Biomedical Applications of Chitosan-Coated Nanosheets

Abstract

Nanotechnology paves the way for the synthesis of novel nanomaterials in one or more dimensions with a size range of less than 100 nm and enhances its specific application because of its unique properties. Nanosheets are a type of layered nanomaterial mostly designed using graphene, poly(L-lactic acid) (PLLA), and molybdenum disulfide by liquid exfoliation method and are of great interest. Nanosheets fabricated could be employed with other materials to enhance their application in diverse areas. Chitosan, a cationic polymer in conjugation with various nanosheets, was designed for various applications like sensors, cancer treatment, drug delivery, and so on. Chitosan-decorated different nanosheets were formulated by various methods, and their diverse application will be focused.

1. Introduction

Chitosan, a cationic polymer, recently attracted researchers because of its extensive properties such as nontoxic, biocompatible, biodegradable, and so on. In addition, chitosan possesses excellent film-forming ability, hydrogel, scaffold, nanofibers, and so on. Hence, this is preferred for various applications as an antibacterial agent. Similarly, nanosheets also favor various benefits because of their nano dimension. Chitosan polymer in combination with different nanosheets is fabricated nowadays to improve its applicability. Nanosheets synthesized using different approaches decorated with chitosan are evaluated for various applications such as antifungal effects, sensors, cancer-specific antigen detection, photo-thermal effect in cancer, drug delivery, solvent dehydration, and so on. In this present chapter, chitosan polymer in combination with various nanosheets and its diverse application is discussed.

2. Nanosheets

Nanosheet (NS) is a layered material of various and highly unexploited sources in two-dimension of nanometer sizes in the 1–100 nm thickness range. Atoms and molecules are usually arranged in single or multilayer of two dimensions in the nanosheets [1]. Nowadays, nanotechnology offers a wide choice to researchers to fabricate novel 2D materials over 1D and make them more specific and superior while comparing them with bulk materials. Top-down or bottom-up approaches could be employed for the synthesis of 2D nanosheets [2]. In general, 2D materials possess a high surface area-to-volume ratio and strength, making them more appealing and compatible with specific applications in wide areas such as cell culture, platelet adhesion, drug delivery, and so on [3].

2.1 Different types of nanosheets

Nanosheets of various forms such as graphene oxide (GO), poly(L–lactic acid), and molybdenum disulfide (MoS₂) are available up-to-dately and are currently used in cellular research, drug delivery, multimodal contrast agents, sensors, membranes, optical and electronic devices, nanomedicine, and other specific applications [4, 5, 6].

Graphene nanosheets are monolayers of carbon atoms arranged in honeycomb crystal assembly [7] with high electron mobility, thermal stability and conductivity, surface area, and biocompatibility [8] and hence could be used as electrochemical immunosensors for disease diagnosis [9]. Graphene oxide nanosheets help DNA cargo functions and study cellular interactions in vivo [10].

Poly(L-lactic acid) (PLLA) nanosheets are known to have anti-adhesive properties and find their application in wound dressing [11]. Better transparency, flexibility, and adhesiveness make it more specific for PLLA nanosheets for burn wounds [12].

Molybdenum disulfide (MoS₂)-based nanosheets are arranged in two-dimensional hexagonal lattices with a single Mo atom. Two S atoms sandwich the Mo atom and result in the formation of a hexagonal honeycomb structural appearance with three layers [6]. Covalent bond formation occurs between the S-Mo-S and makes the nanosheet more stable and compatible with various applications. High surface area, band gap nature, thin size, and better load bearing make it more appealing and enhance its usability.

2.2 Properties of nanosheets

Large contact area, hetero-functionality, noncovalent adhesion, flexibility, and minimum mass introduction are the unique properties of the nanosheet. In addition, high surface area, good mechanical stability, strength, sensitivity, selectivity, and flexibility are of major concern [13].

2.3 Advantages of nanosheets

Nanosheets can be subjected to surface modification very easily, and hence, various alterations could be favored to enhance its property for various applications. Surface structure, charge, hydrophilicity, and hydrophobicity could be altered by surface modification [14].

2.4 Application of nanosheets

Ease of use, flexibility, and modification enhance the specific application of nanosheets as surface-sensitive, substrate material, and in regenerative medicine. In addition, nanosheets are being preferred for other applications such as drug delivery, cell culture, imaging, and sensing [15, 16]. Recent insight into the use of nanosheets with polymer chitosan has received much attention since it has several advantages. Several materials have been combined with chitosan for the fabrication of nanosheets for specific applications [17].

3. Chitosan

Chitin (β-(1–4)-poly-N-acetyl-D-glucosamine) is one of the natural polymer present in the cell walls of exoskeletons of crabs, cuticles of insects, shrimps, and cell walls of fungi. Chitin is deacetylated into chitosan for biomedical applications, using chemicals or with the help of enzymes [18].

Chitosan comprises β-1, 4-linked 2-amino-2-deoxy-β-D-glucose (deacetylated D-glucosamine) and N-acetyl-D-glucosamine units of lower molecular weight (MW) and crystallinity than chitin (MW > 100 kDa) [19]. Moreover, it is structurally similar to cellulose except for the hydroxyl group (∙OH) at the C-2 position and is exchanged with the amino group (∙NH₂) in chitosan. Chitosan is a biocompatible, approved polymer for biomedical applications [20].

Different methods have been followed so far for the preparation of chitosan-based nanocomposites. The solvent casting method, electrospinning, and other methods could be employed for the use of nanocomposites [21]. Chitosan, a cationic deacetylated polymer from chitin, has recently found its application in diverse fields such as drug delivery; antimicrobial activity; excellent film-forming ability, tissue engineering; antitumor, antioxidant, and antifungal activities; wastewater treatment; wound healing; cosmetics; textile and paper; pharmaceutical; and agriculture because of its biocompatibility, nontoxic nature, and structural similarity to natural glycosaminoglycans [22]. Chitosan finds its application in the food industry as an antimicrobial packaging material. In addition, it is added as an emulsifier in the food materials [23].

4. Preparation methods for chitosan-coated nanosheets

4.1 Molybdenum disulfide nanosheets

Sodium molybdate dehydrate (50 mg) and thiourea prepared in deionized water of 50 ml were sonicated at room temperature for 15 minutes, and the pH was maintained at 3.5 using 0.1 M HCl. The mixture was then placed in a Teflon-lined autoclave and subjected to a heating process at 200°C for a period of 18 hours in a hot air oven. After sufficient reaction time, the contents were centrifuged for 10 minutes at 8000 rpm, and the pellet was washed with millipore water and ethanol several times to obtain the black color precipitate and further dried at 80°C under vacuum condition. The final product obtained is molybdenum disulfide nanosheets [24]. Figure 1 illustrates the proposed methodology of synthesis of molybdenum disulfide nanosheets.

Figure 1.

Proposed strategy of MoS₂ nanosheet synthesis.

4.2 Chitosan-based molybdenum disulfide nanosheets

Molybdenum nanosheet (0.3 g) and L–cysteine (30 mg) dissolved in 25 ml of millipore water were stirred continuously for 5 h, and 1% chitosan suspended in acetic acid was added slowly to this mixture at room temperature for the development of chitosan molybdenum disulfide nanosheets. The resultant product obtained after 5 h was collected by centrifuging followed by washing with water and ethanol several times. The recovered product was dried under a vacuum overnight. Figure 2 represents the strategy involved in the preparation of chitosan molybdenum disulfide nanosheets. Figure 3 denotes the chitosan molybdenum disulfide nanosheets.

Figure 2.

Strategy involved in the fabrication of chitosan MoS₂ nanosheet.

Figure 3.

Chitosan molybdenum disulfide nanosheet.

4.3 Graphene nanosheets

Graphene possesses exciting properties such as thermal, optical, electrical, and mechanical properties in its 2D form and is thus preferred for various applications. Graphene in the form of nanosheet decorated with chitosan has multiple benefits. The steps involved in the synthesis of graphene nanosheets are discussed in Figures 4 and 5 represents graphene nanosheets.

Figure 4.

Steps involved in the synthesis of graphene nanosheet.

Figure 5.

Graphene nanosheet.

4.4 PLLA nanosheet

PLLA suspended in 1,1,1,3,3,3-hexafluoropropan-2-ol or dichloromethane at 5–50 mg/mL concentration was poured onto silicon dioxide substrate and spin coated for 20 s at 4000 rpm. It was then allowed to heat at 70°C for 90 s. PVA solution was prepared by dissolving in distilled water at 100 mg/mL concentration and dried up at 70°C for 15 min [11]. Figure 6 represents the fabrication of PLLA nanosheet.

Figure 6.

Proposed mechanism of fabrication of PLLA nanosheet.

5. Characterization of chitosan-coated nanosheets

Nanosheets fabricated can be characterized by various spectroscopic methods such as FTIR, X-ray Diffraction, and EDX and other microscopic methods such as SEM, FESEM, AFM, TEM, and so on. Nanosheets dimensions could be confirmed while we characterize using different spectroscopic techniques.

6. Applications of chitosan-coated nanosheets

6.1 Antibacterial/antifungal activity

Molybdenum disulfide (MoS₂) nanosheets fabricated using the liquid exfoliation method were laden with chitosan polymer and silver nanoparticles to test the antifungal activity in a study conducted by Zhang et al. [25]. Nanosheets were further characterized by UV-Vis absorption spectroscopy, SEM, and EDS [25]. In vivo and in vitro studies were conducted to check the efficiency of the synthesized nanosheets. MoS₂-CS-Ag nanosheets were tested against the selected strains such as Aspergillus niger and Saccharomyces uvarum. Results revealed that better antifungal activity was exhibited with the use of nanosheets both in vivo and in vitro, and its subsequent application in fruit protection was studied.

Nanosheets of molybdenum disulfide with chitosan and silver nanoparticles exhibit better antifungal activity in a study conducted by Zhang et al. [25]. In this present study, MoS₂ nanosheets were produced by the liquid exfoliation method, and subsequently, chitosan was functionalized with this nanosheet and decorated with Ag NPs. The thickness of the nanosheet was found to be approximately 10 nm. Fungal cultures such as Aspergillus niger (ATCC 6275) and Saccharomyces uvarum (ATCC 9080) were selected for the antifungal activity assay. Experiments were carried out at different concentrations, and the results revealed that MoS₂ nanosheets loaded with chitosan and Ag-NPs exhibit better antifungal effects when compared with control, and their application in fruit models such as apples and bananas was also examined. Cell morphology, membrane integrity, and cytotoxicity assays are evidence that MoS₂-CS-Ag-NPs nanosheets were found to have promising effects in food preservation.

In another study conducted by Saha et al. [26], chitosan and molybdenum diselenide (MoSe₂) nanosheets were synthesized using liquid exfoliation, and their subsequent application in fungal eradication was studied. Antifungal activities of MoSe₂/CS nanosheets against variety of unicellular and filamentous fungal strains such as Cryptococcus gattii (C. gattii, ATCC MYA-4071), Candida parapsilosis (C. parapsilosis, ATCC 22019), Issatchenkia orientalis (I. orientalis, ATCC 6258), Aspergillus fumigatus (A. fumigatus ATCC, MYA-4609), Candida albicans (C. albicans, ATCC 76485), Fusarium verticillioides (F. verticillioides, ATCCMYA-3629), Saccharomyces cerevisiae (S. cerevisiae, ATCC 9763), Cryptococcus neoformans (C. neoformans, ATCC 208821), Fusarium falciforme (F. falciforme, ATCC MYA-3636). C. auris (0389), Candida duobushaemulonii (C. duobushaemulonii, 0394), Candida auris (C. auris, 0386), Candida haemulonii (C. haemulonii, 0395), Candida krusei (C. krusei, 0397), C. auris (0388), Krusei ohmeri (K. ohmeri, 0396), Saccharomyces cerevisiae (S. cerevisiae, 0399), and Candida lusitaniae (C. lusitaniae, 0398) were studied [26].

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using the MoSe₂/CS nanosheets. MIC values for most of the unicellular fungal strains lie in the range of 0.78 and 37.5 μg/ml. Similarly, MFCs range between 0.5 and 75 μg/ml for various filamentous and unicellular strains. Candida auris strains were also tested against the prepared nanosheets. MICs and MFCs were recorded as 25–50 μm/ml and 37.5 to 150 μm/ml. MoSe₂/CS nanosheets were found to have good antifungal activity than graphene-based nanosheets. Cell morphology and the mechanism of killing were also investigated for the selected strains A. fumigatus and C. albicans. Cell membrane damage, cytoplasmic leakage, depolarization of the membrane, and metabolic inactivation occur as a result of exposure to MoSe₂/CS nanosheets. The present study showed promising evidence for the use of MoSe₂/CS nanosheets as an antifungal agent.

CS/MoSe₂ nanosheets also exhibit better antibacterial activity against selected gram- positive and gram-negative bacteria in a work carried out by Roy et al. [27]. Electrostatic forces and Vanderwaal’s interaction between chitosan and molybdenum disulfide nanosheet enhances their application as an antibacterial agent. Strains such as Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were tested against the synthesized nanosheets [27] to prove its efficacy. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against the selected strains were also evaluated. Membrane attachment, membrane puncturing, ROS generation, intracellular oxidative stress, membrane depolarization and disruption, and metabolic arrest are the proposed mechanisms that lead to cell death [28].

6.2 Drug delivery

Chitosan functionalized with molybdenum disulfide has promising applications in drug delivery. Chitosan/graphene nanosheets were loaded with doxorubicin for drug delivery in a study conducted by Mousavi et al. [29]. Graphene/N-phthaloylchitosan-graft-poly (methylmethacrylate-block-(poly ethylene glycol methacrylate-random-dimethylamino ethyl methacrylate) GO/CS-g-PMMA-b-(PEGMA-ran-PDMAEMA) nanocomposite was prepared and loaded with the drug to check its efficacy. In vitro release, kinetics were also assessed. Promising results revealed that novel nanosheets functionalized with chitosan exhibited better drug delivery efficacy [29].

6.3 Photothermal therapy of cancer

In a study conducted by Rajasekar et al. [30], MoS₂ nanosheet was fabricated at 200°C using the hydrothermal route for 24 h. 1% chitosan solution was coated onto the fabricated nanosheets to form chitosan-coated molybdenum disulfide (CS-MoS₂). Tantalum oxide (TaO₂) was further incorporated into the nanosheets to synthesize TaO₂-CS-MoS₂. Electrostatic interactions between the nanosheets make them suitable for photothermal treatment (PTT). The prepared nanosheets were tested for their biocompatibility, photostability, and cytotoxic effects on the breast cancer cell lines [30].

6.4 Sensors

MoS₂ nanosheets were initially synthesized by ultrasonic exfoliation and subsequently conjugated with chitosan and bismuth film and GC electrode (MoS₂/GC electrode). Further, the prepared nanosheets were modified by functionalization with thiolated poly (aspartic acid) (TPA-MoS₂/GC electrode) in a study conducted by Cao et al. [31]. The TPA-MoS₂/GC sensor was developed to focus on the detection of cadmium ions (Cd²⁺). TPA-modified MoS₂ nanosheets were found to be an effective and reliable tool to monitor the Cd²⁺ in food and water [31].

6.5 Detection of prostate specific antigen

In a study conducted by Duan et al. [32], MoS₂ quantum dots (MoS₂ QDs) and two-dimensional graphitic carbon nitride (g-C₃N₄) nanosheets were decorated with chitosan and gold nanoparticles and further fabricated with aptamers (MoS₂QDs@g-C₃N₄@CS-AuNPs) to detect prostate-specific antigen (PSA). The construct displayed acts as an electrochemical aptasensor and surface plasmon resonance (SPR) sensor. The limit of detection was found to be 0.71 pg/mL. Enhanced stability, good selectivity, and reproducibility were achieved with the sample tested. Results revealed that it could be used for sensing applications and cancer biomarker prediction [32].

6.6 Dehydration of solvents

Xu et al. [33] fabricated two-dimensional Ti₃C₂T_x MXene nanosheets, which were incorporated with chitosan for efficient solvent dehydration. Organic solvents such as dimethyl carbonate, ethyl acetate, and ethanol were used for evaluating the dehydration performance. Interactions between the chitosan polymer and the hydroxyl groups of MXene improve the infiltration of water molecules across the membrane [33].

6.7 Nitrite oxidation

An inorganic pollutant, nitrite poses a severe hazard to the environment, and it influences the oxidation of hemoglobin and affects the oxygen transfer in the blood. Electrochemical oxidation of nitrite is achieved via the graphene nanosheets and chitosan polymer in a work carried out by Chi et al. [34]. Carbon nanospheres and graphene nanosheet mixture were synthesized and then fabricated with chitosan Prussian blue nanocomposites and glassy carbon electrode (CS@PB/GNS-CNS/GCE) for the nitrite oxidation. Electrostatic interactions between the chitosan/Prussian blue nanocomposite and graphene nanosheets/carbon nanospheres make it an excellent redox mediator for nitrite detection. It is evident from the results that sensors could be developed based on their detection limit and could be used in food samples [34].

6.8 Implantation in heart

In a study conducted by Saravanan et al. [35], graphene oxide nanosheets loaded with gold nanoparticles were incorporated into a chitosan scaffold to check its efficacy in infarcted hearts. Ventricular contractility and its function subsequently improved after implantation. The synthesized scaffold exhibited a better swelling nature and controlled degradation. The attachment of the cells to the scaffold and its growth is enhanced upon the use of nanosheets. The novel scaffold and its application in heart implantation for ventricular function are assessed. Results revealed that the expression of connexin 43 also significantly improved. Immunohistochemistry and immunocytochemistry studies showed prominent results for the implantation of the scaffolds [35].

6.9 Corrosion resistance

Huang et al. [36] developed a chitosan (CS)/boron nitride nanosheet (BNNS) composite, which was fabricated on the surface of Mg-Zn-Y-Nd-Zr alloy by electrodeposition strategy. The construct was designed to diminish the magnesium alloy degradation so that it could be used in bone implants since it possesses antibacterial activity. BNNS has significant changes while coated with chitosan and affords better shelf life to substrate. E. coli and S. aureus were tested to prove the fabricated nanosheet has antibacterial activity. Cytocompatibility tests revealed that the CS-BNNS coating has no adverse effect on the adhesion and proliferation of mouse osteoblasts cells. Thus, the fabricated CS-BNNS composite coating could be a promising application in surface modification for Mg-based bone implants [36].

6.10 Membranes as proton exchangers

In a study carried out by Divya et al. [37], MoS₂ nanosheets were fabricated initially by exfoliation method, and chitosan in different ranges were used for the development of the chitosan-MoS₂ nanocomposites acting as proton exchange membranes because of their unique nature such as proton conductivity, water absorbing capacity, and ion exchange capacity. Characterization of the chitosan-MoS₂ nanosheet membranes was performed using FT-IR, XRD, FESEM-EDX, and AFM. Contact angle, uptake of water, swelling ratio, ion exchange, and proton conductivity were analyzed. Among the various composition selected, 0.75% of chitosan membranes exhibited high membrane selectivity and proton conductivity [37].

6.11 Detection of mutated DNA

Graphene nanosheets modified with chitosan were placed on the surface of the carbon electrode (CMG electrode) for the electrochemical analysis of mutations in DNA. Nanosheets were initially prepared by Hummers methods and subsequently characterized by FT-IR, Raman spectroscopy, and transmission electron microscopy by Alwarappan et al. [38]. The main advantage of this nanosheet is that attachment of single-stranded polynucleotide in the CMG electrode helps in the detection of complementary strands and finds the mutation [38].

Carboxymethyl cellulose-chitosan-montmorillonite nanosheets were synthesized for the remediation of dye effluent by Wang et al. [39]. TiO₂@MMTNS/ CMC/CS nanocomposite was studied for its absorption of methylene blue dye [39]. Cyanometallate/chitosan nanosheet acts as a catalyst to enhance the polysulfide redox reaction in batteries in a work conducted by Fang et al. [40]. Chitosan nanosheets/Honey compounds were tested for wound healing effects in male BALB/c Mice. The wound injury model was developed in adult mice. Animal models were divided into five different groups for the experimental study. Control group, polyethylene glycol (PEG), CNs treated, honey, and CNs dissolved in PEG or honey. While compared with the control group, CNs-treated mice model showed significant results. Better wound healing is achieved upon treatment especially when chitosan nanosheets treated with honey [41].

7. Conclusion

In today’s world field of nanotechnology, especially nanosheets, has a promising impact in cellular research, biomedicine, regenerative medicine, and tissue engineering. Fabrication of nanosheets and their application in in-vivo conditions is still challenging. Novel nanosheets with advanced features can be fabricated to address the various applications. Despite the challenges faced, nanosheets could be employed with various modifications and can be used for the modern era. Chitosan-coated nanosheets could be the alternate choice for targeting multiple applications because of their attractive features.

Acknowledgments

The authors would like to acknowledge the institution for their support.

Conflict of interest

The authors declare no conflict of interest.