Synthesis, Characterization and Efficacy of Antibiotic Coated Chitosan Nanoparticles on Human Pathogens

T. Ramesh¹, M. Thangaraj¹, R. Kumaran², D. Annadurai¹, J. Subramanian¹,

S. Purushothaman¹, M. Shenbagam³

¹CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamilnadu - 608502

²Department of Marine Science, Sethupathy Govt. Arts College, Ramanathapuram, Tamilnadu- 623502

³Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar,

Chidambaram - 608002

*Corresponding Author E-mail: rameshcasmb@gmail.com

ABSTRACT:

The biodegradable nanoparticles of chitosan have received a significance attention as potential drug delivery vehicles in the recent decades. The present study was carried out to extract chitosan nanoparticles from shrimp shell waste and characterized followed by its potential antibacterial activity. Pure chitosan nanoparticles (CNP) and tetracycline coated nanoparticles (TCNP) were prepared by the ionotropic gelation process. The prepared nanoparticles were characterised by UV visible spectrophotometer, SEM, XRD, FTIR, NMR, Nano size - Zeta potential analysis. Antibacterial activity was assessed using CNP and TCNP against 7 different strains of human pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus,
Salmonella typhi, S. paratyphi, Pseudomonas aeruginosa and Bacillus subtilis.  In the present study, the CNP and TCNP had exhibited a growth inhibition activity on bacteria at lower concentration and proved as a remarkable drug carrier.

KEYWORDS: chitosan, nanoparticles, crustaceans, antagonistic assay, drug carrier.

INTRODUCTION:

The seafood processing industries over the world releases more than 60,000 tonnes of waste every year. The head and shell of crustaceans such as shrimp, lobster, crab, prawn, krill etc. are thrown out as wastes. The crustacean wastes contain about 10% of chitin on dry weight basis. The proper utilization of shell wastes not only resolves the difficulty of its disposal but also sets a base track for many beneficial products that might play a key role in several fields such as textiles, photography, medicine, agriculture, food processing etc. The global attention on the importance of chitin and chitosan has been demonstrated in the last two decades to its maximum.

Value added products from chitin had been accepted worldwide. Chitosan is a natural nontoxic biopolymer derived by the deacetylation of chitin and its derivatives have attracted considerable interest due to its antibacterial, antiviral and antifungal activities. Chitosan has received considerable attention as a possible pharmaceutical recipient in recent decades, due to its good biocompatibility biodegradability, non toxicity and adsorption in novel applications such as drug carrier in micro particle systems, films controlling drug release, bioadhesive polymer, site-specific drug delivery and absorption enhancer^1,2,3,4.

Chitosan nanoparticles can be prepared instantly by incorporation of poly anionic substances such as tripolyphosphate (TPP) on chitosan solution under continuous stirring condition. These nanoparticles are harvested and utilized for drug delivery applications and gene therapy⁵. Chitosan nanoparticles acts as an excellent drug carrier with enlarged potential and has major advantage of controlled release of drugs, that improves drug solubility and stability with enhanced efficacy and reduced toxicity⁶. Very few reports are available on the usage of combinations of antibiotics and chitosan and its derivates as potential antimicrobial agent⁷. Disease causing microbes that become resistant to drug therapy are an increasing public health problem. One of the measures to combat this increasing rate of resistance is to have continuous investigations into new, effective and safe antimicrobial agents as an alternate to replace the less effective ones⁸. In this context the present study was carried out to extract chitosan nanoparticles from shrimp shell waste and analyzing its potential antimicrobial property against human pathogens.

MATERIALS AND METHODS:

Sample collection and preparation:

The shrimp shell waste was collected from Mudasalodai landing centre and washed several times to remove the adhering sand particles. The samples were oven-dried for 4 days at 65°C and crushed into finer particles with the help of morter and pestle⁹.

Extraction of chitin:

500g of powdered shell was placed in beakers and soaked in 4 % NaOH in boiling water bath for 1 hour to solubilize proteins and sugars. Then the sample was allowed to cool down to room temperature for 30 minutes^10,11.

Demineralization

The sample was soaked in 1% HCl for 24 h to remove the minerals¹². The demineralized sample was treated with 2% NaOH solution to ensure the decomposition of albumen into water soluble amino-acids. The remaining chitin was washed with deionized water and the final crude chitin was dried at 50ºC for 24 h and used for further analysis. 

Deacetylation:

The chitosan was prepared by deacetylation of chitin¹³.  The deacetylation was done by adding NaOH (50%) and then boiled for 2 h at 100°C. The sample was then cooled for 30 min at room temperature. Afterwards the samples were washed continuously with the 50% NaOH and filtered in order to retain the solid matter, which is the chitosan. The samples were oven dried for 6 hr at 110°C.

Preparation of CNP:

Chitosan nanoparticles were prepared according to the ionotropic gelation process. Chitosan nanoparticles were obtained upon the drop wise addition of a tripolyphosphate (TPP) aqueous solution to a chitosan solution (3mg/ml) and stirred at room temperature. Nanoparticles were collected by centrifugation at 16,000 rpm for a period of 30 minutes and supernatants were discarded and pellet (nano particles) were freeze dried¹⁴.

Preparation of TCNP:

The modified procedure¹⁵ was followed for the antibiotic coated CNP preparation by ionic cross linking technique. Tetracycline (0.5%,) was dispersed in distilled water and acetic acid {concentration 1.5 time greater than chitosan solution (20ml)} containing chitosan (1%) was stirred vigorously for 30min. At room temperature, 20ml of TPP aqueous solution was added drop wise to the chitosan solution under magnetic stirring for 30min. followed by sanitation for 25min and the resulting antibiotic coated chitosan nanoparticles suspension was centrifuged at 19,000rpm for 20min. The supernatant was discarded and pellet was resuspended in de-ionized water followed by sanitation, centrifugation and the process was repeated three times and after drying the nanoparticles were collected.

Characterization of CNP and TCNP:

Ultra-Violet and Visible Spectroscopy:

To verify the formation of nanoparticles¹⁶, Absorbance readings of the samples were determined using Pharma Spec UV-1700 ultraviolet-visible spectrophometer (Shimadzu, Kyoto, Japan). All spectrophotometric measurements were conducted at an operating wavelength of 200 to 600nm in optically homogeneous quartz cuvettes and water was used as the reference.

Scanning Electron Microscope:

The powdered polymer sample was spread on a double-sided conducting adhesive tape pasted on a metallic stub, were coated with 100µg titanium in a sputter coating unit for 5 min and observed under SEM (Olympus) at 20 kV.

Fourier-Transform Infrared Spectroscopy:

RX-1 IR spectrophotometer using 5mg of dry polymer samples and KBr discs under dry air at room temperature. Each FTIR spectrum represents 16scan with 4cm^-1 spectral resolution.

Zeta potential of Nanoparticles:

Zeta potential of chitosan was measured by using zeta potentiometer (Zeta sizer 4, Malvern Instruments, UK) to confirm the polydispersity and surface charge for better interaction to bacterial cells. LMW chitosan nanoparticles in deionized water were used to assess the zeta potential using DTS1060C type clear possible zeta cell at 25ºC temperature. The Smoluchowsky approximation was applied in the calculation of the zeta potential.

X-Ray Diffraction Study:

X-Ray Diffraction Study was performed in Brucker D 8 Advanced X-ray diffractometer using Cu K 2 α ray with a voltage of 40 kV and current of 25mA. Samples were scanned for 2 θ from 10 to 600C. Diffraction pattern for nanoparticles was obtained.

Antibacterial activity:

The antibacterial activity was studied using CNP and TCNP against 7 different strains of human pathogenic bacteria viz., S. aureus, E. coli, V. parahaemolyticus, S. typhi, S. paratyphi, P. aeruginosa, and B. subtilis. In vitro antibacterial assay was carried out by disc diffusion technique¹⁷. Whatmann No.1 filter paper discs with 6 mm diameter were impregnated with known amount of test samples and standard antibiotic disc served as a positive control were placed on the Nutrient agar plates seeded with test bacterial strains. The antibacterial activity was expressed in terms of radius of zone of inhibition and was measured in mm. 125µg/ml of standard Tetracyclin coated commercial discs were used as a control antibiotic against the bacterial strains.

RESULTS:

In the present study, the physic chemical properties of CNP and TCNP were analyzed. In the UV-spectrum, the chitosan nanoparticles recorded the absorption peak ranging 228.13, 245.2, 276.73 and 294.25nm indicating the presence of chitosan nanoparticles. Whereas the tetracycline loaded chitosan nanoparticles recorded the absorption peak 367.22 nm indicated the combination of nanoparticles and tetracycline (Fig. 1.). The morphology of chitosan showed globular in shape (Fig. 2.A.) and the chitosan nanoparticles appear spherical in shape (Fig. 2.B.). Whereas the tetracycline loaded chitosan nanoparticles showed sphere-shaped and triangular in shape (Fig. 2.C.).

Fig.1. UV spectrum analysis of Chitosan and Tetracycline loaded nanoparticles

Fig.2.A and B.. SEM images of Chitosan

Fig.2.C and D. SEM images of Chitosan nanoparticles

Fig.2.E and F.  SEM images of Tetracycline loaded Chitosan nanoparticles

The FT-IR spectrum of chitosan extracted from the shell waste was obtained and compared with that of standard chitosan. The FT-IR spectrum of chitosan shows 15 major peaks lying between 3425.58 and 669.30 cm^-1 (Fig.3. A.).  The absorption peaks of 3425.58, 2924.09, 2881.65, 1654.92 and 1431.18 cm^-1 indicated the N-H stretching, Symmetric CH₃ stretching and asymmetric CH₂ stretching, CH stretching, C=O stretching in secondary amide (amide I) and C–N– stretching in secondary amide (amide II), respectively. The major absorption band is observed between 1072.42 and 1031.92 cm^-1 which represents the free amino group (-NH2) at C₂ position of glucosamine, a major group present in chitosan.

The FT-IR spectrum of chitosan nanoparticles showed 10 major peaks lying between 3431.36 and 671.23 cm^-1 (Fig.3.B.). The broad peak at 3431.36 cm^-1 indicates the NH2- bonded OH stretching. The peak at 1645.28 cm^-1 indicates the amide bending vibrations. The peak at 1417.68 and 1382.96 cm^-1 indicates the pyranose ring vibrations; whereas the peak at 1078.21 and 1024.20 cm^-1 represents the C-O-C stretching vibrations of glucosidic linkage.

The FT-IR spectrum of tetracycline loaded chitosan nanoparticles showed 12 major peaks lying between 3429.43 and 559.36 cm^-1 (Fig.3.C). The broad peak at 3429.43 cm^-1 indicates the NH2- bonded OH stretching. The peak at 2924.09 cm^-1 indicates the symmetric CH₃ stretching and asymmetric CH₂ stretching. The peak at 1643.35 and 1620.21 cm^-1 represented the combination of carbohydrate group. The peak at 1382.96 cm^-1 showed C–N– stretching in secondary amide (amide II).  The peak at 1076.28 1026.13 cm^-1 demonstrated the stretching vibrations of glucosidic linkage.

Fig.3. A. FTIR spectrum of Chitosan

Fig.3. B. FTIR spectrum of Chitosan nanoparticles

Fig.3 C. FTIR spectrum of Tetracycline loaded Chitosan nanoparticles

The zeta potential of chitosan nanoparticles and drug loaded chitosan nanoparticles (Fig. 4.A and 4.B.) were 0.361 mV and -1.37 mV respectively. Crystallographic structure of chitosan nanoparticles was determined in XRD between 0 and 90 of 2θ.

Fig.4. A. Zeta Potential of Chitosan nanoparticles

Fig.4. B Zeta Potential of Tetracycline loaded Chitosan nanoparticles

The XRD pattern of chitosan nanoparticles exhibited its crystalline peak at 2θ = 18.89˚ and 26.19˚ (Fig. 10). Whereas the drug loaded chitosan nanoparticles showed its crystalline peak at 2θ = 18.59˚ (Fig. 5.A and 5.B).

Fig.5. A. XRD pattern of Chitosan Nanoparticles

Fig.5. B. XRD pattern of Tetracycline loaded Chitosan Nanoparticles

In the present investigation, the chitosan nanoparticles (CNP) and tetracycline coated chitosan nanoparticles (TCNP) exhibited a promising antibacterial activity. In this assay the nanoparticles were screened for their ability to suppress/inhibit the growth of 7 human bacterial pathogens. The respective zone of inhibition was recorded in Table 1.

Table 1. The zone of inhibition effect of CNP and TCNP against various bacterial pathogens

S. No	Name of the Bacterial strains	Tetracyclin standard discs 125 µg /ml	CNP 100 µg /ml	TCNP 100 µg /ml
1	Vibrio parahaemolyticus	26mm	14mm	20mm
2	Salmonella paratyphi	31mm	13mm	15mm
3	Salmonella typhi	30mm	11mm	18mm
4	Escherichia coli	31mm	11mm	23mm
5	Pseudomonas aeruginosa	9 mm	13mm	20mm
6	Bacillus subtilis	27mm	20mm	20mm
7	Staphylococcus aureus	30mm	20mm	21mm

The CNP exhibited a maximum growth inhibition against B. subtilis (20mm) and
S. aureus (20mm). They showed moderate activity against V. parahaemolyticus (14mm),
P. aeruginosa (13mm) and S. paratyphi (13mm). Minimum zone of inhibition were recorded in   E. coli and S. typhi (11mm) at a concentration of 100µg. The TCNP exhibited a prominent susceptibility against E. coli (23mm), S. aureus (21mm), P. aeruginosa (20mm), B. subtilis (20mm) and V. parahaemolyticus (20mm). They revealed a good inhibition against Salmonella typhi (18mm) and minimum activity against S. paratyphi (15mm), at a concentration of 100µg.

DISCUSSION:

The characterization of the chemical structure of any extracted compound can be confirmed by using Fourier transform infra-red (FT-IR) technique. The specific chemistries and orientation of the structure will be known from the IR spectrum. FT-IR spectroscopy identifies the molecular structures that are present in a substance based on their respective absorption bands in the infrared spectrum. As a consequence characteristics of bioactive compound are usually performed by using FT-IR. The absorption peaks clearly indicates the presence of N-H stretching, NH₂ bonded OH stretching, symmetric CH₃ stretching, asymmetric CH₂ stretching and stretching vibrations of glucosidic linkage in the tested samples^18,19,20. The chitosan exhibited a wide range of antibacterial activity against all the pathogens tested. The antibacterial mechanisms of chitosan suggested being, the positive charge of the amino group at C-2 resulted in a polycationic structure which can be expected to interact with the predominantly lipopolysaccharides, proteins (anionic components) of the microbial surface²¹. It results in alteration of the structure of outer membrane²², consequently leads to the release of proteinaceous material from the cells²³. Thereby the chitosan was easily able to interact with the anionic components on cell surface, and exhibited higher inhibitory activities. There are several studies that support the current results²⁴, had reported that the chitosan possesses antimicrobial activity against a number of Gram-negative and Gram-positive bacteria. In this study, when compare to control disc and CNP, TCNP showed a significant results against the gram negative bacterial strains and almost equal effect to gram positive bacteria. Normally, P.aeruginosa is resistant to tetracycline antibiotic disc, where as in this study, CNP and TCNP showed a remarkable activity against P.aeruginosa at 100µg/ml concentration when compare to control test. This indicates that the CNP and TCNP will be used as a drug carrier against the bacterial diseases.

In addition to that the particle size and surface charge of nanoparticles were positively correlated with their inhibitory effect. In the present study, the nanoparticles had exhibited a good activity on bacteria at lower concentration.

CONCLUSION:

From this study it can be concluded that the marine waste that possess a major threat over environment can be reduced to a certain extent by employing them for the production of value added products such as chitosan and their derivatives which shows promising potentials in various disciplines. The synthesized chitosan and tetracycline coated nanoparticles revealed a prospective antibacterial activity that illustrates that they could be used as an antimicrobial drug against various clinical pathogens. Further the analysis of development of these nanoparticles as a commercial drug carrier will hopefully open the way for significant antimicrobial agent in near future.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.10.2019           Modified on 29.11.2019

Accepted on 22.01.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(8):3903-3908.

DOI: 10.5958/0974-360X.2020.00691.5