Synthesis and characterization of chitosan nanoparticles of Achillea millefolium L. and their activities [version 1; peer review: awaiting peer review]

Background: Achillea millefolium L. is an herbal aromatic plant of family Asteraceae reported to have various medicinal activities in the literature. The current study evaluated the potential of chitosan nanoparticles of A. millefolium as an effective strategy for targeted treatment of bacterial diseases and urolithiasis. Methods: A. millefolium was collected from Poonch, Jammu and Kashmir, and its inflorescence extracted in water by maceration. Chitosan nanoparticles of A. millefolium (AMCSNPs) were prepared by ionic gelation method using 0.1% chitosan, different concentrations of the cross-linking agent sodium tripolyphosphate (STPP; 0.5%, 1%, 1.5%, 2%) and different concentrations of A. millefolium extract (0.5%, 1%, 1.5%, 2%). Characterization of AMCSNPs was done using UV-Vis spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Antibacterial screening of AMCSNPs was performed by welldiffusion method. Antiurolithiatic screening of AMCSNPs was done by nucleation and aggregation assay. Results: The best chitosan nanoparticles of A. millefolium (AMCSNPs) were obtained with 0.1% chitosan, 1% STPP and 20% A. millefolium. These AMCSNPs showed maximum zone of inhibition of 30±0.5 mm using the well-diffusion method against both Bacillus subtilis (Grampositive) and Pseudomonas aeruginosa (Gram-negative) and maximum antiurolithiatic activity with 68% inhibition shown at aggregation stage. Conclusions: The current study suggests that AMCSNPs are an excellent strategy for targeted drug delivery for treatment of bacterial diseases and urolithiasis.


Introduction
Natural biopolymers are attractive products of living organisms as they serve a number of different applications for human health due to their biodegradability, such as vaccine delivery, drug development, and food preservatives 1 . Chitosan (CS) is a natural biopolymer and a derivative of chitin. It is obtained from different sources of chitin and differs on the basis of its degree of deacetylation 2 . In the last few years, CS nanoparticles (CSNPs) have drawn much attention due to their biodegradability, biocompatibility, quantum size effects, large surface to volume ratios, and their simple and inexpensive production [3][4][5] . Different biological activities of CSNPs have been reported, such as antimicrobial, antioxidant, anticancer 6 , drug delivery, tissue engineering, carbon nanotube, food preservative, and purification of water 7 . CSNPs successfully used in drug delivery for treatment of various diseases, including ocular drug delivery 8 , per-oral drug delivery 9 , nasal drug delivery 10 , pulmonary drug delivery 11 , mucosal drug delivery 12 , gene delivery 13 , buccal drug delivery 14 , vaccine delivery 15 , vaginal drug delivery 16 , and cancer therapy 17 have been reported. Achillea millefolium is a perennial herbal aromatic plant belonging to the family Asteraceae with characteristically finely divided leaves and inflorescence in corymbose cluster. It has been reported to have different medicinal activities, including antibacterial and diuretic 18,19 . The current study was designed to evaluate the potential of CSNPs of A. millefolium (AMCSNPs) as an effective alternative of targeted drug delivery and treatment of various diseases, including bacterial infections specifically urolithiasis.

Methods
Collection, identification, and extraction of A. millefolium A. millefolium was collected from Pathanteer Village, Mendhar Tehsil, Poonch District(Jammu and Kashmir, India; GPS coordinates 33° 39' 40" N-74° 11' 11" E) and identified by Raw Materials Herbarium and Museum (RHMD), National Institute of Science Communication and Information Resources (NISCAIR), Pusa with reference IDNISCAIR/RHMD/consult/2018/3293-94. 5g powder of inflorescence of A. millefolium was extracted in 50 ml of water by maceration at 90° C using water bath.
Crude extract of the plant was evaporated using rotary evaporator (Khera KI-102), which resulted in the semi solid form of extract. This was then weighed and dissolved in a known amount of solvent for making a stock concentration of the plant extract. Different concentrations were made by serial dilution.

Bacterial strains and chemicals
Strains of Bacillus subtilis (MTCC 441) and Pseudomonas aeruginosa (MTCC 1688) were procured from MTCC Chandigarh. All the chemicals used (chitosan, acetic acid, sodium tripolyphosphate (STPP), Tween-80, calcium chloride, sodium oxalate, tris buffer and NaCl) were of good quality and purchased from Fisher Scientific International, Inc.

Synthesis of chitosan nanoparticles of A. millefolium
CSNPs were prepared by ionic gelation method. 10ml 0.1% chitosan solution was made in 1% acetic acid with different percentages of the cross-linking agent STPP (0.5%, 1%, 1.5% and 2%). 5ml of STPP was added drop wise to the chitosanacetic acid solution, which was magnetically stirred at room temperature. An opalescent color was observed, and stirring was continued for 60 min.
To obtain AMCSNPs, variable concentrations of plant extract (5%, 10%, 15% and 20%) were added to the 10 ml chitosan solution by magnetic stirring prior to adding the 5 ml STPP drop wise. This solution was stirred for a further 2 h followed by centrifugation at 10000g for 10 min and then the AMCSNPs were washed three times with distilled water. The pH of the nanoparticles was maintained at 4.8, and 1-2 drops of 1% Tween-80 was used to prevent agglomeration.
Percentage encapsulation efficiency of each concentration of extract was determined using the following formula 20-22 : Encapsulation efficiency (%) = (total amount -free amount/ total amount) *100.
Characterization of chitosan nanoparticles of A. millefolium UV-Vis spectroscopy using SPUV-1000 spectrophotometer attached to Mwave professional software 2.0 (or any software used to obtain the UV-Vis absorption spectra) and spectrum between 200 nm-700 nm was obtained for determining the main absorbing region. FTIR (Fourier Transform-Infrared) Spectroscopy using spectrometer (Brukers) in the range of 1000 cm -1 -3500 cm -1 to identify the peaks of main functional groups, DLS (Dynamic Light Scattering) in the range between 0 nm to 1000 nm using zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at room temperature for particle size distribution and TEM (Transmission electron microscopy) at an accelerating voltage of 200 kV using Tecnai G2 30U-twin kV Ultra-twin microscope to study the morphology.
Antibacterial screening of chitosan nanoparticles of A. millefolium Primary culture of bacteria was obtained from lyophilized culture by inoculating in LB broth, which was incubated in an incubator shaker at 120 rpm and 37°C for 12-16 h. Pure colonies of each bacterium were obtained from primary culture by streak plate method using LB agar plates, which were inoculated in LB broth, incubated in an incubator shaker at 120 rpm 37°C for 12-16 h. Absorption of bacterial culture was adjusted to 0.1±0.02 at 600nm using SP-UV1000 spectrophotometer to reach the concentration of 10 8 CFU/ml for final use, which is equal to 0.5 McFarland standards, as previously performed in the literature [23][24][25] to obtain a similar concentration of each bacterium. Each reading was taken thrice.
Antibacterial screening of AMCSNPs was done using welldiffusion method 31 . 1.5% LB agar plates were used and a 5mm cork-borer made four wells in each plate. 20 µl of B. subtilis and P. aeruginosa culture was added to the plates and spread using a glass spreader. 100 µl of AMCSNPs was poured in the wells. Plates were sealed with parafilm, incubated at 28° C for 12-16 h and the zone of inhibition (ZOI) recorded.
Antiurolithiatic screening of chitosan nanoparticles of A. millefolium Nucleation and aggregation assays were used to determine the antiurolithiatic potential of AMCSNPs.
Nucleation assay: The method of Hennequin et al. 27 was used with some minor modifications. Solutions of calcium chloride and sodium oxalate were prepared at a final concentration of 3mmol/l and 0.5mmol/l, respectively, in a buffer containing Tris 0.05mol/l and NaCl 0.15mol/l at pH 5.5. A total of 1.9 ml of calcium chloride solution mixed with 200 µl of AMCSNPs was incubated for 30 minutes in a 37° C water bath. Crystallization was started by adding 1.9 ml of sodium oxalate solution. Equal volume of water has used for a control instead of AMCSNPs. The optical density of the solution was recorded at 620 nm for 420 sec using spectrophotometer SPUV-1000. % Inhibition = {(Abs. Control-Abs. Sample)/ Abs. Control} * 100 Aggregation assay: The method of Hess et al. 28 was used with some minor modifications. `Seed' CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50mmol/l. Both the solutions were equilibrated in a 60° C water bath for 1h and then cooled at 37° C overnight. The crystals were harvested by centrifugation at 10000g and then evaporated at 37° C. COM crystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/l and NaCl 0.15 mol/l at pH 5.7. A total of 1 ml of AMCSNPs were added in a test tube to 3 ml COM crystal solution and incubated at 37°C. Equal volume of water was used for a control instead of AMCSNPs. Absorption at 620 nm was recorded at different time intervals (30 min, 60 min, 90 min, and 120 min).

% Inhibition = {(Slope Control-Slope Sample)/ Slope Control} * 100
Statistical analysis Statistical analysis was performed using Microsoft Excel 2007. One-way ANOVA was used followed by t-test to determine the significant difference of antibacterial activity between different samples and regression analysis was used to plot graphs of nucleation and aggregation assays.

Synthesis of chitosan nanoparticles of A. millefolium
On addition of STPP to chitosan solution, an opalescent color was observed, which indicates the formation of CSNPs. Different concentrations of STPP (0.5%, 1%, 1.5%, and 2%) were used for nanoparticle preparation and 1.0% STPP was found to be most suitable with sharpest peak shown by UV spectroscopy, indicating the most CSNPs made (Figure 1). Therefore, 1.0% STPP was used to obtain AMCSNPs. Different percentages of A. millefolium water extract (5%, 10%, 15% and 20%) in 0.1% of chitosan solution were used to make AMCSNPs and excellent loading efficiency was observed, i.e. 94%, 94.7%, 94.7%, and 95.2% for 5%, 10%, 15% and 20% A. millefolium respectively. A standard graph for absorbance of A. millefolium extract at 417 nm (maximum absorption verses concentration of extract) was obtained. The amount of loaded extract was determined using the standard graph as a decrease in the absorption values of the supernatant of AMCSNPs indicated the loading of extract of the nanoparticles. Loading efficiency was calculated using the above encapsulation efficiency formula for each concentration. Hence 20% AMCSNPs have been used for further analysis.

Characterization of chitosan nanoparticles of A. millefolium
A broad absorption band between 200 to 300 nm was shown for the UV spectrum of AMCSNPs ( Figure 2 Figure 3). DLS revealed the size range of nanoparticles with Z average of 118nm having characteristic peaks at 10 nm, 122 nm and 712 nm, and highest intensity was recorded at size 10nm ( Figure 4). TEM was used to study the morphology of the nanoparticles, which revealed a spherical shape with smooth surface. TEM also revealed the size of AMCSNPs: <100 nm with smallest size of 4.15 nm ( Figure 5).     Antibacterial screening of chitosan nanoparticles of A. millefolium AMCSNPs exhibited excellent antibacterial activity against both Gram-positive B. subtilis and Gram-negative P. aeruginosa. AMCSNPs showed three-times the increase in antibacterial activity as compared with A. millefolium extract only (control); ZOI increased from 10 mm to 30mm against both B. subtilis and P. aeruginosa with a statistically significant difference between A. millefolium, CSNPs and AMCSNPs ( Figure 6).

Antiurolithiatic screening of chitosan nanoparticles of A. millefolium
AMCSNPs showed significant antiurolithiatic activity with 68% inhibition in the aggregation assay and 51.26% inhibition in the nucleation assay as compared to 55.132% and 9.09% inhibition by A. millefolium extract (control). In the nucleation assay, the % inhibition is nearly equal in the case of A. millefolium and AMCSNPs, but CSNPs did not show any inhibition. In the aggregation assay there is a significant increase in % inhibition with 9.09%, 63.63% and 68% inhibition by A. millefolium, CSNPs and AMCSPs, respectively, (Figure 7).

Discussion
For characterization of nanoparticles, different techniques used in the literature including UV-Vis spectroscopy, FTIR spectroscopy, DLS and TEM 29,30 . In our study, a UV-Vis absorption band for AMCSNPs of 200-300 nm indicates the presence of a CO group in the CSNPs, as reported by Vaezifer et al. 31 . A shift of     32 , 216 nm by Agarwal et al. 33 , and size range of 135-729 nm by Rasaee et al. 34 and 6.5-1331.2 nm by Iswanti et al. 35 . TEM of AMCSNPs in our study revealed a spherical shape with a smooth surface, which was also reported by Da Silva et al. 21 .
Chitosan is a positively charged macromolecule, which interacts with the negatively charged microbial membrane, and results in the breakage of intracellular components. Chitosan acts as a chelating agent and limits toxin production and microbial growth [36][37][38] . Antibacterial screening of Ocimum basilicum CSNPs against E. coli and Bacillus vallismortis have been reported by Rasaee et al. 34 , chitosan-tripolyphosphate nanoparticles against Staphylococcus aureus and P. aeruginosa have been reported by Bangum et al. 39 , and against phytopathogens of tomato Xanthomonas and Erwinia strains by Oh et al. 29 . Gallic acid-chitosan conjugates have been reported to inhibit the formation of calcium oxalate crystals by Queiroz et al. 40 . Antiurolithiatic activity of Aerva lanata chitosan nanoparticles at 0.8 µg/ml concentration through nucleation assay have been reported by Chandirika et al. 41 and Tridax procumbens by Chandirika et al. 42 . In our study, AMCSNPs showed excellent antibacterial activity against both B. subtilis and P. aeruginosa, and significant antiurolithiatic activity at aggregation stage of urolithiasis.
This project contains the following underlying data: • Output files of chitosan nanoparticles with different concentrations of STPP • Raw data for Figures 1, 2 and 4 • Unedited and uncropped FT-IR graphs and TEM images of AMCSNPs • ZOI of antibacterial activity of AMCSNPs • Absorption values of antiurolithiatic activity AMCSNPs Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).