-shell nanoparticles.
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Fig. 5. High resolution scanning electron microscopic images showing the morphology of (a)
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IONP and (b) CSNP; (c) High resolution transmission electron microscopic image showing
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the morphology of CSNP.
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3.7. Effect of temperature and pH
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The aminated GG attains a strong gel structure by interpenetrating polymeric networking
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without using any toxic crosslinking agents. The digital photographs of AGG, AGG-CSNP
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and AGG-CSNP-DOX hydrogel systems at 27 and 37°C are shown in Fig. 6a and 6b,
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respectively. When the AGG solution is sonicated and heated at 37°C, the gelation occurs 14 Page 14 of 22
and hydrogel forms. However, it does not form hydrogel at 0, 5 and 27°C. It is also observed
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that the formed hydrogel systems show remarkably high stability up to 95°C. Also, the
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hydrogel systems do not show any significant change in their structure at different pH
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conditions (pH 2 to 12). The results revealed that the prepared injectable hydrogel system is
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highly stable when compared to various other hydrogel systems reported based on synthetic
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polymers and toxic cross linking agents (Kim et al., 2012; Tan et al., 2012). Hence, it is
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possible to use the prepared thermo-responsive and thermo-stable hydrogel system for solid
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tumor treatment applications such as hyperthermia.
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3.8. In-vitro hydrogel stability and drug release studies
The injectable biodegradable hydrogel can offer a long-term theranostic and drug release
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to the infection site within the tumor tissues. Recently, it evolved as a direct treatment for the
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tumor tissues by preventing the side effects and providing long term diagnosis to the patients
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(Al-Abd, Hong, Song, & Kuh, 2010; Chun et al., 2009). Hence, the de-gelation studies are
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important to understand the stability of the hydrogel during the tumor treatment process. The
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in-vitro de-gelation studies of the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel
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systems are shown in Fig. 6c. It is seen that the maximum de-gelation for all the AGG, AGG-
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CSNP and AGG-CSNP-DOX hydrogel systems is around 90% after 20 day incubation. It is
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important to note that the AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel systems do not
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show any significant difference in the de-gelation rate. This implies that the CSNP and DOX
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materials have evenly dispersed and interlaced in the interpenetrating polymeric network of
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the AGG hydrogel system. The results confirm that the CSNP and DOX do not alter the
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hydrogel system and provide long term stability, drug delivery and theranostic properties for
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the solid tumor treatment.
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Fig. 6. Digital images showing the physical state of AGG, AGG-CSNP and AGG-CSNP-
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DOX systems as a function of temperature at (a) 27°C and (b) 37°C; (c) In-vitro de-gelation
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patterns of AGG, AGG-CSNP and AGG-CSNP-DOX hydrogel systems; (d) In-vitro drug
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release pattern of AGG-CSNP-DOX hydrogel.
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Fig. 6d shows the release of DOX from AGG-CSNP-DOX hydrogel system determined
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using UV-Visible spectrophotometer. A progressive linear release pattern of DOX is seen
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from 0th day up to 21st day of incubation demonstrating the sustained drug delivery. It is seen
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that the mean cumulative amount of DOX released was up to 92% after 21st day of
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incubation. It should be noted that the long term DOX release was primarily due to the strong
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interlacing of DOX within the aminated GG polymeric network chains. It is seen that the de-
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gelation rate is directly proportional to the rate of DOX release from the hydrogel. Hence, the
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results show a sustained CSNP and DOX release from the hydrogel. Hence, the results
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suggest that the prepared hydrogel can be used directly for drug delivery, hyperthermia and
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theranostic applications.
332 4. Conclusions
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A stable injectable magnetic nanoparticle incorporated hydrogel system was prepared as
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an alternative for the long-term drug release and diagnosis to the cancer patients. The
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injectable hydrogel system consisted of AGG as the biodegradable polymer and gelling
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agent, DOX as the anticancer drug and CSNP as the imaging agent without using any toxic
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crosslinkers. Studies reveal that a strong gel was formed by the reaction between NH3+
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groups and water molecules at 37°C and stable over a wide temperature and pH range. The
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in-vitro drug releasing tendency of DOX is seen up to 21st day of incubation demonstrating
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the sustained delivery over long period. Hence, the prepared thermoresponsive injectable
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hydrogel system has promising applications such as sustained drug release, hyperthermia and
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theranostic for the solid tumor treatment.
344 Acknowledgements
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Authors thank SERC, Department of Science and Technology, India for providing
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financial assistance under the Fast Track Scheme for Young Scientists (SERC/ET-
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0359/2011). We also thank SAIF, IIT Madras for providing the VSM and SEM facility.
17 Page 17 of 22
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Graphical Abstract
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Highlights • Aminated guar gum and core shell nanoparticles were synthesized via simple method. • Injectable hydrogel system was prepared without using toxic crosslinkers. • Hydrogel forms at body temperature and is stable up to 95oC. • In-vitro drug release shows the sustained release of DOX for more than 21 days. • Inclusion of core shell nanoparticles would help in diagnosis of lesion sites.
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