Dibutyrylchitin nanoparticles as novel drug carrier

International Journal of Biological Macromolecules 82 (2016) 1011–1017

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Dibutyrylchitin nanoparticles as novel drug carrier Tanvi Jain a,b , Sushil Kumar a , P.K. Dutta b,∗ a b

Department of Chemical Engineering, M N National Institute of Technology, Allahabad 211004, India Department of Chemistry, M N National Institute of Technology, Allahabad 211004, India

a r t i c l e

i n f o

Article history: Received 30 September 2015 Received in revised form 27 October 2015 Accepted 12 November 2015 Available online 29 November 2015 Keywords: Nanoparticles Chitin Drug delivery 5-Fluorouracil Dibutyrylchitin

a b s t r a c t Chitin is a ubiquitous renewable biopolymer that is significantly distributed in the natural world. Biopolymeric nanoparticles (Nps) have been developed for various biomedical applications by researchers. Here, chitin derivative, dibutyrylchitin Nps (DBC) was synthesized as a nanocarrier for drug delivery using butyric anhydride and perchloric acid as a catalyst under heterogeneous conditions. The structural characterization was analyzed by FT-IR and FE SEM study showed spherical particles in a size range of 80–90 nm. The physiochemical evaluation involves swelling behavior and in vitro biodegradation studies. The results of in vitro hemolytic assay validate the blood compatibility of the prepared system. Drug release profiles indicate that 5-flourouracil (Fu) loaded dibutyrylchitin Nps (DBC-Fu) gives the enhanced drug release in acidic pH when compared to neutral pH. The encapsulation efficiency of DBC-Fu was found to be 90%. The confocal analysis also confirmed the uptake of both DBC and DBC-Fu Nps by A549 cell lines. Hence, this study shows that the DBC have the potential to be used as a drug carrier and also for other biomedical applications. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, a widespread range of biomaterials, such as dendrimers, lipids, amphiphilic polymers and surfactants, have been engaged as drug delivery carriers [1,2]. Among these, polysaccharides have established an increasing attention due to its stupendous physicochemical and biological characteristics [3–6]. Nps have been utilized broadly as carriers for drug delivery, which aim to enhance the bioavailability, solubility and permeability of many potent drugs which are difficult to deliver orally. The nanoparticle technology used in the recent decades has a great impact for improving the efficacy of the drug and their release profile studies. The foremost goal in designing Nps as delivery system is to release of pharmacologically active agents for site-specific encounter of drug at optimal proportion and dosages [7,8]. Nps are particulate dispersions or solid particles with a size range of 10–100 nm. The natural polymers such as chitin, chitosan, glycosaminoglycan, gelatin, alginate, etc. are more useful than any other synthetic polymer like PVA, PEG because they are having highly biocompatible, bioavailability, solubility and permeability of many potent drugs which are difficult to deliver orally especially for cancer treatment. A large number of researchers exist

∗ Corresponding author. Tel.: +91 532 2271283. E-mail address: pkd [email protected] (P.K. Dutta). http://dx.doi.org/10.1016/j.ijbiomac.2015.11.031 0141-8130/© 2015 Elsevier B.V. All rights reserved.

today on the use of chitin and its derivatives as it is safe biomaterial for various biomedical functions: there are recent review articles in biomedical sciences [9–13], engineering sciences [14,15] and in pharmaceutical sciences [16]. Chitin is such a polymer, and its bioactive impact on human organisms has been proved by many investigations [17–19]. Chitin and its derivatives are being broadly utilized for the synthesis of nanocarriers for drug delivery applications. It is also being used in the fabrication of a variety of medical devices, and regenerative medical components, due to its high crystallinity, biochemical significance and biocompatibility [20]. The inherent and unique properties of large and active surface area are being explored in the fields of engineering, technology and medicine like drug delivery [21]. It is necessary to deliver a drug through a nanocarrier that has to be efficiently removed from the body after delivering drugs and does not cause any harmful effects in the body. In other words, it must not accumulate in the body nor must it be toxic [22–24]. Chitin derivatives such as carboxymethyl chitin, chitosan hydrogel, hydroxyethyl chitin etc., have been shown to hold such features. The main limitation of chitin is the insolublilty in water due to its intermolecular hydrogen bonds. Due to the difficulty in processing of chitin various chitin derivatives such as chitosan, carboxymethyl chitin, sulfated chitin, aminoethylchitin, oxychitin and dibutryl chitin were formed for biomedical engineering applications. One of the most important traits of chitin is its ability (flexibility) to be shaped into different forms such as fibers, nanoparticles, hydrogels,

1012

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

continuously stirred overnight under fume hood at room temperature and thus isolated by using vacuum drying at 70 ◦ C. 2.3. Preparation of dibutyrylchitin Nps (DBC)

Fig. 1. Synthesis of dibutrylchitin from chitin.

and films. Dibutyrylchitin is a fresh entry in the short list of modified chitins of interest in medicine; it is soluble in common solvents such as ethanol, methanol and preserves the filmogenic characteristic of plain chitin; for all these reasons, dibutyrylchitin can be easily spun to manufacture threads, filaments and non-woven fibers [25] and for all other biomedical applications as well. It is a chitin derivative which is created during the esterification process of chitin using butyric anhydride where 72% perchloric acid is used as a catalyst. Decent solubility of DBC in presence of numerous organic solvents (ethanol, dimethyl sulfoxide (DMSO), acetone, etc.) results from the presence of bonding butyryl groups in chitin [26]. The initial investigations of the biological properties of DBC materials showed good biocompatibility of the polymer [27–32]. Dibutyrylchitin, which is not been utilized in the field of nanomedicine or not used for drug delivery. Hence, this study will focus on synthesis of dibutyrylchitin and its Nps used for drug delivery of 5-Flourouracil and performing physiochemical evaluation, antibacterial evaluation, drug release profile and their characterization. 2. Material and methods 2.1. Materials Chitin powder, 5-flourouracil (Fu) and butyric anhydride were obtained from Sigma Aldrich Pvt. Ltd. (USA), 70% perchloric acid was obtained from Spectrochem Pvt. Limited (Mumbai). Sodium hydroxide was obtained from Ranbaxy laboratories. Ethanol was purchased from Merck India. Lysozyme was purchased from Alfa Aesar, Muller Hinton Agar (MHA) was obtained from Himedia Pvt. Ltd. Mumbai (India) for antibacterial activity. All chemicals were used without additional purification. The test strain, Escherichia coli (MTCC 739) a gram negative bacteria, Staphylococcus aureus (MTCC 3160) a gram positive bacteria, were obtained from IMTECH, Chandigarh, India. Dulbecco’s modified eagle medium: nutrient mixture Ham’s F-12 powder (DMEM. F12, Fetal Bovine Serum (FBS), Antibiotic Antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin, 25 ␮g/mL amphotericin-B), Trypsin–EDTA (1 × 0.25%), (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye, 2 ,7 -dichlorofluorescin diacetate was purchased from Invitrogen Co. (Carlsbad, CA, USA). Human lung carcinoma cell lines (A549) were received from the National Center for Cell Sciences, Pune, India. All the chemicals used are of AR grades.

Briefly, 50 mg of dibutyrylchitin was dissolved in 20 mL of ethanol for 1–2 h at room temperature. The solution was then filtered to remove unmixed traces if found in the solution [34]. Next, the Nps were obtained by repeated centrifugation and probe sonication at least 5–6 times so that the particle size comes into nanoregime. The prepared DBC Nps was then washed with distilled water and isolated by drying in vacuum. 2.4. Preparation of drug loaded dibutyrylchitin Nps (DBC-Fu) Drug-loaded (Fu) Nps were prepared by the emulsion crosslinking process. DBC solution was prepared after adding 50 mg of DBC in 20 mL of ethanol. Then, 5-fluorouracil (Fu) (1 mg/mL) was dissolved into the above prepared DBC solution under continuous sonication. This initial emulsion was stirred for 2 h. The prepared solution was then stirred for 2–3 h so that colorless solution changes to whitish color and thus pellet were then resuspended in water with the help of ultrasonication. Repeated centrifugation and sonication of the pellet in distilled water removes the ethanol. The prepared sample is then kept for drying in vacuum at 50 ◦ C. 2.5. Characterization In the present studies Fourier transform infrared spectroscopy spectra (FT-IR) of desired samples have been carried out using Perkin Elmer Spectrum RXI-FTIR spectrophotometer with diffuse reflectance accessory in the wave-number range 400–4000 cm−1 . The surface morphologies of the synthesized Nps were studied by field emission scanning electron microscopy (FE SEM) after the gold sputter coating of the samples. The absorbance for various tests was analyzed by using UV–vis spectrophotometer (Shimadzu UV spectrophotometer, USA). 2.6. Swelling study The swelling behavior of the prepared DBC Nps was examined under three different pH as acidic, neutral and basic, since then drug carrier was a nanoparticle, which alters the physical traits with respect to its pH. All the dried Nps were pelletized by using a hydraulic pelletizer. The dry weight of the formed pellet was taken (Wd). The pellet was then positioned in a corresponding pH solution for 1 h, and afterwards removed from the medium and the adsorbed medium at surface was then removed using Whatman filter paper and wet weight was taken (Ww). Three different pellets were utilized for this test [35]. The swelling ratio was determined from the following equation: Swelling ratio (SR) =

Ww − Wd Wd

2.7. In vitro biodegradation study 2.2. Preparation of dibutyrylchitin from chitin The synthesis of dibutyrylchitin was carried out under heterogeneous environment using butyric anhydride, chitin and 70% perchloric acid in approximate amount equal to 1:10:1 (g/g) [33] was shown in Fig. 1. Briefly, 1 g of chitin powder was dissolved into 10 mL of butyric anhydride under fume hood continuously for 4 h. After that, 1 mL of 70% perchloric acid was added while stirring which acts as a catalyst. Then the prepared solution was

Lysozyme is abundantly present in all body secretions. Therefore, it can be used to study the degradability of the prepared Nps in vitro. The pellets of the synthesized Nps of known weight were put in 1 mL PBS pH 7.4 containing hen egg white lysozyme and one without lysozyme, in the concentration of 1 mg/mL of the buffer. The PBS solution was kept for several days at 37 ◦ C in an incubator and changed every day to compensate for the degradation of the enzyme at such a high temperature. After regular intervals of days,

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

the samples were dried and weighed to observe change in weight [35,36].

1013

in a well which was created in the center of the agar plate. This incubation was done for 12 h at 37 ◦ C and finally zone of inhibition was estimated and analyzed.

2.8. In vitro drug release 2.12. Tagging of Rhodamine-B dye with DBC (Rhod-DBC) & DBC-Fu (Rhod-DBC-Fu)

Since the normal pH of the blood is 7.4, phosphate buffer saline (PBS) of the same pH was used to perform the study. DBC-Fu Nps of known weight were kept in 10 mL PBS buffer and in acidic medium and put on a shaker at 37 ◦ C. 1.5 mL of aliquot were removed from the solution at regular time intervals and its absorbance recorded on Shimadzu UV–vis spectrophotometer, USA at ambient temperature after 1, 2, 3, 4, 5, 6, 7 and 24 h at wavelength 262 nm for Fu. The same amount of buffer and acid was replaced in the solution at regular intervals.

The fluorescent dye Rhodamine-B was tagged with the prepared Nps by the method [34–36]. Briefly, add 40 ␮L solution of 1 mg/mL concentration of Rhodamine-B to 3 mL of DBC and DBC-Fu solution under continuous stirring in dark conditions for 2–3 h. This was then sonicated for 10 min and continued magnetic stirring for another 2 h and further centrifuged for 30 min at 10,000 rpm to remove the unbound Rhodamine-B.

2.9. Drug encapsulation efficiency

2.13. Cell culture

A 10 mL of the freshly prepared nanosuspension was centrifuged at 3000 rpm for 20 min. The amount of unincorporated drug was calculated by taking the absorbance of properly diluted supernatant solution 262 nm for Fu using single beam UV spectrophotometer against blank/control nanosuspension. By subtraction from the initial amount of drug taken, encapsulation efficiency was calculated with the help of drug absorbance curve [37]. Using above method, Drug encapsulation efficiency was determined by the following equation:

A549 cell line (Human lung carcinoma, NCCS Pune) was maintained in Dulbecco’s modified Eagles Medium (DMEM) complemented with 10% fetal bovine serum (FBS). The cells were then incubated in CO2 incubator with 5% CO2 . The cells were detached from the flask with Trypsin (1 mL) after reaching confluency. This cell suspension was centrifuged at 800–1000 rpm for 5 min and then resuspended in the growth medium for further study.

EE =

Total amount of Drug − Amount of free Drug × 100 Total amount of Drug

2.10. In vitro hemolysis assay of dibutyrylchitin Nps In vitro hemolytic assay of DBC and DBC-Fu Nps were done based on the protocol as explained in the literatures [34,38]. Fresh blood was taken and collected in to acid citrate dextrose containing vials. Different concentrations ranging from 1to10 mg/mL were then added to 1 mL of blood and incubated it for 3 h at 37 ◦ C. Then, all the samples were centrifuged at 4000 rpm for 20 min to attain the plasma. This plasma was thus accumulated and was added in 1 mL of Na2 CO3 . The OD values were finally taken at 450, 380 and 415 nm. The plasma hemoglobin can be calculated using the following equation. Plasma Hb =





(2 A415 ) − [A380 + A450 ] × 76.25

The OD values attained for all the samples were then evaluated with that of Triton X-100 as a positive control and normal saline as a negative control. 2.11. Antibacterial assessment The antibacterial assessment of the Nps was estimated through agar diffusion method [39,40]. Briefly, nutrient agar (14 g in 500 mL water) and nutrient broth (1.3 g in 100 mL water) were prepared and autoclaved at 121 ◦ C, 15 psi for 15 min. Nutrient Broth was used as growing medium for the bacteria E. coli (Gram negative) and Staphylococcus aureus (Gram negative). Nutrient agar was poured in petridish and loopful of both the bacterial strains was streaked on nutrient agar and thus incubated at 37 ◦ C for 24 h to generate the single colonies. A typical bacteria colony was taken off with the help of a inoculating loop, placed in already sterilized nutrient broth and then incubated overnight at 37 ◦ C for 12 h. By appropriately diluting with sterile distilled water and nutrient broth, the cultures of bacteria containing ∼107 CFU/mL were formed. Then prepared bacteria medium was dispensed on to agar plate and finally 1 mL of each sample solution was placed on to agar plate

2.13.1. Cell uptake studies For cell uptake studies, Rhod-DBC and Rhod-DBC-Fu were used. 24 well plates were loaded with A549 cells with a density ∼ of 20,000 cells per well incubated for 24 h for the cells to grow and get attach to the walls of the wells. Then the media was removed after incubation and the wells were cautiously washed with filtered PBS buffer solution several times. Then the sample particles, at a concentration of 1 mg/mL, were added along with the media to the wells and incubated for a period of 6 h. After incubation time, media with sample was removed from the wells and wells can directly processed for confocal microscopy. Preprocessing involves the washing of the wells with PBS and then fixing the cells in 4% paraformaldehyde (PFA). The wells were then analysed under confocal microscope (EVOS Microscope) to study the internalization of DBC and DBC-Fu by the cells. 2.13.2. Cell viability: MTT assay Cell viability was carried out by MTT assay [41] using A549 cells. MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium] assay for cytotoxic evaluation is a colorimetric test based on the selective ability of viable cells to reduce the tetrazolium component of MTT to purple-colored formazan crystals. The cells were grown on a 96 well plate with a density of 10,000 cells/cm2 . Different concentrations of the prepared Nps (DBC & DBC-Fu) were prepared. Then the cells were washed with PBS buffer and different concentrations of all the prepared samples were added and incubated after reaching 90–95% confluency. After 24 h, 10 ␮L MTT (5 mg MTT/mL) was added and incubated for 2 h. After, incubation 200 ␮L dimethylsulfoxide was added in each well and incubated at room temperature for 1 h to suspend the formazan crystals. The optical density of the solution was finally measured at a wavelength of 570 nm using Microplate Reader (Omega Fluostar). 3. Results and discussion 3.1. FT-IR spectra analysis The potential chemical interaction within the constituents DBC and Nps were studied by FT-IR spectra. FT-IR spectra of DBC-Fu nanoparticle was recorded and compared with DBC Nps and Fu. In Fig. 2(a), Fu showed its characteristic absorption peaks in the

1014

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

1658 cm−1 (DBC) and 1639 cm−1 (Fu) due to the formation of weak intermolecular hydrogen bonding between both of them. 3.2. FE SEM characterization The FE SEM images of prepared DBC Nps is shown in Fig. 3. The size of the prepared DBC Nps is around 80–90 nm is shown in Fig. 3(a) and (b). The FE SEM image confirms that the particle shape is spherical in shape and very dense. The figure also confirms that the synthesized Nps has smooth surface whereas the size of the DBC-Fu Nps. Fig. 3(c) and (d) is around 100–110 nm which may help in skin permeation. 3.3. Swelling ratio Swelling ratio is a property of the Nps which indicates its behavior in the presence of body fluids. It also plays a vital role in studying the drug uptake and release. The comparative analysis shows that DBC Nps show the highest swelling ratio in acidic medium than neutral and basic medium. Fig. 4(a) shows the analysis of swelling of prepared Nps. The reason is that all the polyelectrolytes like chitin/chitosan and its other derivatives swells at pH below pKa [35]. Fixed charges are established on Nps when exposed to acidic pH. 3.4. In vitro biodegradation study Fig. 2. FT-IR spectra (a) 5-Fu (b) DBC nanoparticles (c) DBC-Fu nanoparticles.

region 3000–2900 cm−1 due to C H stretching, 1425 cm−1 corresponding to the C N and C C ring stretching vibrations, bands at 1344 cm−1 due to interaction and vibration in the pyrimidine compounds [42,43]. In the spectra of DBC shown in Fig. 2(b), the strong absorption peak at 1744 cm−1 showed the overlapped the ester in DBC butyl anhydride. DBC characteristic peaks at 1658 cm−1 , 1552 cm−1 , 1456 cm−1 and 1206 cm−1 was shown with the cumulative DBC contents, and the broad peak was further disturbed by adding Fu [30,32]. Fig. 2(c) shows the FT-IR spectra of DBC-Fu. Here, there was a heightening in peak intensity and also a shift in peak at

It is well known fact that chitin and its derivatives are biopolymers that are biodegradable even though analysis of lysozyme assisted in vitro biodegradation was performed to verify the biodegradability of the synthesized Nps. Lysozyme is a protein digesting enzyme that is commonly found in body fluids. It is the main protease which degrades chitin. Hen egg white lysozyme was used for the study since it is most similar to human lysozyme. In general, chitin and its derivatives can be easily degraded with lysozyme in human body as lysozyme is an enzyme which directly attacks glycosidic linkages from which N-acetyl glucosamine & dglucose amine monomers can be formed as its degrading product. Without lysozyme, cleavage begins in amorphous area that is auto catalyzed by carbonyl groups [44]. The degradation of DBC Nps in

Fig. 3. SEM images of nanoparticles: (a and b) DBC (c and d) DBC-Fu.

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

1015

Fig. 4. (a) Swelling study of DBC nanoparticles (b) In vitro biodegradation analysis.

Fig. 5. Drug Release Profile of DBC-Fu nanoparticles in neutral and acidic medium.

PBS showed that at the regular intervals of around 30 days, samples with lysozyme degrade at faster rate as compared to samples without lysozyme. Fig. 4(b) shows the in vitro biodegradation of DBC Nps. DBC Nps degrade upto 91% and 63% with lysozyme and without lysozyme, respectively, which clearly indicates that the prepared drug carrier is biodegradable compound and it can easily degraded in in vivo conditions. 3.5. Drug release profile study Fig. 5 shows the drug release profile of prepared DBC-Fu Nps in acidic as well as neutral medium which shows immediate release in 1–2 h. After about 24 h, the prepared Nps showed a sustained release of the drug. The drug release profile shows 70% of the drug release in neutral medium whereas in acidic medium, the total drug release was found to be around 80%. This analysis show that 5flourouracil under in vitro conditions illustrates quick degradation in acidic pH than in neutral pH and which may support the site specific release of the drug through the synthesized carrier as pH of the tumor site is also acidic in nature [35,42].

Fig. 6. Antibacterial activity (ZOI in mm) of prepared nanoparticles: DBC (a) E. coli (b) B. subtilis; DBC-Fu (c) E. coli (d) B. subtilis.

3.6. In vitro hemolysis assay and encapsulation efficiency of dibutyrylchitin Nps The hemolytic ratio of the prepared sample for DBC and DBCFu Nps was found to be 4.15% & 5.76% which is significantly below 10%, the safe hemolytic ratio for bio-materials according to ISO/TR 7406. This signifies that the risk of hemolysis by all the prepared Nps are not alarming or harmful. The encapsulation efficiency was also calculated which was found to be 90% in DBC-Fu Nps.

3.7. Antibacterial assessment The antibacterial tests analysis shown in Fig. 6 depicts the clear zone of inhibition (in mm) of all prepared Nps. Here, DBC-Fu Nps exhibits more effective zone of inhibition whereas DBC Nps exhibits slightly lesser antibacterial activity. Chemically prepared derivatives of chitin from shrimp shell is highly active antibacterial agent that native chitin [45]. Here, the former consists of drug (Fu) which enhances the inhibition zone as shown in Fig. 6(c) and (d) and thus responsible for effectual and improved antibacterial properties.

1016

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

Fig. 7. Cellular localization on A549 cells after a 6 h incubation period by confocal microscopy of (a) DBC (b) DBC-Fu nanoparticles.

4. Conclusion The biodegradable and biocompatible dibutyrylchitin had been successfully prepared from chitin and its Nps had been synthesized. Fu was effectively encapsulated in DBC Nps. FT-IR and FE SEM confirmed the successful formation of DBC and DBC-Fu Nps as both are nanospherical in shape and very dense. Swelling and in vitro drug release studies revealed that the Nps show the increased and improved swelling and it release in acidic medium in comparison to neutral and basic medium. The drug release was found to be 70% in neutral medium whereas in acidic medium, the total drug release was found to be around 80%. The hemolysis assay also shows that the prepared Nps are hemocompatible. The antibacterial assessment proves that the prepared Nps are more antibacterial as when compared to native chitin. Cell culture studies also confirm the internalization of the Nps inside A549 cells and showing more cell deaths in case of DBC-Fu Nps. The overall analysis of all the evaluation lead to the conclusion that DBC Nps showing good characteristics of becoming a new carrier for the drug delivery systems. Fig. 8. Cell cytotoxicity on A549 Cells of DBC and DBC-Fu nanoparticles.

Acknowledgements 3.8. Cellular uptake studies Fig. 7(a) and (b) shows the internalization of DBC and DBC-Fu Nps in A549 cells after an incubation period of 6 h and thus has observed by confocal microscopy. It was clear from the fluorescence images of the cells that there is an internalization of both the Nps in the cell and the cells are alive.

3.8.1. Cell cytotoxicity: MTT assay The MTT assay analysis shown in Fig. 8 indicates that higher rate of cell death in case of DBC-Fu Nps whereas decent toxicity was seen in case of DBC Nps. The reason for the more cell death in case of DBC-Fu NPs due to the presence of Fu which itself is toxic and thus causing cell apoptosis. Fu is a totally a S-phase active drug with null activity when cells are in either G0 or G1 phase. Fu has a convoluted mechanism of action with various enzymes included in its metabolic activation. It inhibits thymidylate synthase as its key mechanism of action leading to depletion of dTTP which ultimately cause harm to DNA of the cell because of the due to addition of FdUTP into DNA which ultimately stops the cell proliferation and its survival [42].

One of the authors (TJ) wishes gratefulness to Director Prof. P. Chakrabarti, Motilal Nehru National Institute of Technology, Allahabad, India, for providing the stipend. TEQIP II Grant of the Institute who provides financial support for the research work. The author is also thankful to Material Research Centre (MRC) MNIT Jaipur for FE SEM and FTIR analysis along with Ms. Sushma Chaudhary, PhD scholar, CSIR-IITR Lucknow for cell cytotoxicity tests.

References [1] V.V. Torchilin, Antibody-modified liposomes for cancer chemotherapy, Exp. Opin. Drug Delivery 5 (2008) 1003–1025. [2] N.M. Dand, P.B. Patel, A.P. Ayre, V.J. Kadam, Polymeric micelles as a drug carrier for tumor targeting, Chron. Young Sci. 4 (2013) 94–101. [3] Z. Liu, Y. Jiao, Y. Wang, C. Zhou, Z. Zhang, Polysaccharides-based nanoparticles as drug delivery systems, Adv. Drug Delivery Rev. 60 (2008) 1650–1662. [4] N. Deepa, R. Jayakumar, K.P. Chennazhi, Versatile carboxymethyl chitin and chitosan nanomaterials: a review, WIREs Nanomed. Nanobiotechnol. 6 (2014) 574–598. [5] M. Yadollahia, S. Farhoudiana, H. Namazi, One-pot synthesis of antibacterial chitosan/silver bio-nanocomposite hydrogel beads as drug delivery systems, Int. J. Biol. Macromol. 79 (2015) 37–43. [6] P. Garg, S. Kumar, S. Pandey, S. Hoon, P.H. Choung, J. Koh, J.H. Chung, Triphenylamine coupled chitosan with high buffering capacity and low

T. Jain et al. / International Journal of Biological Macromolecules 82 (2016) 1011–1017

[7]

[8] [9]

[10] [11]

[12]

[13]

[14] [15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

viscosity for enhanced transfection in mammalian cells, in vitro and in vivo, J. Mater. Chem. B 1 (2013) 6053–6065. R.A.A. Muzzarelli, Chitosan composites with inorganics, morphogenetic proteins and stem cells, for bone regeneration, Carbohydr. Polym. 83 (2011) 1433–1445. P.K. Dutta, R. Srivastava, J. Dutta, Functionalized nanoparticles and chitosan-based functional nanomaterials, Adv. Polym. Sci. 254 (2013) 1–50. D. Archana, B.K. Singh, J. Dutta, P.K. Dutta, Chitosan-PVP-nano silver oxide wound dressing: in vitro and in vivo evaluation, Int. J. Biol. Macromol. 73 (2015) 49–57. P.L. Kashyap, X. Xiang, P. Heiden, Chitosan nanoparticle based delivery systems for sustainable agriculture, Int. J. Biol. Macromol. 77 (2015) 36–51. S. Kumar, J. Dutta, P.K. Dutta, Preparation and characterization of N-heterocyclic chitosan derivatives based gels for biomedical applications, Int. J. Biol. Macromol. 45 (2009) 330–337. C. Deng, F.F. Li, M. Griffith, M. Ruel, E.J. Suuronen, Application of chitosan-based biomaterials for blood vessel regeneration, Polym. Org. Chem. 297 (2010) 138–146. H. Kumar, R. Srivastava, P.K. Dutta, Highly luminescent chitosan-l-cysteine functionalized CdTe quantum dots film: synthesis and characterization, Carbohydr. Polym. 97 (2013) 327–334. B.K. Park, M.M. Kim, Applications of chitin and its derivatives in biological medicine, Int. J. Mol. Sci. 11 (2010) 5153–5165. T. Jain, S. Kumar, P.K. Dutta, Theranostics: a way of modern medical diagnostics and the role of chitosan, J. Mol. Genet. Med. 9 (2015) 1–5. R. Srivastava, D.K. Tiwari, P.K. Dutta, 4-(Ethoxycarbonyl) phenyl-1-amino-oxobutanoic acid-chitosan complex as a new matrix for silver nanocomposite film: preparation, characterization and antibacterial activity, Int. J. Biol. Macromol. 49 (2011) 863–870. R.A.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and insulin: a tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial, Carbohydr. Polym. 87 (2012) 995–1012. G.A. Morris, M.S. Kok, S.E. Harding, G.G. Adams, Polysaccharide drug delivery systems based on pectin and chitosan, Biotechnol. Genet. Eng. Rev. 27 (2010) 257–283. R.A.A. Muzzarelli, in: R. Ito, Y. Matsuo (Eds.), Chitosans: New Vectors for Gene Therapy in Handbook of Carbohydrate Polymers: Development, Properties and Applications, NOVA Publishers Hauppauge, New York, NY, 2010, pp. 583–604. A. Chaudhury, S. Das, Recent advancement of chitosan-based nanoparticles for oral controlled delivery of insulin and other therapeutic agents, AAPS Pharm. Sci. Tech. 12 (2011) 10–20. C.J. Knill, J.F. Kennedy, in: C.N.R. Rao, A. Müller, A.K. Cheetham (Eds.), Nanomaterials Hemistry: Recent Developments and New Directions, Wiley-VCH, Weinheim, Germany, 2007, pp. 403–418. T. Saito, Y. Nishiyama, J. Putaux, M. Vignon, A. Isogai, Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose, Biomacromolecules 7 (2006) 1687–1691. M. Ishihara, K. Obara, S. Nakamura, M. Fujita, K. Masuoka, Y. Kanatani, B. Takase, H. Hattori, Y. Morimoto, M. Ishihara, T. Maehara, M. Kikuchi, Chitosan hydrogel as a drug delivery carrier to control angiogenesis, J. Artif. Organs. 9 (2006) 8–16. T. Jain, P.K. Dutta, Chitin and carboxymethylchitin nanoparticles for drug delivery: preparation, characterization and evaluation, Asian Chitin J. 8 (2012) 7–12. K. Van de Velde, P. Kiekens, Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state C-13 NMR, Carbohydr. Polym. 58 (2004) 409–416.

1017

[26] D. Paluch, S. Pielka, L. Szosland, J. Staniszewska-Ku´s, M. Szymonowicz, L. ˙ Biological investigation of regenerated chitin fibres, Eng. Solski, B. Zywicka, Biomater. 12 (2000) 17–22. [27] Y. Oshimoma, Y.K. Nishino, Y. Yonekura, S. Kishimoto, S. Wakabayashi, Clinical application of chitin non-woven fabric as wound dressing, Eur. J. Plast. Surg. 10 (1987) 66–69. [28] R.A.A. Muzzarelli, M. Guerrieri, G. Goteri, C. Muzzarelli, T. Armeni, R. Ghiselli, M. Cornellisen, The biocompatibility of dibutyryl chitin in the context of wound dressings, Biomaterials 26 (2005) 5844–5854. [29] D. Paluch, L. Szosland, J. Staniszewska-Ku´s, L. Solski, M. Szymonowicz, E. ˛ The biological assessment of chitin fibres, Polym. Med. 5 (2000) Gebarowska, 3–32. ´ [30] L. Szosland, I. Krucinska, R. Cisło, D. Paluch, J. Staniszewska-Ku´s, L. Solski, M. Szymonowicz, Synthesis of dibutyrylchitin and chitin for medical applications, Fibres Text. East. Eur. 9 (2001) 54–57. [31] S. Pielka, D. Paluch, J. Staniszewska-Ku´s, B. Zywicka, L. Solski, L. Szosland, A. ´ Wound healing accelerating by a textile dressing Czarny, E. Zaczynska, containing dibutyrylchitin and chitin, Fibres Text. East. Eur. 11 (2003) 79–84. [32] I.H. Jeon, J.Y. Mok, K.H. Park, H.M. Hwang, M.S. Song, D. Lee, M.H. Lee, W.Y. Lee, K.Y. Chai, S.I. Jang, Inhibitory effect of dibutyryl chitin ester on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 cells, Arch. Pharm. Res. 35 (2012) 1287–1292. [33] A. Chilarski, L. Szosland, D. Binias, J. Szumilewicz, Novel dressing materials accelerating wound healing made from dibutyrylchitin, Fibres Text. East. Eur. 63 (2007) 77–81. [34] M. Sabithaa, N.S. Rejinold, A. Nair, V.K. Lakshmanan, V.N. Shantikumar, R. Jayakumar, Development and evaluation of 5-fluorouracil loaded chitin nanogels for treatment of skin cancer, Carbohydr. Polym. 91 (2013) 48–57. [35] S. Mangalathillam, N.S. Rejinold, A. Nair, V.K. Lakshmanan, S.V. Naira, R. Jayakumar, Curcumin loaded chitin nanogels for skin cancer treatment via the transdermal route, Nanoscale 4 (2012) 239–250. [36] H. Liu, Y. Du, X. Wang, Y. Hu, J.F. Kennedy, Interaction between chitosan and alkyl ␤-d-glucopyranoside and its effect on their antimicrobial activity, Carbohydr. Polym. 56 (2004) 243–250. [37] T. Thanpitch, A. Sirivat, A.M. Jamieson, R. Rujiravanit, Polyaniline nanoparticles with controlled sizes using a cross-linked carboxymethyl chitin template, J. Nanopart. Res. 11 (2009) 1167–1177. [38] A. Dev, J.C. Mohan, V. Sreeja, H. Tamura, G.R. Patzke, F. Hussain, S. Weyeneth, S.V. Nair, R. Jayakumar, Novel carboxymethyl chitin nanoparticles for cancer drug delivery applications, Carbohydr. Polym. 79 (2010) 1073–1079. [39] P.K. Dutta, S. Tripathi, G.K. Mehrotra, J. Dutta, Perspectives for chitosan based antimicrobial films in food applications, Food. Chem. 114 (2009) 1173–1182. [40] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Preparation and physicochemical evaluation of chitosan/poly(vinyl alcohol)/pectin ternary film for food-packaging applications, Carbohydr. Polym. 79 (2010) 711–716. [41] D. Angelis, D.H. Svendsrud, K.L. Kravik, T. Stokke, Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery, Mol. Cancer 18 (2006) 5–20. [42] E.B. Yassin, M.K. Anwer, H.A. Mowafy, I.M. Elbagory, M.A. Bayomi, I.A. Alsarra, Optimization of 5-flurouracil solid-lipid nanoparticles: a preliminary study to treat colon cancer, Int. J. Med. Sci. 7 (2010) 398–408. [43] F.H. Lin, Y.H. Lee, C.H. Jian, J.M. Wong, M.J. Shieh, C.Y. Wang, A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier, Biomaterials 23 (2002) 1981–1987. [44] S. Saravanan, D.K. Sameera, A. Moorthi, N. Selvamurugan, Chitosan scaffolds containing chicken feather keratin nanoparticles for bone tissue engineering, Int. J. Biol. Macromol. 62 (2013) 481–486. [45] M.S. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M.F.A. Goosen, N. Mameri, Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste, Food Hydrocoll. 29 (2012) 48–56.