hydroxypropyl-β-cyclodextrin inclusion complex with enhanced solubility and antimicrobial activity

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Development of Anacardic Acid/hydroxypropyl-β-cyclodextrin inclusion complex with enhanced solubility and antimicrobial activity Md Meraj Anjum a, Krishna Kumar Patel a, Nidhi Pandey b, Ragini Tilak b, Ashish Kumar Agrawal a,⁎, Sanjay Singh a,⁎ a b

Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, Uttar Pradesh, India Department of Microbiology, Institute of Medical Sciences (Banaras Hindu University), Varanasi 221005, Uttar Pradesh, India

a r t i c l e

i n f o

Article history: Received 10 September 2019 Received in revised form 1 November 2019 Accepted 6 November 2019 Available online xxxx Keywords: Anacardic Acid HP-β-CD Inclusion complex Antimicrobial Biofilm

a b s t r a c t Anacardic Acid (AnAc), a key constituent of cashew nut shell liquid (CNSL), has been reported to exhibit antibacterial activity against S. aureus but its bioactivity and clinical applicability are limited owing to its poor water solubility and physicochemical stability. The aim of the present study was to develop inclusion complex of AnAc (C15:3) and hydroxypropyl-β-cyclodextrin (HP-β-CD) to improve its aqueous solubility and antimicrobial activity. The inclusion complex was prepared using the co-evaporation method considering different molar ratios of AnAc (C15:3) and HP-β-CD. The prepared inclusion complex was characterized using FT-IR, DSC, XRD, SEM, and 1 H NMR, which provided appropriate evidence to confirm the formation of the inclusion complex. The prepared inclusion complex demonstrated ~2009 times improved aqueous solubility. The developed complex maintained the antimicrobial activity of pure AnAc against S. aureus. AnAc/HP-β-CD complex exhibited excellent biofilm dispersal potential against mature biofilms. Confocal laser scanning microscopy (CLSM) demonstrated a remarkable decrease in bacterial biomass and thickness upon treatment with the inclusion complex. Conclusively, the developed inclusion complex of Anacardic Acid (C15:3) can be effectively used to overcome the poor aqueous solubility and improve the therapeutic efficacy for many other indications. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Anacardic Acid is a long chain phenolic acid obtained from cashew nut shells of the plant Anacardium occidentale belonging to family Anacardiaceae; it is the major component of natural cashew nut shell liquid (CNSL). The solvent extracted CNSL is a rich source of many long-chain phenolic compounds like cardanol (10%), cardol (15–20%), and Anacardic Acid (60–65%) [1]. The long aliphatic side chain of Anacardic Acid is found as a mixture of monoene (C15:1), diene (C15:2), and triene (C15:3), with the saturated component (C15:0) being present in lesser amount (Fig. 1-A). Anacardic Acid has been proved for its wide pharmacological activities, including antimicrobial [2], antifungal, antimalarial, anticancer [3], anti-inflammatory, and antioxidant [4] activities. However, the compound shows poor water solubility [5], which in turn limits its wider pharmaceutical applications in formulation development and drug delivery. Therefore, in order to make the drug suitable for delivery in the body, it is necessary to increase its water solubility, which in turn improves bioavailability and pharmacological activity [6]. Several ⁎ Corresponding authors. E-mail addresses: [email protected] (A.K. Agrawal), [email protected] (S. Singh).

approaches have been implemented so far to enhance solubility and bioavailability of poorly aqueous soluble drugs such as the particle size reduction, drug dispersion in suitable carriers [7] as well as complex formation with cyclodextrins. The preparation of cyclodextrin inclusion complexes is a key technique to boost the biopharmaceutical properties of bioactive compounds [8]. Cyclodextrins possess the ability to form noncovalent associates like “guest–host” inclusion complexes with hydrophobic drug molecules as well as they tend to crystallize easily and have a well-designed chemical composition as well as physicochemical stability [9]. β-Cyclodextrin possesses limited aqueous solubility owing to its rigid structure due to the incurrence of intermolecular hydrogen bonds in the midst of the secondary hydroxyl groups [10]. For further improving the solubility, β-cyclodextrin may be chemically altered to different forms. 2Hydroxypropyl-β-cyclodextrin (HP-β-CD) (Fig. 1-B) is produced by substitution of the hydroxyl group with hydroxypropyl substituents in the positions 2, 3, and 6. HP-β-CD possesses better solubility and lower toxicity than β-cyclodextrin [11]. Thus, the objective of the current work was to enhance the water solubility and thus the therapeutic efficacy of Anacardic Acid against antimicrobial-resistant bacteria, as antimicrobial drug resistance has been progressively prevalent over the last decade and become the key threat to public health. To have the adequate therapeutic efficacy of

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Fig. 1. Chemical structure of Anacardic Acid showing its various forms (A) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) (B).

the antimicrobials against bacterial infections with resistant strains, either a higher dose of antibiotics or the combination of multiple antibiotics may be needed, which ultimately leads to severe toxicity and enhanced mortality rate. There is a long list of antimicrobial-resistant bacteria, including penicillin-resistant, sulphonamide resistant, vancomycin-resistant, and MRSA. Methicillin-resistant S. aureus (MRSA) is concomitant with more mortality than methicillin-sensitive S. aureus (MSSA) [12]. Moreover, these microbes invent strategies to safeguard their existence and adaptation to tense environments. This persuades the development of a cohesive and tough community of possessing intercellular communication, termed as biofilm [13]. Nature of biofilms includes heterogeneous communities (demonstrating species diversity) of amassed, organized, and active microbial cells that stay embedded into the matrix of extracellular polymeric substances (EPS) that allow irreversible adhesion to the biotic or abiotic surfaces [14]. The biofilm predominantly forms the matrix of EPS, which is composed of nucleic acid, exopolysaccharides, phospholipids, extracellular proteins, and teichoic acid [15]. Thus, in the present work, we hypothesized that improving the solubility of Anacardic Acid will also enhance the therapeutic efficacy even against antimicrobial-resistant bacteria. To prove our hypothesis, we developed the inclusion complex of Anacardic Acid (C15:3) with HP-βCD and evaluated for its antimicrobial efficacy against Staphylococcus aureus (S. aureus). The rationale behind the selection of Anacardic Acid (C15:3) among different forms is based upon earlier reports in which Anacardic Acid (C15:3) has shown the highest antimicrobial activity among all the forms [16]. In this study, the inclusion complex of AnAc was prepared with HP-β-CD, and further stability constant of the inclusion complex was determined by performing a phase solubility study.

Furthermore, the solid inclusion complex was characterized using different analytical techniques and evaluated for the solubilization effect. Finally, the antimicrobial and antibiofilm activities of AnAc (free and complexed) were examined to establish the potency of the developed complex in retaining bioactivity. 2. Materials and methods 2.1. Materials Anacardic Acid extraction, isolation, and characterization were done as per the previous report [17]. Further, Anacardic Acid (C15:3) (denoted by AnAc further) was isolated from the mixture of all the forms of Anacardic Acid using the previously reported protocol [18]. Hydroxypropyl-β-cyclodextrin was purchased from Himedia Laboratories Pvt. Ltd., Mumbai. Ethanol was obtained from Merck Life Science Pvt. Ltd., Mumbai. S. aureus (ATCC 29213) was purchased from Himedia Laboratories Pvt. Ltd., Mumbai. All other chemicals and reagents unless otherwise mentioned used in the study were of analytical grade. 2.2. Preparation of AnAc/HP-β-CD inclusion complex Anacardic Acid Inclusion Complex (AnAc-IC) of molar ratio 1:1 was formulated by the co-evaporation method [19]. Briefly, AnAc (87 mg) and HP-β-CD (344 mg) were dissolved completely in ethanol (10 ml) and water (10 ml), respectively. Further, both the solutions were stirred in a sealed glass vial and retained at room temperature with mild stirring for 30 min. The temperature of the mixture was then increased up to 50 °C and maintained constant for 2 h while stirring at 500 rpm. Further, the resultant solution was dried by evaporation under low

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pressure using a rotary evaporator (IKA, Germany) at 40 °C to get the final product in the form of solid inclusion complex (AnAc-IC). AnAcIC with AnAc/HP-β-CD ratio of 0.5:1, 1:1, 1:2, 1:3, and 1:4 were also prepared using the same technique. A physical mixture of AnAc and HP-βCD having a molar ratio of 1:1 was also developed for comparison with prepared inclusion complexes. The physical mixture was prepared by mixing AnAc (solid) and HP-β-CD continuously in a ceramic mortar for 5 min until the formation of a homogeneous mixture. 2.3. UV–Vis spectroscopy UV–Visible Spectra of AnAc and AnAc-IC were recorded using UV– Visible spectrophotometer (UV-1700 PharmSpec, Shimadzu) with 1 cm quartz cell. The scans were registered in the wavelength range of 210 to 400 nm. We did not find any interference in the analysis of AnAc due to the presence of HP-β-CD. AnAc (10 mg) was dissolved in ethanol (100 ml) in an amber-colored volumetric flask. Further, the resulting solution was diluted to various concentrations ranging from 5 to 40 μg/ml using ethanol, and the calibration curve was made by measuring the absorbance at wavelength 312 nm. 2.4. Solubility study An excess quantity of AnAc (20 mg) and Anacardic Acid/HP-β-CD inclusion complex (100 mg) were added into a glass vial containing 10 ml of water and kept for stirring at temperature 25 ± 0.1 °C for 72 h. For solubility determination, both the suspension samples were filtered using a 0.22 μm filter (Millipore syringe filter, PTFE) to separate the undissolved solid phase followed by suitable dilution with ethanol and UV analysis at λmax 312 nm. 2.5. Phase solubility study Phase solubility studies of a physical mixture of AnAc and HP-β-CD were conducted as per the previous report [20]. Briefly, excess quantity (20 mg) of AnAc was mixed with 10 ml of HP-β-CD hydrous solutions with different concentrations (0, 5, 10, 15, 20, 25 mM) followed by ultrasonication for 30 min. The sample suspensions were incubated at a thermal vibrator having vibration rate of 100 r/min for 72 h at 25 °C followed by filtration using 0.22 μm membrane filter (Millipore syringe filter, PTFE) suitable dilution with phosphate buffer (pH 6.8) and analysis by UV spectroscopy at 312 nm. The study was also performed at 35 °C and 45 °C by following the same procedure described above. All the experimental results are expressed in terms of the mean value of the minimum of three repeated experiments. The apparent stability constants (K) were determined using the phase solubility diagrams as per the Higuchi–Connors equation, where S0 refers to the intrinsic solubility of the solute in the absence of HP-β-CD and slope corresponds to the slope obtained from the phase-solubility plot data from the equation: The phase solubility curves were plotted using the molar concentration of HP-β-CD on Х-axis and the molar concentration of AnAc on the Y-axis. The stability constants (K) were calculated from phase solubility curves as per the Higuchi-Connors equation: K ¼ Slope=ðS0  ð1−SlopeÞÞ

ð1Þ

where S0 is the intrinsic solubility of AnAc and slope can be expressed as slope obtained from phase solubility curves. 2.6. Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra of AnAc, HP-β-CD, physical mixture, and AnAc/HPβ-CD inclusion complex were obtained using FTIR, Bruker Alpha II FTIR Spectrometer (Bruker Corporation, Germany). The samples were scanned at a spectral range 500–4000 cm−1 with resolution 2 cm−1 and 128 scans for each run. The samples were first diluted with KBr

3

powder and compressed to form pellets for performing the measurements. 2.7. X-ray diffraction (XRD) study The X-ray diffraction characteristics of the pure compound AnAc, HP-β-CD and the AnAc/HP-β-CD inclusion complexes were examined using Rigaku Miniflex 600 Desktop X-Ray Diffraction system (Rigaku Corporation, Japan) using HyPix-400 MF 2D hybrid pixel array detector (HPAD). The current and voltage applied were 40 mA and 40 kV, respectively. The results were recorded in the 2θ range of 5–40° with scan rate 10°/min and step size 0.03°. 2.8. Differential scanning calorimetry (DSC) The thermal behavior of the AnAc, HP-β-CD and inclusion complex were analyzed using differential scanning calorimeter (DSC-60 Plus, Shimadzu, Japan). The DSC thermograms were recorded in argon environment (with 99.99% purity) using typical aluminum pans for samples while keeping empty pan as reference. Samples were weighed accurately into DSC sample pans, hermetically sealed, and heated at a rate of 10 °C/min. The samples were heated, ranging between 25 and 250 °C under a constant nitrogen flow of 100 ml/min. 2.9. Scanning electron microscopy (SEM) The morphological investigation of the test samples was carried out using EVO - Scanning Electron Microscope MA15/18 (Carl Zeiss Microscopy Ltd.) equipped with 51N1000 – Energy Dispersive Spectroscopy (EDS) System (Oxford Instruments Nanoanalysis). The sample preparation involved fixing of sample on a brass stub followed by thin layer gold coating to avail good electrical conductivity while performing microscopic scans. Images were recorded at 1.0 KX magnification using secondary electrons (SE) to obtain images with good clarity. 2.10. 1H NMR Proton (1H) spectra of individual compounds and AnAc/HP-β-CD inclusion complex were recorded using AVH D 500 AVANCE III HD 500 MHz one bay NMR Spectrometer (Bruker BioSpin International AG, Germany). The test samples were dissolved in dimethyl sulfoxide (DMSO) at 298 ± 0.1 K, and chemical shifts (in ppm) were recorded using tetramethylsilane (TMS) as the internal standard. 2.11. Antimicrobial activity Antimicrobial activity of the AnAc and inclusion complex was assessed by estimating MIC against S. aureus. MIC for AnAc and inclusion complex were determined using the broth dilution method. Microbial suspensions were cultured at 37 °C for 24 h and further diluted to produce 1 × 105 CFU/ml. Further, 20 μl of microbial suspension (1 × 105 CFU/ml) was mixed with 180 ml Muller Hinton Broth (MHB) comprising different concentrations of AnAc and inclusion complex ranging from 0.097–50 μg/ml in microtiter 96 well plate. The inclusion complex was diluted with the culture media directly, while AnAc sample was obtained as an aqueous suspension, including 1% w/v DMSO [21,22]. Well plates were further incubated at temperature 37 °C for 24 h, and further turbidity of the test samples was determined using microplate reader (MR 680 Bio-Rad) at 450 nm. The MICs of AnAc and inclusion complex were considered as the minimum concentration with no observable growth (≤0.05 difference in OD 450) examined following the incubation for 24 h. All the experiments were performed in triplicates.

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3. Results and discussion 3.1. UV–Vis spectroscopy The UV–Vis spectra of AnAc in phosphate buffer (pH 6.8) having different concentrations of HP-β-CD in the wavelength range of 210–400 nm (Fig. 2). The UV-spectrum of AnAc in phosphate buffer presents bands showing absorption maxima at 243 and 312 nm. The first peak corresponds to benzene absorption, while the other peak indicates a typical pattern of polyphenols. Besides the buffer, HP-β-CD significantly enhances the optical density of AnAc and the bathochromic shift of the absorption bands was observed. This phenomenon demonstrated the noncovalent correlation of AnAc with HP-β-CD and was similar to the earlier reports [28]. 3.2. Preparation of Anacardic Acid/HP-β-CD inclusion complex

Fig. 2. UV absorption spectra Anacardic Acid (C15:3) in phosphate buffer pH 6.8 at different HP-β-CD concentrations: (1) 0.00 mM, (2) 10.00 mM, (3) 20.00 mM and (4) 25.00 mM.

2.12. Effect of AnAc and AnAc-IC on S. aureus biofilms The biofilm dispersal potential of free AnAc and AnAc-IC was determined by using the biofilm developed on the peg lids [23]. Concisely, 96 culture plates (made of polystyrene, transparent, sterile plates) were plated with 200 μl (0.5 × 106 CFU/ml) of S. aureus culture in LB media sheathed with peg lids followed by incubation for 48 h at 37 °C on the rotary incubator shaker to yield the biofilm which was further confirmed by methylene blue dye test [24]. Following the 48 h, peg lids having the biofilm were washed with PBS thrice to eliminate the spliced planktonic bacteria, and further, the peg lids containing biofilm were kept over the culture plate (96 well) comprising serially diluted test samples viz. 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390, 0.195, and 0.097 μg/ml diluted with the LB media. The plates were incubated for 24 h at 37 °C, and adhered biofilm was removed by positioning the lid on another 96 well plates comprising fresh LB media followed by sonication. The obtained cells were serially diluted and kept on LB agar plates for counting the CFU. Further, the biofilms were treated with recurrent dose (5 × MIC value) for 72 h to examine the biofilm eradication efficacy of the test samples.

The AnAc-ICs were formulated using co-evaporation method, and the effects of different variables on complexation efficiency were also investigated. It was clear from the results that the molar ratio of AnAc (guest) and HP-β-CD (host) significantly affects the preparation of the inclusion complex. Enhancement in the complexation efficiency was observed with an increase in the molar ratio of HP-β-CD to AnAc up to 2:1 which could be ascribed to the more number of host molecules available to accommodate the AnAc molecules. The effect of reaction temperature was also studied and had a positive impact on entrapment efficiency up to 45 °C while further increase in temperature did not show a significant increase. The reaction time and stirring speed were also optimized and found to be 2 h and 500 rpm, respectively. The present study revealed 2009 times enhancement in the solubility of Anacardic Acid/ HP-β-CD inclusion complex (38.17 ± 0.38 mM) than AnAc (0.019 ± 0.005 mM) in pure water at 25 °C. 3.3. Phase solubility study Phase solubility study of AnAc and AnAc-IC was conducted in phosphate buffer (pH 6.8) at a temperature range from 25, 35, and 45 °C (Fig. 4). The solubility of AnAc continued to increase with the increasing concentration of HP-β-CD and might be attributed to the more and more inclusion of AnAc in the cavity of HP-β-CD. The driving force of inclusion complex formation is non-covalent interactions, out of which dipole-dipole interaction plays key role in releasing high energy water molecules from the HP-β-CD cavity into the solvent media. It may be stipulated that with an increase in HP-β-CD concentration, more AnAc

2.13. Microscopic study of biofilm Biofilms were developed on the coverslip (12 mm diameter) inside the culture plate (12 well) as per the preceding study [25]. Concisely, coverslips comprising biofilms were recovered from the culture plate after 48 h and gently washed with PBS so as to eliminate the loosely attached bacterial cell. Additionally, the coverslips were relocated to another well plate (12 well) for inducing treatment with various test samples (5 × MIC value) in LB media. Treatment was continued for 24 h followed by removal of coverslips from the well plate. The cells were then fixed with paraformaldehyde (4%w/v), and then the biofilms were stained using LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fischer, India) in the dark environment following the manual guidelines (6 μM SYTO9 and 30 μM Propidium Iodide). The samples were examined using confocal laser scanning microscopy (CLSM) (Zeiss, Germany). The sample sections were scanned at two excitation wavelengths, i.e. 488 and 560 nm employing 40× magnification. The z stack images were acquired at z step size 2.63 μ. Furthermore, the obtained images were analyzed using COMSTAT-2 software to calculate the biomass and thickness of biofilms for different treatment groups [26,27].

Fig. 3. Phase solubility diagram of Anacardic Acid (C15:3) as a function of HP-β-CD concentrations in phosphate buffer pH 6.8 at different temperatures (25, 35, and 45 °C).

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Table 1 Various parameters for calculation of stability constant. Temp (°C)

Slope

Intrinsic solubility (S0) (mM)

Intercept

K (M−1)

Correlation coefficient (R2)

0.2038 ± 0.058

0.09

0.0971

2830

0.9998

0.2795 ± 0.077

0.141

0.1417

2750

0.9991

0.3103 ± 0.091

0.17

0.1848

2647

0.9998

25 35 45

molecules enter the HP-β-CD cavity which in turn increases the overall solubility of the AnAc. Enhanced solubility observed at higher temperatures could be attributed to the enhanced intrinsic solubility of AnAc at higher temperatures [29]. Phase solubility diagram (Fig. 3) was plotted and found to be linear, indicating AL type as per Higuchi and Connors [20]. Since the slope of the plot is b1, therefore it can be predicted that solubility enhancement might be attributed to the development of the first-order complex of AnAc with HP-β-CD molecule, and also it was assumed that the stoichiometry of the complex to be 1:1. The stability constant is of great importance, as it indicates the possibility of using inclusion complexes for drug delivery applications [30]. Considering the data obtained from phase solubility diagrams, the stability constants (K) of the AnAc-IC was calculated using Eq. (1). The stability constants (K) obtained from phase solubility plots were 2830, 2750, and 2647 M−1 at temperatures 25, 35, and 45 °C, respectively (Table 1). The results of the stability constants were between 200 and 5000 M−1, demonstrating its stability and also suitability for improving solubility and bioavailability of the poorly water-soluble drugs. Inverse relation of stability constant was found with temperature indicating the formation of inclusion complex to exothermic process, and stability of the complex reduces with an increase in temperature [31]. It was observed that at a HP-β-CD concentration of 25 mM, the AnAc solubility increases from 0.09 Mm to 5.18 mM indicating approximately 58 folds increase in solubility in pure phosphate buffer (pH 6.8).

3.4. Fourier transform infrared spectroscopy (FT-IR) The formation of inclusion complexes of drugs with cyclodextrins can be easily assessed using FT-IR spectroscopy. This technique helps in determining the change in the bands of vibrations of the individual moiety in the process of complex formation. Inclusion complex formation is generally identified by a change in intensity, disappearance, and widening as well as shifting of peaks. The FT-IR spectra of AnAc, HP-β-CD, physical mixture and AnAc-IC are shown in Fig. 4. The FT-IR spectra of HP-β-CD showed the presence of peaks at 3600–3000 cm−1 (O\\H stretching vibration), 1038 cm−1 (C_O stretching vibration), and 2917 cm−1 (C\\H stretching vibrations). The FT-IR spectra of AnAc revealed the presence of peaks at 3420 and 1304 cm−1 (Ar\\OH), 3400–2400, 1645 cm−1 and 1304 cm−1 (\\COOH), 3009 cm−1 (Ar\\H and vinyl-H), 2924 and 2849 cm−1 (aliphatic C\\H), 1607 cm−1 (aliphatic C_C), and 1446 cm−1 (aromatic C_C). The FT-IR spectra of the physical mixture (Fig. 5-C) were characterized by a superimposed spectrum of AnAc and HP-β-CD. However, wider peaks in the fingerprint region were observed, which might be due to a higher weight ratio of HP-β-CD, leading to the prominence of HP-β-CD in the physical mixture. The inclusion complex revealed a distinct FT-IR spectrum with the absence of absorption peaks at 2400–3400 cm−1. The loss of aromatic peaks of AnAc in the inclusion complex could be an indication of inclusion of AnAc inside the HP-βCD cavity. Moreover, most of the peaks of AnAc were observed to be

Fig. 4. The FT-IR spectra of HP β-CD (A), Anacardic Acid (C15:3) (B), Anacardic Acid (C15:3)/HP-β-CD physical mixture (C) and inclusion complex (D).

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Fig. 5. X-ray diffraction patterns of HP-β-CD (A), Anacardic Acid (C15:3) (B), and inclusion complex (C).

smoothed, suggesting the prevalence of high intermolecular interaction between AnAc and HP-β-CD. The IR band at 1636 cm−1 of the HP-β-CD relates to the water crystallization, which is absent in the inclusion complex spectra. This phenomenon is strongly attributed to the replacement of water molecules inside the CD cavity with AnAc molecules and thereby validating the inclusion complex formation. 3.5. X-ray diffraction (XRD) study The formation of inclusion complex was further confirmed by comparing the X-Ray diffraction pattern of different samples. XRD analysis is prominently used as a tool to study cyclodextrins and its complexation with various compounds in powder form. Interaction between components in the formation of the inclusion complex can be assessed by comparing XRD patterns of individual compounds and inclusion complex. The appearance of new diffraction peaks, as well as the shift of drug (guest molecule) peaks or variations in their relative intensities, are the indications to confirm the formation of inclusion complex [32]. The XRD patterns of AnAc, HP-β-CD, and AnAc-IC are shown in Fig. 5. Evidence of several peaks was observed in the XRD pattern of AnAc (Fig. 5-B) at diffraction angles (2θ) 9.02, 13.4, 17.9, 20.2, and 22.8, indicating crystallinity while XRD patterns of HP-β-CD did not show the presence of sharp peaks suggesting amorphous nature. However, HPβ-CD depicted broad diffraction peaks between 2θ = 15.52 and 22.12, suggesting disordered crystal structure, which is a characteristic feature of amorphism. Furthermore, AnAc-IC displayed broad diffused peaks with decreased intensities as well as the complete absence of primary diffraction peaks corresponding to AnAc. This, in turn, providing conclusive evidence to favor the formation of inclusion complex consisting of solid phase and also transition from crystalline to amorphous form.

cyclodextrins, as well as inclusion complexes [34]. Information regarding solid-state modifications and interaction among components can be obtained to compare the thermograms of individual compounds and complex [35]. The DSC thermal curves of AnAc, HP-β-CD, and inclusion complex are depicted in Fig. 6. The DSC thermogram of AnAc (Fig. 6-B) revealed a wide peak at around 167 °C, which is characteristic of the crystalline nature of the compound [36]. DSC curve of HP-β-CD (Fig. 6-A) shows an endothermic peak in the region between 33 °C to 113 °C indicating amorphous nature which might be associated with the release of water from the cavity of

3.6. Differential scanning calorimetry (DSC) DSC provides exhaustive data related to physical changes [33]. It has also been widely used to investigate the interactions between drug,

Fig. 6. DSC thermograms of HP-β-CD (A), Anacardic Acid (C15:3) (B), and the inclusion complex (C).

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Fig. 7. Scanning electron microscopy image of HP-β-CD (A) physical mixture (B) and inclusion complex (C).

HP-β-CD. In the thermogram of inclusion complex (Fig. 6-C), the complete absence of melting peaks corresponding to AnAc was observed. However, a broad endothermic peak in the region between 30 °C–132 °C was seen, which is an indication of the possible inclusion of AnAc in the HP-β-CD cavity.

3.7. Scanning electron microscopy Microscopic surface morphology was assessed using SEM. SEM images of HP-β-CD, physical mixture, and AnAc-IC are shown in Fig. 7. The surface morphology of HP-β-CD revealed its spherical shape with

Fig. 8. Comparative 1H NMR spectrum of HP-β-CD (A), Anacardic Acid (C15:3) (B), and inclusion complex (C).

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Table 2 Variation of the 1H NMR chemical shifts (δ/ppm) of Anacardic Acid (C15:3) and HP-β-CD in inclusion complex formation. Compound

Proton

δ δ (free) (complex)

Anacardic Acid (C15:3) H4 H5 H3 Aliphatic (alkenyl proton) Methylene CH2 Aliphatic CH2 Aliphatic CH2 HP-β-CD H1 H3 H5

7.012 6.503 6.428 5.829 3.052 2.804 1.484 5.874 4.836 3.742

H6

3.745

H2 CH3

3.613 1.022

Table 3 Biofilm parameters of S. aureus biofilms without treatment (control), AnAc, and AnAc-IC treatment.

Δδ

6.969 0.043 6.455 0.048 6.374 0.054 5.937 −0.108 3.034 0.018 2.792 0.012 1.472 0.012 5.828 0.046 4.833 0.003 Overlapped with HP-β-CD Overlapped with HP-β-CD 3.632 −0.019 Overlapped with HP-β-CD

the presence of the cavity inside. However, the SEM image of the physical mixture (Fig. 7-B) demonstrated distorted spherical particles as well as some crystal structures indicating the presence of both AnAc and HP-β-CD. Further, the inclusion complex (Fig. 7-C) exhibited particles possessing bulky prismatic shape.

Control (without treatment) AnAc AnAc-IC

Biomass (μm3/μm2)

Thickness (μm)

27.59 ± 2.2b 22.16 ± 2.1a,b 17.69 ± 1.8a

49.8 ± 4.17b 38.89 ± 3.34a,b 27.6 ± 2.8a

p b 0.005; Student's t-test. Data mentioned represents the mean ± standard deviation of three replicated experiments. a Represents the significant difference in control (without treatment) biofilm. b Denotes a significant difference in comparison with AnAc-IC.

majority of the variations of chemical shifts of AnAc (Fig. 8-B) were at the aromatic region and aliphatic region, which was distinct from the variation of HP-β-CD. The chemical shifts of HP-β-CD (Fig. 8-A) were observed at δ values 5.87 due to H1, 4.84 due to H3, 3.74 due to H5 & H6, 3.61 due to H2 and 1.02 due to the CH3 group. All the characteristic chemical shifts of HP-β-CD and AnAc were present in the proton NMR spectra of the AnAc inclusion complex (Fig. 8-C) except the absorption band of \\COOH group of AnAc which was observed in spectra AnAc at δ value of 15.93. This indicates the possible inclusion of AnAc into the HP-β-CD cavity.

3.9. Antimicrobial activity 3.8. 1H NMR (proton) spectroscopy Formation of inclusion complex can be explained using 1H NMR spectroscopy by considering changes in the chemical shift of protons of the AnAc (guest) and HP-β-CD (host) molecules. However, changes in a chemical shift in this process are relatively small due to noncovalent interaction. The inclusion behavior of AnAc in HP-β-CD can be explained by comparing the chemical shift changes among 1H spectra (Fig. 8) of AnAc, HP-β-CD, and AnAc-IC (Table 2). The 1H NMR spectra of AnAc depicts aromatic moiety with absorption bands at ~7.012 ppm, ~6.503 ppm, and ~6.428 ppm due to H-4 (t), H-5 (d) and H-3 (d), respectively. Another absorption band was observed at 15.93, which was due to proton present in \\COOH group. The hydrogens present in double bonds of alkyl chain showed absorption at different values of δ. Alkenyl protons showed absorption at 5.829–4.953, while methylene (CH2) absorptions were observed at ~3.052, ~2.804, ~2.015, and ~1.484. Aliphatic CH2 showed absorption at δ = 1.252. As demonstrated in Fig. 8, the

As per the results obtained from MIC assay against S. aureus, both AnAc alone and AnAc-IC were effective against planktonic bacteria. The MIC value of both free AnAc and AnAc-IC was 0.78 μg/ml indicating that the complexation process of AnAc with HP-β-CD does not alter the antibacterial activity of Ana against the planktonic S. aureus rather it maintains the antibacterial activity as reported earlier [37]. Maintaining the efficacy of drugs while developing a novel formulation may also be considered as the foundation of other useful applications [38]. The unchanged MIC value upon complexation is not surprising, as at this lower concentration of AnAc, its water solubility is somehow not affected significantly. Moreover, only free AnAc out of the inclusion complex is available for its activity [39]. The antibacterial action of AnAc is primarily due to the physical disruption of the bacterial cell membrane. It has also been reported to act via entering into the cytoplasmic membrane lipid bilayers and thereby disrupting energy converting systems such as electron transport system (ETS) as well as ATPase [40]. AnAc also inhibits bacterial resistance

Fig. 9. Disruption effect of free AnAc and AnAc on mature biofilm was grown in 96 well plates. The study demonstrates a reduction of 48 h grown S. aureus biofilm on single dose (A) and repeated dose for three recurrent days (B) of different AnAc concentrations. The data represent a mean ± standard deviation (n = 3). ap b 0.005 as compared to AnAc (Two way ANOVA followed by Bonferroni test). ns indicates the results are not significant as compared between the two groups. The viable count for the control experiment without AnAc was 2.39 × 109 ± 1.19 × 108 CFU/ml.

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possible due to its action on the extracytoplasmic region and thereby avoiding cellular pump based resistance mechanisms [41]. 3.10. Effect of AnAc and AnAc-IC on S. aureus biofilms The antibiofilm activity of free AnAc and AnAc-IC on 48 h grown mature biofilm at different concentrations is displayed in Fig. 9. At the highest concentration, both free AnAc and AnAc-IC exhibited N80% biofilm reduction. It can be observed that at concentrations below MIC, the antibiofilm activity of AnAc and AnAc-IC does not reveal significant difference while at concentrations above MIC value, the AnAc-IC displayed excellent biofilm dispersal potential and there was a significant difference between the activity of free and complexed AnAc. This could be attributed to the fact that at concentrations below MIC value, AnAc shows better aqueous solubility, and hence, it exhibits biofilm dispersal potential similar to the complexed AnAc. Moreover, the treatment of biofilms requires a high dose of the drug (more than MIC value), and due to poor aqueous solubility of AnAc, it does not demonstrate enough potential at higher concentrations as the insoluble fraction does not get access to the biofilms. Moreover, the inclusion complex formation with HP-β-CD results in a drastic enhancement in water solubility of AnAc, and thereby, the AnAc-IC exhibits better biofilm eradication potential at higher concentrations. Furthermore, to attain the long term potential of AnAc and AnAc-IC on mature biofilms, the antibiofilm activity with consecutive treatment for 72 h was assessed. The free AnAc exhibited biofilm reduction by 92.11% at the highest concentration (50 μg/ml) while AnAc-IC demonstrated complete biofilm dispersal at 25 μg/ml concentration. The improved biofilm dispersal potential of complexed AnAc might be attributed to the enhancement of aqueous solubility of AnAc, as discussed earlier. The other factors involved in such a phenomenon could be the slow release of AnAc from the complex so that a gradient of the free and complexed drugs is maintained throughout. HP-β-CD is bioadhesive and biocompatible biomaterial, which could have facilitated the adhesion of inclusion complex with the biofilms.

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dispersal potential, but the complexed AnAc showed better biofilm eradication and hence exhibited lower biomass and thickness. Anacardic Acid has earlier been reported to inhibit biofilm, the distinguishing property of AnAc might be attributed to its unique features like it can penetrate the bacterial cell membrane and disrupt the lipid bilayers. AnAc also interferes with the bacterial respiratory system by blocking its electron transport chain as well as NADH oxidase activity. Since AnAc acts upon multiple targets and hence, it is effective against not only planktonic bacteria but also bacterial biofilms. Moreover, the complexation of AnAc with HP-β-CD resolves the fundamental issue of water solubility and thereby making it potent even at lower concentrations. Thus, we evaluated the antibiofilm activity on the glass surface, and the results indicated that AnAc-IC could potentially dismantle the adhered biofilm, which therefore suggested the new dimension in the treatment of biofilm-related infections. Biofilm forming bacteria communicate among its counterparts employing a specific mechanism known as quorum sensing. Potential of AnAc to inhibit the quorum sensing makes it more effective in dismantling the bacterial biofilms [42]. 4. Conclusion Anacardic Acid has been extensively studied for a variety of indications, yet poor aqueous solubility restricts its use to the fullest of its potency. In the present study, we hypothesized that the formation of the inclusion complex of Anacardic Acid with HP-β-CD would not only overcome the solubility issue but also improve the efficacy in the eradication of S. aureus biofilms. In line with our hypothesis, the developed inclusion complex demonstrated N2009 fold improvement in water solubility. The developed complex was further tested against resistant bacterial (S. aureus ATCC 29213) strain and demonstrated its potential. Anacardic Acid was also found effective in the eradication of biofilms associated with S. aureus. The findings collectively support the hypothesis to improve the solubility and thus the activity of Anacardic Acid and can be further extended to overcome the poor solubility and to improve the efficacy of other antimicrobial agents.

3.11. Microscopic study of biofilm Declaration of competing interest For, further confirmation of the antibiofilm potential of AnAc and AnAc-IC on S. aureus biofilm, the biofilm was grown (48 h) and treated on the coverslip in culture plate (12 well). The data (Table 3) depicts the biomass and thickness, which were determined from z stack CLSM images (Fig. 10A–C) using COMSTAT 2 software. The biofilm not receiving any treatment was considered as a control group which demonstrated highest biomass (27.59 ± 2.2 μm3/μm2) and thickness (49.8 ± 4.17 μm). Furthermore, both free AnAc and AnAc-IC displayed biofilm

The authors declare no conflict of interest in the current work. Acknowledgments The authors acknowledge financial support from Department of Science & Technology (DST), Ministry of Science and Technology (grant no: DST/SSTP/UP/2013-14/12TH PLAN/132), Government of India. The

Fig. 10. CLSM image of S. aureus biofilm formed on cover slip surface with media supplementation. The images demonstrate dense biofilms formed without treatment (A) or with AnAc (B) and AnAc-IC (C) treatment.

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