Cotton fabric incorporated with β-cyclodextrin/ketoconazole/Ag NPs generating outstanding antifungal and antibacterial performances

The influence of ketoconazole and β-CD/ketoconazole on cotton fabric as fungal skincare was previously reported however the impact of nanosilver on the antifungal and antibacterial properties of the same products is unknown. Here, silver NPs were synthesized on β-CD/KZ composite and then loaded on cotton fabric by using a cross-linking agent. The nanocomposite and treated fabrics were analyzed by diffenent means such as UV–vis, dynamic light scattering, zeta-potential and FE-SEM. The nanocomposite antimicrobial efficiency was examined on Candida albicans and Aspergillus niger as fungi and E. coli and S. aureus as bacteria. The synthesis of Ag NPs on β-CD/KZ amplifies both antifungal and antibacterial efficiencies. Having tremendous antimicrobial activities without cytotoxicity effects and drug release regularity with excellent washing durability makes the product suitable for medical applications as well as wound dressings and sportswear designed for sensitive skin.


Introduction
Human skin covers the whole body but its contacts with the environment generates most injuries such as wounds, burns and skin fungi (Benita 2006;Farokhi et al. 2018;Kim et al. 2019;Wu et al. 2018). This immense surface is easily accessible for drug delivery as a painless and non-invasive method. Topical drug delivery has advantages such as high drug concentration on the skin, lower side-effects, ease of transfer, more sustainability and water solubility (Benita 2006;Farokhi et al. 2018;Kim et al. 2019;Lee et al. 2017). Therefore, diverse chemical and physical techniqes were adapted to improve drug delivery via the skin. On this base, nanoparticles were used to extend half-life, decrease drug dosage and improve drug properties (Farokhi et al. 2018;Kim et al. 2019;Lee et al. 2017;Veerakumar et al. 2020;Wang et al. 2015). Nevertheless, a dressing substrate with drug release properties such as cotton fabric treated with cyclodextrin (CD) can be useful for this purpose (Ning et al. 2020;Pinho et al. 2014;Radu et al. 2016;Sharaf and El-Naggar 2019;Yao et al. 2019). Thus, b-cyclodextrin (b-CD) with its hydrophobic cavity and hydrophilic surface can control the release of aromatic compounds (Abarca et al. 2016).
Ketoconazole (KZ), as a water-insoluble antifungal drug with high molecular weight, often used topically to treat superficial fungal infection (Che et al. 2015), especially Candida albicans (Kaur and Kakkar 2010). The entrapped KZ in b-CD showed antifungal properties without antibacterial effects (Kaushik et al. 2017;Rameshkumar et al. 2003). However, introducing nanoparticles to the antimicrobial drugs has a significant synergistic effect due to entering into the cell wall causing cell death (Durán et al. 2016;Jiang et al. 2019;Rana et al. 2016;Sedighi et al. 2014). Further, capping Ag-NPs with b-CD improves the efficacy and reduces cytotoxicity (Gupta et al. 2020). Nevertheless, KZ/Ag-NPs showed the most synergistic effects in comparing with the common antifungal drugs such as nystatin, fluconazole and clotrimazole (Falahati et al. 2014;Jiang et al. 2019;Rana et al. 2016;Sedighi et al. 2014).
In our previous study, KZ and b-CD/KZ were loaded on cotton fabric via padding by immersing the fabric in the prepared solution, squeezing and finally drying at 80°C. This was also done through exhaustion by immersing the fabric in the prepared solution heating for 1 h at 80°C along with stirring and final drying at 80°C. The drug release, antimicrobial activities and cytotoxicity of the products were already reported (Hedayati et al. 2020). The KZ release was prolonged, especially in the exhaustion method compared with the other drugs. Also, b-CD/KZ showed antifungal properties with no inhibition zone against bacteria (Hedayati et al. 2020). Although the b-CD/ KZ treated fabric with great antifungal activity and slow drug release was suitable for wound dressing, it has no antibacterial activity. Hence, it was tried to produce cotton fabric with the both antifungal and antibacterial features with low cytotoxicity and drug release rate alogside good washing durability for medical applications, likewise wound dressings and sportswear designed for sensitive skin. Here, Ag NPs were synthesized on b-CD/KZ to improve the antimicrobial properties and moderate the release rate thus KZ was first entrapped into b-CD, then Ag NPs were synthesized on b-CD/KZ composite successively loaded on the cotton fabric through exhaustion and padding methods.
The sample with the most antimicrobial activities and slow/regular drug release, besides the good washing durability, breathability and moisture absorption was selected as the best sample that can be used as a safe product for medical purposes and clothing applications.

Materials and experimental procedures
Materials b-CD was obtained from X'ian Hong Chang Pharmaceuticals Co. (China) and KZ was gifted from Arasto Pharmaceutical Chemicals Inc. Silver nitrate 99%, acetic anhydride, citric acid, sodium hypophosphite (SHP), methanol, ethanol and cell culture medium were used from Merck Co. (Germany). Aspergillus niger-ATCC 9642, Candida albicans-ATCC 10,231, E. coli-ATCC 25,922 and Staphylococcus aureus-ATCC 2592 were also used. Sodium dioctyl sulfosuccinate and phosphate-buffered saline were purchased from Sigma-Aldrich Co. (Germany). Nonionic surfactant was procured from a local market in Tehran, Iran. Also, the bleached cotton fabric with 118 g.m -2 weight was used from Brojerd Textile Co, Iran.

Preparation of nanocomposite solution and indicator
b-CD/KZ was prepared according to the previous report; briefly, KZ and b-CD in a molar ratio of 1:1 were stirred in water at 25 ± 2°C and pH = 7 ± 0.5 for 3 days in dark and the remaining solid was separated by paper filter (Hedayati et al. 2020).
In order to synthesize silver nanoparticles on b-CD/ KZ nanocomposite, silver nitrate (Ag ? :b-CD with molar ratios of 1:50 (2% w/w), 1:100 (1% w/w), 1:200 (0.5% w/w) and 1:500 (0.2% w/w)) was added to 100 mL b-CD/KZ solution at pH = 11.5 ± 0.5 that was adjusted with sodium hydroxide. The solution was stirred on a magnetic stirrer and heated for 3 h keeping the temperature around 80°C. The clear color of the solution was changed to dark brown confirmed the action of b-CD as a reducing agent to synthesis Ag NPs and also as a stabilizer for controlling the size of synthesized Ag NPs.
On these bases the synthesized Ag NPs can be entrapped inside the b-CD cavities (small size) and also Ag can be nucleated outside the b-CD and grown.
Also, KZ was dissolved in ethanol (pH = 7 ± 0.5) with the same portion used in b-CD/KZ without b-CD.
As previously reported, the k max of KZ was obtained at 243 nm with a slope of 0.0282 and R 2 = 0.994 (Hedayati et al. 2020). The obtained k max conflicted with the absorbance peaks of the other used compounds; thus, it was shifted to the visible range by using a saturated solution of citric acid in acetic anhydride (CAA) as an indicator. Briefly, 100 mL of acetic anhydride was added to the excess solid citric acid (preparing a saturated solution) stirred for 2 h and filtered, immediately used (Omar et al. 2006). The stock solutions of KZ were produced with different concentrations of KZ in absolute methanol (Hedayati et al. 2020). 10 mL of each solution transferred to a beaker and evaporated to dry in a Bain-Marie. Then 2.5 mL of CAA indicator was added and put in Bain-Marie for 20 min kept at room temperature (20 ± 2°C) to cool down and then pure ethanol introduced to obtain the volume of 10 mL (Omar et al. 2006). The k max of KZ was obtained at 535 nm with a slope of 0.3615 and R 2 = 0.9916 (Y = 0.3615X-0.0056) (Fig. 1).

Preparation of samples
The loading of nanoparticles on cotton fabric was carried out by using two diverse methods (Hedayati et al. 2020). Before loading of nanocomposite, the scouring of samples was done with 1 g.L -1 nonionic detergent for 30 min at 50°C. Then the nanocomposite was loaded on the fabric through exhaustion (1 h at 80°C with stirring and drying at 80°C similar to dyeing) and padding (three times imperegnation then squeezing and finally drying at 80°C) procedures. Further, citric acid (10% w/w) as a cross-linking agent and sodium hypophosphite (6% w/w) as a catalyst were added to the nanocomposite solution and then the fabric immersed in the solution squeezed and dried at 80°C finally cured at 140°C for 4 min followed by rinsed with cold water. Moreover, the blank sample without b-CD was prepared through treating of the cotton fabric with the same amount of dissolved KZ in ethanol. One piece of the fabric sample cut and immersed in 10 mL pure methanol (as solvent) along with stirring to study the amount of KZ entrapped in b-CD and the release rate. At a specific time (up to 30 days), the sample was removed from the solution and the solution was put in the bath to completely dry. Then 2.5 mL CAA indicator was added and put in the bath for 20 min to cool down; finally, pure ethanol was added to adjust the voluome in 10 mL. The KZ content was found by using the absorbance of the solution at 535 nm (Optizen, Mecasys, Deajoen, Korea, 2120UV) (Omar et al. 2006).
Antifungal activity against A. niger (FTTS-FA-006 method): 50 mg sodium dioctylsulfosuccinate as a wetting agent was dissolved into 1 L water (0.005%) and the concentration of A. niger was adjusted to 10 6 ± 2 9 10 5 CFU/mL in a wetting agent followed by thoroughly shaking. The sterilized circle samples in 3.8 ? 0.5 cm were placed in solid mineral salt agar medium (MSA) then 1 ± 0.1 mL of the fungi suspension was evenly distributed over the specimen by micropipette. All plates were incubated at 28 ± 1°C and humidity above 85% for 14 days. The samples were assessed by the number of clonies produced on their surfaces.
Antifungal activity against C. albicans (AATCC 100-2004 method): The raw and treated samples were cut circular in 4.8 ± 0.1 cm and sterilized in an autoclave then 1.0 ± 0.1 mL of 10 5 CFU/mL microorganisms in sabouraud dextrose broth medium were added on untreated and treated fabrics and incubated at 30°C for 24 h. There after, 100 mL of normal saline was added to each jar and shaken vigorously for 1 min. 1 mL of each sample was taken and introduced to the plates (in duplicate) containing 15 mL sabouraud dextrose agar and mixed gently followed by incubation at 37°C for 24 h.
Antibacterial activity against E. coli and S. aureus (AATCC 100-2004 method): The raw and treated circle samples with 4.8 ± 0.1 cm diameter sterilized in an autoclave then 1.0 ± 0.1 mL of 10 5 CFU/mL microorganisms in triptic soy broth medium was poured on untreated and treated fabrics and incubated at 37°C for 24 h. The 100 mL of neutralizing solution (normal saline) was added to each jar and shaken vigorously for 1 min then 1 mL from each sample was taken and added to plates (in duplicate) containing 15 mL triptic soy agar medium and mixed gently. The plates were incubated at 37°C for 24 h and the number of colony forming units for untreated and treated samples (A and B) were counted on the agar plates. The reduction percentage of microbial colonies (R) was finally recorded according to Eq. 1 (Hedayati et al. 2020;Navik 2017;Pivec et al. 2017: Also, the durability of nanocomposite on cotton fabric was studied through 10, 20 and 30 washes with a nonionic surfactant at 60°C for 20 min. These samples were considered for antifungal activities against C. albicans (Ghayempour and Montazer 2017; Hedayati et al. 2020).
The cell culture, cytotoxicity and metabolic activity were evaluated on the normal human skin fibroblasts by MTT assay (Ahmed et al. 2017;Martinez-Gutierrez et al. 2010;Korrapati et al. 2016;Hedayati et al. 2020). The fibroblasts were cultured in DMEM (1X), GlutMAXTM (GibcoTM) and 10% fetal calf serum. In order to grow and multiply natural fibroblast cells, passage number 3 was used and added to the 96-well microplates, then incubated with 5% CO 2 at 37°C for 48 h. The raw and treated fabrics in 1 9 1 inch 2 sterilized in an autoclave (121°C, 15 lb/inch 2 for 20 min) then soaked in 2 mL of cell culture medium and incubated for 24 h at 37°C. The culture medium of multiplied cells in a 96-well microplate was slowly drained and the samples were replaced. Finally, the microplate was incubated at 37°C for 24 h and examined by MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in phosphatebuffered saline. The absorption of the solution in each well was recorded by Hiperion microplatereader (MPR4 ?) at 570 nm, and the number of living cells were measured. This was repeated 3 times and the percentage of cell viability was obtained through the absorption ratio of treated and control samples.
The detailed information of nanocpmposite was determined by 1 H-NMR in CDCl 3 as a solvent for KZ and D 2 O for b-CD and b-CD/KZ using Ultrashield 400, BRUKER, Germany.
The dynamic light scattering (DLS) and zeta potential were used at 25°C by HPPSv42, Malvern Instrument, Malvern, UK, to investigate the size and surface charge of the nanocomposite.
The Fourier transform infrared spectroscopy (FTIR-Thermo Nicolet Nuexus 870 with the range 400-4000 cm -1 , USA) was also used to analyze the powder and treated samples such as KZ, b-CD/KZ and b-CD/KZ/Ag. The fabric was finely chopped and mixed with KBr in a ratio of 1:100.
The FE-SEM, MIRA3TESCAN-XMU (3-30 kV in the low vacuum) Hitachi, S-4160 was used with resolution of lower than 1 nm through a magnification of 100000X, with prime covering of samples with Au. This is also equipped with EDS microanalysis and E-map, which allows the detection of smallest phase, with the possibility of adjusting pressure and high resolution with an accelerating voltage of 15 kV and beam current of 1.200 nA.
The mechanical properties of 2.5 cm 9 15 cm samples with the same thickness were measured using an Instron 5566 tensile tester according to ASTM D882-02 standard at a tensile speed rate of 25 mm min -1 .
Due to the high importance of textile hydrophilicity for medical and clothing usages, the water absorption test was performed by dropping 3 droplets of water at a distance of 1 cm from the treated samples and measured the water spreading time three times, reported the average time.
Air permeability test was performed on the raw and treated fabrics using Shirley air permeability tester (SDL, Atlas, USA) based on BS 5636 (Horrocks and Anand 2000).
The statistical analysis of tensile and air permeability of raw and treated samples was performed in seven versions with five repetitions of individually treated samples, and the mean values along with their standard deviations (SD) are reported. One-way analysis of variance (ANOVA) was performed, and the significance of each mean value was determined (P-values \ 0.05) with Tukey's multiple range test using version 6.0 of GraphPad Prism (GraphPad Software, La Jolla, CA).

Results and discussion
The entrapment efficiency of KZ in b-CD The efficiency of KZ entrapment in b-CD was considered according to the total weight of KZ and b-CD and unloaded KZ in b-CD. The amount of KZ entrapped in b-CD with a molar ratio of 1:1 along shaking at 25 ± 2˚C for 3 days was about 73.6% due to the large molecular size of KZ and a lower rate of penetration that is an acceptable and justified value (Hedayati et al. 2020). Here, the KZ entrapment efficiency was similarly evaluated at 65°C ± 3 in 3 days. After filteration of the solution and drying, the residual deposit was weighed, and the efficiency of KZ entrapment in b-CD was determined by using Eq.
M t and Mu are the total weight of b-CD/KZ and unloaded KZ, respectively.
The entrapment efficiency of KZ in b-CD was about 73.71% however due to the very high absorption in UV-Vis spectrum possibly due to KZ degradation, the testing was not carried out at 65°C.

H-NMR analysis of the complex
The best method to obtain a more in-depth insight into the molecular interactions between b-CD and drugs is spectroscopic and especially NMR studies (Conceicao et al. 2018;Demirel et al. 2011;Varga et al. 2019;Yuan et al. 2018). In this analysis, only up and downfield shifts occurred therefore, the upfield shift of b-CD protons and downfield shift in guest protons happened because of KZ entrapment into b-CD cavity Ventura et al. 2006;Xu et al. 2015). This was only performed on KZ, b-CD and b-CD/KZ powder not on b-CD /KZ/Ag-NPs due to possible toxicity. The b-CD and KZ 1 H-NMR spectra in Fig. 2 verified the prominent peaks of KZ in 1 H-NMR spectra of b-CD/KZ nanocomposite confirmed KZ entrapment. Thus, the KZ molecules entrapped into b-CDs cave mostly from -CH 3 side due to the downfield shift of H 1 (-CH 3 ) from 2.15 to 2.07, H 11-13 , H10-14 and upfield shift of H 28 , H 17 , H 24 and H 27 benzene ring of KZ (Kundu and Roy 2017;Zoppi et al. 2019).

Dynamic light scattering (DLS)
DLS was recently used to express more details on cyclodextrins accumulation (Ishiguro et al. 2010;Sajomsang et al. 2011). The colloidal stability can also be determined by the surface charge as a very effective method via zeta potential that can only be used on the nanocomposite solutions. Table 1 demonstrates the surface charge and particle size of the synthesized nanocomposite. The hydrodynamic size and surface charge of KZ, b-CD and b-CD /KZ were already reported (Hedayati et al. 2020) as the bigger hydrodynamic size related to b-CD and creation of hydrogen or weak physical bonding (Attarchi et al. 2013;Hedayati et al. 2020;Myles et al. 2018).
Further, KZ with two active chlorine groups caused to the bigger hydrodynamic diameter of particles due to the possible interactions between KZ and b-CD and also hydrolysis of KZ in alkali media according to Reaction 1in Fig. 3, (Reichle 1970). Nevertheless, KZ in an alcoholic or alkaline environment underwent different reactions as illustertaed in Reaction 1 and 2 in Fig. 3 (de Almeida et al. 2019; Reichle 1970). The high temperature initiated the bigger particles with a hydrodynamic size of about 2570 nm (PDI = 1.000) and surface charge of -11.3 mV (Zeta Deviation = 10.2) for b-CD/KZ nanocomposite when KZ loaded at 65 ± 3°C in 3 days. The size of Ag NPs was larger than b-CD cavity thus interacted with hydroxyl groups of b-CD surface reducing the particles agglomeration (Attarchi et al. 2013;Ouyang et al. 2015). Also, Ag NPs in the solution showed the smaller size in the nanometer range.
Zeta potential, as an important indicator of the colloidal solution stability, was considered (Rajamanikandan and Ilanchelian 2018). Both KZ and b-CD indicated a negative surface charge in an unstable range however, b-CD/KZ/Ag NPs nanocomposite specified a more negative charge, greater stability and repulsion of the particles provided the lower aggregation and particle size (Hedayati et al. 2020).

Synthesis of Ag NPs in b-CD/KZ
One of the proposed methods for synthesis Ag NPs from AgNO 3 is using alkaline media with heating in presence of stabilizer and reducing agent such as cellulose or b-CD (Reaction 3) (Aladpoosh et al. 2014;Prasher et al. 2018;Rajamanikandan and Ilanchelian 2018;Ouyang et al. 2015). In this research, b-CD was considered as a reducing agent with stabilizing effect to synthesis Ag NPs. b-CD in sodium hydroxide solution formed b-CD-ONa ? . The Na ? ions can be replaced with Ag ? producing b-CD.O-Ag due to the higher electronegativity of silver (1.93) than sodium (0.93) in an aqueous solution. This was also confirmed previously by X-ray photoelectron spectroscopic (XPS) analysis (Ouyang et al. 2015).

Drug release
Drug release was studied three times based on the amount of drug-loaded on cotton fabric by using a color indicator. Similar percentages of KZ release were already reported for samples treated with KZ and b-CD/KZ in ethanol for both methods using the standard curve (Hedayati et al. 2020). However, in order to reduce the possible errors, no color indicator was used for nanoparticle-free solutions. The highest percentage and amount of KZ loaded on the fabric related to the fabrics obtained through exhaustion method. Also, the release behavior of KZ from different samples with or without b-CD was thoroughly discussed previously (Hedayati et al. 2020). Here, only the effect of Ag NPs on the release profile of KZ was investigated.
According to Table 2 and Fig. 4.a, synthesis of Ag NPs in the solution containing b-CD/KZ led to the lower nanocomposite loading or KZ release from the fabric in the both applied methods. This can be related to the decrease in aggregation due to the silver NPs (Attarchi et al. 2013;Ouyang et al. 2015). Figure 4-b showed the slower, regular and more linear KZ release from the samples containing Ag NPs even after 30 days and merely a partial KZ release reported after 40 days. The hydrophobic internal cavity of b-CD (0.78 nm) allows very small synthesized Ag NPs to enter without Ag nucleation. The Ag This proportionate to the state of KZ entry into b-CD cavity as KZ molecule entrapped into b-CD cave mostly from -CH 3 side according to 1 H-NMR spectra. Hence, the rate of KZ release can be slowed down by increasing the size of composite. Moreover, the nucleation mostly formed on hydroxyl groups of b-CD and oriented out of the structure. Also, Ag NPs likely blocked the route of KZ release from b-CD cavity (Fig. 3) reduced the release of KZ from the samples having Ag NPs (Jaiswal et al. 2010). For those samples with Ag NPs, the test was performed on b-CD/KZ/Ag 2% in two application methods.
The cotton fabrics loaded with KZ and b-CD/KZ indicated a very slow release rate because of the unique structure of KZ with two active chlorine groups that can react with hydroxyl groups of b-CD or cellulose fibers creating covalent bonds between KZ and cellulose (Hedayati et al. 2020). Also, the synthesis of Ag NPs in an alkaline environment led to KZ hydrolysis that caused to interactions with Ag or b-CD hydroxyl groups prolonged the KZ release (Hedayati et al. 2020;Lewis 2011]. Further, the negative charge of KZ makes it ready for the placement of Ag ? (Reaction 2 in Fig. 3,)  Antimicrobial activity of treated samples Two fungus and yeasts, A. niger and C. albicans and two Gram negative and positive bacteria, E. coli and S. aureus were selected to study the antimicrobial activities of treated samples (Table 3). In general, the gray columns in Table 3 indicate the percentage of microbial reduction before washing.
The antimicrobial activities of KZ and b-CD/KZ and the influence of loading method have already been reported. As such, the antifungal activities of exhausted samples were much stronger than padded ones because of the high temperature, fibers swelling, greater pore size and more loading of nanocomposite (Hedayati et al. 2020). However, none of the prepared samples with KZ showed antibacterial activity. Ag NPs are sticking to the microorganism membranes disrupting the function and making holes in the membrane and cytoplasmic fluid outflow, causing cell death (Vale et al. 2019;Wenhao et al. 2020).
Hence, Ag NPs were synthesized on b-CD/KZ composite to investigate their effects on the padded and exhausted samples. b-CD/Ag 2% showed 70, 42, 87 and 82% microbial reduction for C. albicans, A. niger, E. coli and S. aureus, respectively. All samples containing KZ were affected on C. albicans however, Ag NPs indicated potential effects on A. niger. Among various samples, b-CD/KZ/Ag 2% indicated the best antifungal and antibacterial effects. Although the amount of KZ in b-CD/KZ was much more than b-CD/KZ/Ag in the exhaustion method, a weaker fungicidal effect was observed on A. niger. Based on the above results, Ag NPs and KZ with great improved effects can advance the final antimicrobial properties (Falahati et al. 2014).

Washing durability
The washing durability of samples was examined against C. albicans and the percentages of growth inhibition are reported in Table 3. The good washing durability has already been shown on treated samples with KZ and b-CD/KZ via exhaustion method (Hedayati et al. 2020). The effect of Ag NPs on the nanocomposite after various cycle of washing exhibited very high durability however, the exhausted samples revealed better durability than padded ones. This can be due to the processing conditions at hightemperature along with mechanical movements that caused swelling of fibers, formation of pores, and greater loading of nanocomposite onto the fibers. Also the covalent bonds were formed between the nanocomposite and the cellulosic chains of fabric. The loaded sample with b-CD/Ag exhibited a good washing durability after 30 washes as the antifungal properties merely decreased from 70 to 65%, due to the fixation of Ag NPs on b-CD and cotton surfaces (Fig. 3).

Cell culture and cytotoxicity
The used dosage of KZ and b-CD indicated no cytotoxic effects on the human fibroblasts skin (Hedayati et al. 2020). The cytotoxicity of Ag NPs in different concentrations was examined due to its importance for wound dressings and underwear. All treated samples illustrated no cytotoxic effects on the human fibroblasts confirming the safety of final products for the skin (Fig. 5). This confirmed no side effects on the human skin with loading of a very low Ag NPs on the samples.

Fourier transform infrared spectroscopy (FTIR)
The exhausted samples were selected for analysis due to the more loading of nanocomposite with the highest Ag NPs on the cotton fabric. Figure 6 displays the FTIR spectrum of KZ powder, raw and treated fabrics with nanocomposite. The prominent peaks of KZ, cotton and cross-linked b-CD on fabric (at 1726 cm -1 ) were thoroughly discussed previously (Hedayati et al. 2020).
The increase in the absorbance intensity of prominent KZ peaks, such as C-N stretching or C = C aromatic rings of the treated samples confirmed KZ entrapment or loading on the fabric. However, the lack of KZ prominent peak at 814 cm -1 related to C-Cl stretching on the treated fabric proved the removal of chlorine groups due to the reactions of chlorine with b Fig. 3 Reaction 1 is poossible hydrolysis of KZ in dichlorophenyl side (Reichle 1970), Reaction 2 is possible hydrolysis of KZ in ethanone side (de Almeida et al. 2019) and Reaction 3 is interactions of KZ, cellulose and b-CD with silver nitrate for producing Ag NPs (Attarchi et al. 2013;de Almeida et al. 2019;Prasher et al. 2018;Rameshkumar et al. 2003)

Field emission scanning electron microscopy (FE-SEM), elemental mapping (E-map) and EDS spectrum
The FE-SEM, EDS and E-map pictures of raw and exhausted samples with the most properties are displayed in Fig. 7 to show the surface topography and identify the elements and uniformity of their distribution.
The image of raw cotton fabric with the fibrils on surface can be realized in Fig. 7, however the treated fabric entirely covered with the nanocomposite. The aggregation of particles can be seen in some parts, especially on b-CD/KZ due to the high b-CD content created hydrogen, covalent or van der Waals bonds between b-CDs or b-CD/KZ. However, Ag NPs potentially reduced the aggregation of nanocomposite. The particle size of KZ and is about 40-65 and 20-40 nm for b-CD/KZ and b-CD/KZ/Ag respectively. The E-map also confirmed the uniformity of KZ and Ag NPs on all samples.
Energy-dispersive X-ray spectroscopy (EDS) was also used to identify elements, including Ag (Urban et al. 2009) (Fig. 7). The peaks of carbon and oxygen are associated with KZ and cellulose of cotton and b-CD. The loading of b-CD on fabric led to the strong peaks of carbon and oxygen elements. The small peaks of chlorine and nitrogen is corresponding to KZ. A very low intensity of chlorine peak compared with nitrogen indicated a possible reaction of chlorine with hydroxyl groups of b-CD or cotton fabric. The amounts of Cl and N elements can be specified to KZ in the following order: Au is also associated with a gold layer for preparing the sample.

Tensile properties
The tensile strength of raw and treated cotton samples with nanocomposite is revealed in Table 4 to screen the effects of nanocomposite and citric acid on the mechanical properties of cotton fabric. Based on the previous study, the more nanocomposite on cotton fabric (without fixation) caused a significant (Pvalues \ 0.05) increase in the fabric strength due to the created bonds within the cellulosic chains of cotton fabric. It was claimed that the reaction of KZ with cotton fabric led to the higher tensile strength. Also, b-CD/KZ with citric acid on cotton fabric caused the lower tensile strength due to the acid degradation of cellulosic chains at high temperature. This can be partially improved with increasing b-CD in the exhaustion method (P-values \ 0.05) (Hedayati et al. 2020).
It is certain that the treated fabric with b-CD (Pvalues \ 0.05) has higher tensile strength (225.6 N) than b-CD/KZ (219.38 N) because of the entrapped KZ into b-CD cavity or bonding with its rim hydroxyl groups and lower b-CD loading. The Ag NPs somehow reduced b-CD gathering and nanocomposite on cotton fabric consequently reduced the tensile strength (196.07 N) (P-values \ 0.05) however, it is within the

Water absorption
The water absorption test was performed and the average of three tests are reported in Table 4. The b-CD, KZ and Ag NPs on the cotton fabrics improved the water absorption. The rate of spreading and absorption of water droplets on all samples was fast recorded zero second.

Air permeability
Air permeability test was performed on the raw and treated fabric samples at a test pressure drop of 50 Pa for 5 cm 2 test area. This was repeated three times, and the averages are reported in Table 4. The nanocomposite treated sample airflow rates are almost similar and somehow more than the raw sample due to reducing the lateral fibers and increasing the pore size for air to pass through the fabric. On the other hand, the application of nanocomposite on cotton fabric at high temperature in some way increased the fabric air permeability (P-values \ 0.05).

Conclusion
In this study, b-CD/KZ/Ag nanocomposite was produced and applied on cotton fabric via two diverse procedures of exhaustion and padding to create a novel antimicrobial drug delivery system. The final products were characterized by various analyzing tools. The exhaustion method is mostly recommended due to the much more nanocomposite loading on cotton fabric. The best antimicrobial effects reported for the treated sample with b-CD/KZ/Ag 2% that indicated 100% reduction in C. albicans and A. niger and about 85% in E. coli and S. aureus with no cytotoxic effects for fibroblasts cell. The presence of Ag NPs in the composite prevented the nanoparticles from aggregation and reduced its loading on the cotton fabric that possibly reduced the drug side effects. The placements of Ag NPs within and around b-CD/KZ nanocomposite may also block the b-CD cavity and reduce the KZ release produced more control release product. Finally, the good durability of b-CD/KZ/Ag nanocomposite on cotton fabric in consecutive washes leads to the superior antimicrobial activities, breathability, water and moisture absorption with reasonable tensile strength that can be offered as a safe product with both the antifungal and antibacterial features.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Informed consent For this type of study formal consent is not required.
Animal and Human rights This work does not contain any studies with human participants or animals performed by any of the authors.