Home  |  About JAPTR |  Editorial board  |  Search |  Ahead of print  |  Current issue  |  Archives |  Submit article  |  Instructions  |  Subscribe  |  Advertise  |  Contacts  |Login 
Users Online: 853   Home Print this page Email this page Small font sizeDefault font sizeIncrease font size
     

 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 4  |  Page : 373-377  

Characterization and cytotoxicity of low-molecular-weight chitosan and chito-oligosaccharides derived from tilapia fish scales


1 Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala, Terengganu, Malaysia
2 Institute of Marine Biotechnology, Universiti Malaysia Terengganu; Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala, Terengganu, Malaysia

Date of Submission30-Apr-2021
Date of Decision26-Jun-2021
Date of Acceptance19-Jul-2021
Date of Web Publication19-Oct-2021

Correspondence Address:
A W. M. Effendy
Faculty of Fisheries and Food Science, University Malaysia Terengganu, 21030 Kuala, Terengganu
Malaysia
Dr. Gul-e-Saba Chaudhry
Institute of Marine Biotechnology, University Malaysia Terengganu, 21030 Kuala, Terengganu
Malaysia
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.japtr_117_21

Rights and Permissions
  Abstract 


The present study evaluated the physicochemical characterization and cytotoxicity activity of chitosan and chito-oligosaccharides (COSs). The extraction of chitosan and COSs was executed by chemical hydrolysis. The physicochemical characterization and deacetylation (DA) value were determined using an FTIR. The molecular weight was determined by using the Mark–Houwink equation. The physical parameters such as solubility, water-binding capacity (WBC), and fat-binding capacity (FBC) were determination as per equation (i), (ii), and (iii) respectively. The cytotoxic activities of chitosan and COS against MCF-7, HepG2, HeLa-6, and 3T3 were performed by MTS assay. The COS induced enhance cytotoxicity with IC50 0.87 and 2.21 mg/ml against MCF-7 and HepG2 respectively. However, COSs seem to be more sensitive toward the cell lines with the relative potential of MCF-7 > HepG2 > HeLa. Hence, the results showed promising future perspectives of chitosan and COS to develop biodegradable, antibacterial, cytotoxic naturally derived polysaccharides for cancer drug delivery and smart wound dressings.

Keywords: Biodegradable polymer, cancer, chito-oligosaccharides, chitosan, cytotoxicity, MTS


How to cite this article:
Chaudhry Ge, Thirukanthan C S, NurIslamiah K M, Sung Y Y, Sifzizul T S, Effendy A W. Characterization and cytotoxicity of low-molecular-weight chitosan and chito-oligosaccharides derived from tilapia fish scales. J Adv Pharm Technol Res 2021;12:373-7

How to cite this URL:
Chaudhry Ge, Thirukanthan C S, NurIslamiah K M, Sung Y Y, Sifzizul T S, Effendy A W. Characterization and cytotoxicity of low-molecular-weight chitosan and chito-oligosaccharides derived from tilapia fish scales. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 Nov 30];12:373-7. Available from: https://www.japtr.org/text.asp?2021/12/4/373/328625




  Introduction Top


Naturally, derived biopolymers have gained much attraction in various fields. Due to biocompatibility, biodegradability, and various physicochemical features, these bio-proteins and bio-polysaccharides mimic inside the extracellular matrix of a biological system. The biopolymers are composed of (i) glycosaminoglycan, (ii) β-linked D-glucosamine, and N-acetyl-D-glucosamine, which possess physicochemical as well as biomedical properties. These various derivatives of biopolymers, having desire molecular weight (MW) and chemical modifications, make them undefine composition to unique and targeted applications. Chitin is a polymer of N-acetylglucosamine, present profoundly from unicellular to multicellular organisms.[1] Chitosan is derived from chitin and is composed of N-acetyl-D-glucosamine and β-(1,4)-linked-D-glucosamine, widely used in various biomedical applications, targeted drug delivery systems, and nanomedicines. Due to its nontoxic, biocompatible, and biodegradable nature chitosan used in drug delivery.[2],[3] The utilization is considered to be safe and approved by the Food and Drug Administration in wound healing applications.[4],[5] Furthermore, the chitosan microspheres and microcapsules have previously been studied in controlled release therapy for drugs and proteins.[6],[7] Chitosan-based nanomedicine also gained massive attention due to its nontoxic and biodegradable nature. The chitosan-based nanoformulation improves the presence of drug in blood circulation as well as sustained drug release.[8],[9],[10] Moreover, the non- immunogenic nature unable to trigger the immune response in body.[11],[12] Chito-oligosaccharide (COS) is short oligomers of chitosan, with degree of polymerisation (DP) <55 and molecular weight (MW) is 10 kDa.[13] COSs have been used as a drug delivery system for disease treatment also as food.[14],[15] Furthermore, chitosan, along with other chemicals/polymers/cross-linkers and receptor ligands, has been shown to improve the efficacy of potential drug therapeutic. In this study, physicochemical characterization of chitosan and COSs was conducted along with cytotoxicity on selected cancer cell lines, including MCF-7, HepG2, HeLa, and fibroblast 3T3.


  Materials and Methods Top


Pretreatment of raw materials and isolation of chitosan

The tilapia fish scales were obtained from a commercial fish processing plant situated in West Malaysia. Chitosan extraction from fish scales was done by using the modified method given by Toan.[16] The fish scales were soaked in 2% HCl at ambient room temperature with a solid-to-solvent ratio of 1:5 (w/v) for 24 h. Then, the dried residues were treated with 4% NaOH at ambient room temperature with a solid ratio of 1:5 (w/v) for 24 h. Then, chitin was treated with 80% NaOH at 120°C for 6 h.

Extraction of chito-oligosaccharides

COS was extracted by chemical hydrolysis following the methods described by Trombotto et al.[17]

Degree of deacetylation

The degree of deacetylation (DA) was determined by the method described by Muzzarelli and Rochetti (1985).[18] The chitosan and COS samples were first pelleted with KBr at a sample/KBr ratio of 1:60.

Physicochemical and functional properties

Crude protein, moisture, and ash were determined using official methods described by the Association of Official Analytical Chemists (1984).

Molecular weight

Firstly, a series of mixture ranging from 0.02% to 0.1% of chitosan and COS in 0.1M acetic acid and 0.2M sodium chloride was prepared. Then, the MWs of the respective samples were determined based on the Mark–Houwink equation.[19]

Solubility

For solubility, 0.5 g of chitosan and COS was placed in a 50 mL tube, dissolved with 50 mL of 1% acetic acid, and mixed in a vortex mixer for 10 min. The residues were weighed, and the percentage of solubility was calculated using the formula as below:[20]



Water- and fat-binding capacity

The water binding capacity (WBC) and fat binding capacity (FBC) of chitosan and COS were determined by previously reported method.[21] The WBC value was determined using the formula below:

WBC (%) = (Precipitated pellet [g]/Initial sample weight [g]) ×100 eq. (ii)

The FBC value was determined using the formula below:

FBC (%) = (Precipitated pellet [g]/Initial sample weight [g]) ×100 eq. (iii)

Cytotoxicity analysis

The cytotoxicity of chitosan and COSs against MCF-7, HepG2, HeLa-6, and 3T3 was determined. The cell proliferation assay was conducted using the CellTiter 96™ Aqueous One Solution Cell Proliferation Assay.[22],[23] The absorbance was then read at a wavelength of 490 nm using an ELISA reader (Multiskan, Thermo Fisher, USA).

Statistical analysis

The experimental data were subjected to two-way ANOVA analysis (ANOVA), using Origin 8 SR4.


  Results Top


Physicochemical and functional properties

The physicochemical properties of chitosan and COSs are given in [Table 1]. The degree of DA for chitosan and COS was reported as 92.235% and 94.65%, respectively. Furthermore, the MW of chitosan and COS was 11.58 and 4.6 kDa, respectively. The degree of degree of acetylation (DA) of chitosan and its derivatives influenced by varous factors such as sample location, extraction method and analytical instrumentation used.[24],[25] Previous studies supported the value of DA; the degree of DA of chitosan ranged from 56% to 99%.[26]
Table 1: The physicochemical properties of chitosan and chito-oligosaccharides extracted from fish scales

Click here to view


Interestingly, the solubility of COS reporting is 98.65%, which is higher than the solubility of solubility chitosan (78.65%). However, protein contaminants have a significant impact since the solubility is associated with the reaction of the amino groups.[27] Furthermore, the WBC for both chitosan and COS was considerably high, reporting 860% and 930%, respectively, whereas for the FBC, chitosan and COS reported 572% and 640%, respectively.

Cytotoxicity analysis

The cytotoxicity of chitosan and COSs against MCF-7, HepG2, HeLa, and 3T3 cell lines [Figure 1] was evaluated. There is a dose-dependent increase in cytotoxicity noticed after chitosan and COS treatment in all cell lines. The COS inhibited the growth of MCF-7 cells at a concentration of 0.25 mg/ml in all three cancer cell lines. Interestingly, HepG2 is more sensitive toward chitosan with cytotoxicity of 26.40% versus COS inhibition of 14.40%. Similarly, HeLa cells are more sensitive toward COS (23.40%) inhibition versus chitosan (13.80%). Moreover, the highest cytotoxicity, 92.80%, was observed in MCF-7 after treatment with 1 mg/ml of chitosan. However, HepG2 seems to be less sensitive toward chitosan. The IC50 values of chitosan against HepG2 were above 4 mg/ml [Table 2]. Furthermore, 3T3 cells exhibited the least growth inhibition against chitosan and COS. Hence, chitosan and COS could be used in wound healing as they are safe against fibroblast even at high concentrations (4 mg/ml). The MCF-7 shown to be more sensitive against COS, followed by HepG2 and HeLa cells. However, the IC50 values for 3T3 unable to achieved in given concentration of 4mg/ml.
Figure 1: The cell viability activity of chitosan and chito-oligosaccharides against MCF-7 (a); HepG2 (b); HeLa (c); 3T3 (d) lines at 72 h. Results are mean ± standard deviation (n = 8)

Click here to view
Table 2: IC50 values of chitosan and chito-oligosaccharides against cell lines (mg/mL)

Click here to view



  Discussion Top


The biopolymers could play a remarkable role in therapeutic drug development. In our study, chitin from fish scales converted into low MW, chitosan, and COSs with a high degree of DA. The chitosan samples were tested for their cytotoxicity properties against four cell lines, including MCF-7, HepG2, HeLa, and 3T3 cell lines. The role of chitosan nanoparticles and the effects of MW in cellular uptake and cytotoxicity activities have been proven in many studies. Huang et al.[28] demonstrated that the increasing DA degree had a much significant impact on the cellular uptake capability of the chitosan samples. The increase in DD% from 61% to 88% significantly increased the cytotoxic activities. Cytotoxic activities were highly dependent upon the three-dimensional arrangement of cationic residues along with the charge density of chitosan. Chitosan with a higher degree of DA binds more readily with cell membranes due to its extended conformation caused by the charge repulsion. A previous cytotoxicity study was done on three different marine sources, including shrimp and crab shells and cuttlefish bones against RT112 bladder cancer cells. The cytotoxicity depended on the chitosan MW.[29] The chitosan and COS used in this study were found not to induce cytotoxic activities on 3T3 fibroblast cells. Even at high concentrations of 4 mg/ml, the cell viability percentage of 3T3 cells treated with chitosan exhibited 67.2%, and COS revealed 78.5% with an undetectable IC50 value. The results from this study were comparable to the ones conducted by Nor et al., Lim et al., and Abdull Rasad et al.[30],[31],[32] The IC50 value of the chitosan nanoparticles used in their study reported was 5.3 μg/mL after 48 h treatment. The fibroblast cells treated with chitosan possessing a higher degree of DA exhibited significantly higher proliferation rates than lower degree DA chitosan.[33] Moreover, chitosan was used as a polymeric vehicle for delivering drugs, i.e. pioglitazone, heparin, and bemiparin, for diabetic wounds.[34],[35] Also, chitosan oligosaccharide nanofiber promotes wound healing by activating TGFβ1 Smad signaling pathway.[36],[37] However, the potential toxicity of chitin limits its utilization due to potentially harmful effects on the human body.[38] Interestingly, chitosan and oligosaccharide have water-soluble nature and have reported minimal toxicity via oral routes.[39],[40] Hence, chitosan and oligosaccharide could be a huge potential in developing targeted drug therapeutics with enhanced efficacy.


  Conclusion Top


Scientific breakthroughs are seen in the last decade in chitosan-based nanomedicines in treating cancer. Unfortunately, both chitosan molecules exhibited comparable cytotoxicity. However, fibroblast cells are not sensitive toward these biopolymers, which show the potential to be used in disease treatment and therapeutics. The in vivo study on the pipeline strengthens the future perspectives of chitosan and COS for biodegradable, antibacterial, cytotoxic naturally derived polysaccharides for cancer drug delivery and innovative wound dressings.

Acknowledgment

The authors would like to acknowledge the funding provided to the Institute of Marine Biotechnology.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tsurkan MV, Voronkina A, Khrunyk Y, Wysokowski M, Petrenko I, Ehrlich H. Progress in chitin analytics. Carbohydr Polym 2021;252:117204.  Back to cited text no. 1
    
2.
Qin Z, Zhao L. The history of chito/chitin oligosaccharides and its monomer. In: Zhao L, editor. Oligosaccharides of Chitin and Chitosan: Bio-Manufacture and Applications. Singapore: Springer; 2019. p. 3-14.  Back to cited text no. 2
    
3.
Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2003;2:347-60.  Back to cited text no. 3
    
4.
Mao S, Sun W, Kissel T. Chitosan-based formulations for delivery of DNA and siRNA. Adv Drug Deliv Rev 2010;62:12-27.  Back to cited text no. 4
    
5.
Chandy T, Sharma CP. Chitosan-as a biomaterial. Artif Cells Blood Substit Biotechnol 1990;18:1-24.  Back to cited text no. 5
    
6.
Martinac A, Filipović-Grcić J, Voinovich D, Perissutti B, Franceschinis E. Development and bioadhesive properties of chitosan-ethylcellulose microspheres for nasal delivery. Int J Pharm 2005;291:69-77.  Back to cited text no. 6
    
7.
Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for antiinflammatory drugs. J Control Release 1996;39:17-25.  Back to cited text no. 7
    
8.
Prabaharan M. Chitosan-based nanoparticles for tumor-targeted drug delivery. Int J Biol Macromol 2015;72:1313-22.  Back to cited text no. 8
    
9.
Khawar IA, Kim JH, Kuh HJ. Improving drug delivery to solid tumors: Priming the tumor microenvironment. J Control Release 2015;201:78-89.  Back to cited text no. 9
    
10.
Wicki A, Witzigmann D, Balasubramanian V, Huwyler J. Nanomedicine in cancer therapy: Challenges, opportunities, and clinical applications. J Control Release 2015;200:138-57.  Back to cited text no. 10
    
11.
Garcia-Fuentes M, Alonso MJ. Chitosan-based drug nanocarriers: Where do we stand? J Control Release 2012;161:496-504.  Back to cited text no. 11
    
12.
Gorzelanny C, Pöppelmann B, Pappelbaum K, Moerschbacher BM, Schneider SW. Human macrophage activation triggered by chitotriosidase-mediated chitin and chitosan degradation. Biomaterials 2010;31:8556-63.  Back to cited text no. 12
    
13.
Muanprasat C, Chatsudthipong V. Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacol Ther 2017;170:80-97.  Back to cited text no. 13
    
14.
Li J, Cai C, Li J, Li J, Li J, Sun T, et al. Chitosan-based nanomaterials for drug delivery. Molecules 2018;23:2661.  Back to cited text no. 14
    
15.
Gallo M, Naviglio D, Armone Caruso A, Ferrara L. Applications of chitosan as a functional food. In: Grumezescu AM, editor. Novel Approaches of Nanotechnology in Food. London, UK: Academic Press; 2016. p. 425-64.  Back to cited text no. 15
    
16.
Toan NV. Production of chitin and chitosan from partially autolyzed shrimp shell materials. Open Biomater J 2009;1:21-4.  Back to cited text no. 16
    
17.
Trombotto S, Ladavière C, Delolme F, Domard A. Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers. Biomacromolecules 2008;9:1731-8.  Back to cited text no. 17
    
18.
Muzzarelli RA, Rochetti R. Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry. Carbohydr Polym 1985;5:461-72.  Back to cited text no. 18
    
19.
Wang W, Bo SQ, Li SQ, Qin W. Determination of the Mark-Houwink equation for chitosans with different degrees of deacetylation. Int J Biol Macromol 1991;13:281-5.  Back to cited text no. 19
    
20.
Fernandez-Kim SO. Physicochemical and Functional Properties of Crawfish Chitosan as Affected by Different Processing Protocols. USA: Louisiana State University, M.Sc. Thesis; 2004. p. 1-99.  Back to cited text no. 20
    
21.
Knorr D. Functional properties of chitin and chitosan. J Food Sci 1982;47:593-5.  Back to cited text no. 21
    
22.
Chaudhry GE, Rahman NH, Sevakumaran V, Ahmad A, Mohamad H, Zafar MN, et al. Induction of cytotoxicity by Bruguiera gymnorrhiza in human breast carcinoma (MCF-7) cell line via activation of the intrinsic pathway. J Adv Pharm Technol Res 2020;11:233-7.  Back to cited text no. 22
  [Full text]  
23.
Chaudhry GE, Akim A, Zafar MN, Abdullah MA, Sung YY, Muhammad TS. Induction of apoptosis and role of paclitaxel-loaded hyaluronic acid-crosslinked nanoparticles in the regulation of AKT and RhoA. J Adv Pharm Technol Res 2020;11:101-6.  Back to cited text no. 23
  [Full text]  
24.
Khan TA, Peh KK, Ch'ng HS. Reporting degree of deacetylation values of chitosan: The influence of analytical methods. J Pharm Pharm Sci 2002;5:205-12.  Back to cited text no. 24
    
25.
No HK, Meyers SP. Crawfish chitosan as a coagulant in recovery of organic compounds from seafood processing streams. J Agric Food Chem 1989;37:580-3.  Back to cited text no. 25
    
26.
No HK, Meyers SP. Preparation and characterization of chitin and chitosan – A review. J Aquatic Food Prod Technol 1995;4:27-52.  Back to cited text no. 26
    
27.
Austin PR, Brine CJ, Castle JE, Zikakis JP. Chitin: New facets of research. Science 1981;212:749-53.  Back to cited text no. 27
    
28.
Huang M, Khor E, Lim LY. Uptake and cytotoxicity of chitosan molecules and nanoparticles: Effects of molecular weight and degree of deacetylation. Pharm Res 2004;21:344-53.  Back to cited text no. 28
    
29.
Hajji S, Younes I, Rinaudo M, Jellouli K, Nasri M. Characterization and in vitro evaluation of cytotoxicity, antimicrobial and antioxidant activities of chitosans extracted from three different marine sources. Appl Biochem Biotechnol 2015;177:18-35.  Back to cited text no. 29
    
30.
Nor AM, Ahmad SH, Shaharum S, Che MC, Zanariah U, Ahmad HA. The effect of chitosan derivatives film on the proliferation of human skin fibroblast: An – in vitro study. J Sustain Sci Manag 2013;8:212-9.  Back to cited text no. 30
    
31.
Lim CK, Halim AS, Lau HY, Ujang Z, Hazri A. In vitro cytotoxicology model of oligo-chitosan and n, o-carboxymethyl chitosan using primary human epidermal keratinocytes cultures. J Appl Biometer Biomech 2007;5:82-7.  Back to cited text no. 31
    
32.
Abdull Rasad MS, Halim AS, Hashim K, Hazri A, Yusof N, Shamsuddin S. In vitro evaluation of novel chitosan derivatives sheet and paste cytocompatibility on human dermal fibroblasts. Carbohydr Polym 2010;79:1094-100.  Back to cited text no. 32
    
33.
Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ. The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials 2001;22:2959-66.  Back to cited text no. 33
    
34.
Cifuentes A, Gómez-Gil V, Ortega MA, Asúnsolo Á, Coca S, Román JS, et al. Chitosan hydrogels functionalized with either unfractionated heparin or bemiparin improve diabetic wound healing. Biomed Pharmacother 2020;129:110498.  Back to cited text no. 34
    
35.
Natarajan J, Sanapalli BK, Bano M, Singh SK, Gulati M, Karri V. Nanostructured lipid carriers of pioglitazone loaded collagen/chitosan composite scaffold for diabetic wound healing. Adv Wound Care (New Rochelle)2019;8:499-513.  Back to cited text no. 35
    
36.
Li ZW, Li CW, Wang Q, Shi SJ, Hu M, Zhang Q, Cui HH, et al. The cellular and molecular mechanisms underlying silver nanoparticle/chitosan oligosaccharide/poly (vinyl alcohol) nanofiber-mediated wound healing. J Biomed Nanotechnol 2017;13:17-34.  Back to cited text no. 36
    
37.
Li CW, Wang Q, Li J, Hu M, Shi SJ, Li ZW, et al. Silver nanoparticles/chitosan oligosaccharide/poly (vinyl alcohol) nanofiber promotes wound healing by activating TGFβ1/Smad signaling pathway. Int J Nanomed 2016;11:373-87.  Back to cited text no. 37
    
38.
Van Dyken SJ, Garcia D, Porter P, Huang X, Quinlan PJ, Blanc PD, et al. Fungal chitin from asthma-associated home environments induces eosinophilic lung infiltration. J Immunol 2011;187:2261-7.  Back to cited text no. 38
    
39.
Mohammed MA, Syeda JT, Wasan KM, Wasan EK. An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics 2017;9:53.  Back to cited text no. 39
    
40.
Bellich B, D'Agostino I, Semeraro S, Gamini A, Cesàro A. “The Good, the Bad and the Ugly” of Chitosans. Mar Drugs 2016;14:99.  Back to cited text no. 40
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
   Abstract
  Introduction
   Materials and Me...
  Results
  Discussion
  Conclusion
   References
   Article Figures
   Article Tables

 Article Access Statistics
    Viewed350    
    Printed12    
    Emailed0    
    PDF Downloaded38    
    Comments [Add]    

Recommend this journal