In-vitro dissolution and microbial inhibition studies on anticancer drug etoposide with β-cyclodextrin

Materials Science & Engineering C 102 (2019) 96–105

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In-vitro dissolution and microbial inhibition studies on anticancer drug etoposide with β-cyclodextrin

T

Shanmuga Priya Arumugama, Suganya Bharathi Balakrishnana, Vigneshkumar Ganesana, Maniyazagan Munisamya, Sakthi Velu Kuppua, Vimalasruthi Narayanana, ⁎ Vaseeharan Baskaralingamb, Sivakamavalli Jeyachandranb, Stalin Thambusamya, a b

Department of Industrial Chemistry, School of chemical Sciences, Alagappa University, Karaikudi 630003, Tami Nadu, India Department of Animal Health and Management, Alagappa University, Karaikudi, 630003, Tamil Nadu, India

ARTICLE INFO

ABSTRACT

Keywords: Etoposide β-Cyclodextrin Phase solubility Dissolution rate Antimicrobial efficacy

In this article, we have reported the inclusion complex behaviors and their pharmaceutical application of anticancer drug property of Etoposide with β-cyclodextrin. The inclusion complex is used to improve the poor aqueous solubility of the anticancer drug Etoposide. The aqueous solubility and in-vitro dissolution studies support to the anticancer drug Etoposide with β-cyclodextrin complex is significantly improves the aqueous solubility. Etoposide:β-cyclodextrin solid-state complexes were prepared by Physical mixture, kneading and solvent evaporation methods, and were characterized by FT-IR, 1HNMR, XRD, DSC and SEM techniques. In addition, the in-vitro antimicrobial and antibiofilm study of Etoposide drug is a sensible microorganism was significantly increased by an inclusion complexation process. The antibiofilm of anticancer drug Etoposide with β-cyclodextrin studies have been analyzed by confocal laser scanning microscopy.

1. Introduction Anticancer drug Etoposide (EPS), is a chemotherapy medication used for the treatments of many types of cancer [1], the International Agency for Research on Cancer (IARC) of World Health Organization reported that lung cancer is the most commonly diagnosed and cause of death among all cancers. About 1.8 million (13% of total) new lung cancer cases were diagnosed and about 1.6 million (19.4% of total) deaths occurred due to lung cancer [2]. Etoposide [4′-demethylepipodophyllotoxin-9-(4,6-oethllidene-β-D-glycopyranoside)] (EPS) is a semi-synthetic derivative of podophyllotoxin and has been approved by Food and Drug [2] administration for the treatment of small cell lung cancer and testicular carcinoma. EPS also reported some significant cytotoxicity against Kaposi's sarcoma associated with AIDS, Hodgkin's and non-Hodgkin's lymphoma, breast, gastric and ovarian cancers. In addition, the anticancer drug EPS (Scheme S1) is a potent topoisomerase II inhibitor [3] enzyme responsible for unwinding of DNA helix. It acts in the G2 and S phases of cell cycle and prevents entry of cell into the mitotic phase of cell cycle leading to cell death [4]. Where, the poor aqueous solubility, chemical instability and non-specific toxicities to normal tissues are the major limitations of EPS in its clinical applications [5,6]. Currently, EPS is being supplied as oral soft capsules and

⁎

injection formulations [7,8]. However, non-aqueous, organic solvents and solubilizers such as benzyl alcohol, ethanol, polysorbate 80 and PEG 300 are being used to solubilize the EPS. These solubilizers frequently cause hypotension, anaphylaxis and bronchospasm. To avoid solubility issues, different pharmaceutical strategies have been employed which include: particle size reduction, solid dispersion formulation, pH adjustment, supercritical fluid process, manipulation of solid, complexation by complexing agents, solubilization by surfactant systems and complexation with cyclodextrins [9–12]. Particularly, Zhang and their co-researchers [13], reported the interaction of β-Cyclodextrin with etoposide studied by fluorescence spectroscopy and they found the linear regression, correlation coefficient and the detection limit were achieved by the anticancer drug etoposide molecule. Moreover, here, Sun, et al., was reported the Hydroxypropyl-β-cyclodextrin of a substituted degree in 6.7 was investigated the complex forming capability by etoposide [14]. In this report, the inclusion complex was confirmed by IR, DSC and XRD. The 1: 1 M ratio inclusion complex with hydroxypropyl-β-cyclodextrin and improved solubility about 75.1 times. The experimental results indicate the improvement of drug solubility, stability and bioavailability behaviors. The inclusion complex, especially cyclodextrins molecule has been broadly used to improve solubility and dissolution rate of poorly water-

Corresponding author at: Department of Industrial Chemistry, School of chemical sciences, Alagappa University, Karaikudi 630003, Tami Nadu, India E-mail address: [email protected] (S. Thambusamy).

https://doi.org/10.1016/j.msec.2019.04.033 Received 12 December 2017; Received in revised form 4 April 2019; Accepted 12 April 2019 Available online 13 April 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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in the range of 4000–400 cm−1. 1H NMR spectra were taken in an BRUKER-MMR 500 MHz, Two-dimensional rotating-frame Overhauser effect spectroscopy (ROESY) experiments were performed using BRUKER-NMR 400 MHz instrument operating at 300 K and DMSO‑d6 was used as a solvent. X-ray diffraction patterns are taking at room temperature using XPert PRO PANalytical diffractometer with Ni filtered (Cu Kα radiation) operating at a voltage of 40 K and 30 mA current. The surface morphology of the drug and their inclusion complexes were examined with Hitachi S 3000H scanning electron microscope (Tokyo, Japan). DSC measurements were carried out under nitrogen flow (20 K/min) at scanning rate of 10 °C/min and the temperature range is maintaining at 0–400 °C. Antibiofilm study is measuring by the confocal laser scanning microscope CLSM-LSM 710 (objective 40; numerical apertures, 0.6) and the confocal images were obtained by using zen 2009 image software (model 710; carl zeiss Imaging Germany) in the deconvolution mode. The Ultraviolet–visible spectra (absorption spectral measurements) were carried out with Shimadzu UV-2401PC double-beam spectrophotometer (range 1100–200 nm) (Japan) with scan speed at 400 nm min−1.

soluble drugs [15–18]. β-Cyclodextrin (β-CD) (Scheme S1) are oligosaccharides of glucose having central hydrophobic cavity in the structure which is able of forming stable inclusion complex with guest molecules [19,20]. β-CD are useful carrier because of their hydrophilic nature and capability to improve the poorly water-soluble drugs solubility, enhancement in physicochemical properties and chemical stability of drugs [21,22]. The undesired properties of drugs, such as a distinctive smell and taste can be masked with β-CD inclusion complex [23,24]. β-CD have the nature to increase extensive acceptance in pharmaceuticals for their ability to form inclusion complex that increases the aqueous solubility and driving force for diffusion across the biological membrane for lipophilic drugs [25–28]. However, while forming inclusion complex with hydrophobic drugs, they do not change their molecular structure and permeability characteristics. Accounting for the solubility of EPS drug with hydroxypropyl-βcyclodextrin, Avital beig et al., reports a head-to-head comparison of different etoposide's solubility enabling formulations [29]. The EPS drug is binding in the ternary enzyme-drug-DNA complex and altered sites of enzyme-mediated DNA cleavage. Steven L. Pitts and his research group propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex [30]. Recently, Jian-Song Sun group based on the new approach, syntheses of clinically used anticancer reagents etoposide and teniposide were accomplished, and the overall yields reach as high as 18% and 9%, respectively. The novel method used as a broad scope in terms of both glycosyl donors and podophyllotoxin derivative acceptors, providing the excellent yields [31]. Not only that, the interaction Force between Etoposide and Functionalized non-mesporous silica coated with iron based nanoparticle acts as a nanocarrier for drug loading and release processes. Above said phenomena proposed by Weiwei Zhao [32] research groups about the experimental values of the molar entropy and enthalpies in terms of the hydrogen-bond interaction, it deals to the externally controlled drug-delivery system in cancer therapy. For the past more than one decade, corresponding author/my research group has largely been involved in studying the complexation properties [33–38] and their uses in chemosensor and pharmaceutical applications [39–43] of various organic fluorophores. This stimulated us to carry out a study on Etoposide drug to examine the possibility of complex formation with β-CD in aqueous and solid state to improve its dissolution profile. The solubility and the stability constant of the complex were recognized as per the phase solubility studies. Especially, the solid-state inclusion complex of EPS with β-CD was prepared by physical mixture, kneading and solvent evaporation methods. The resulting solid-state complex was characterized by different analytical/ spectroscopic techniques such as Fourier transformation-infrared spectroscopy (FT-IR), 1H NMR, Differential scanning calorimetry (DSC), XRay powder diffractometry (XRD) and SEM techniques. Finally, in addition to the in-vitro antimicrobial and antibiofilm activity of the EPS drug and its inclusion complex systems were carried out against gram positive and gram negative microorganisms.

2.3. Preparation of solid inclusion complex Complexes of EPS and β-CD were prepared in the molar ratio of 1:1 by different methods like Physical mixing, Kneading and Solvent evaporation methods. (a) Physical Mixture Physical mixture was prepared by triturating EPS and β-CD together for 30 min in a clean and dry glass mortar until a homogeneous mixture was obtained. And then was forced through sieve number 100. (b) Kneading Method EPS and β-CD, was mixed separately in glass mortar along with water to obtain a homogeneous paste. The drug (either in powder form or as solution with minimum quantity of methanol) was then slowly added to the paste and the mixture was triturated for 1 h. during the process the water content was empirically adjusted to maintain the consistency of the paste. Methanol was added to assist dissolution of EPS during the process. The paste was dried at room temperature pulverized and forced through sieve number 100. (c) Solvent evaporation method

2. Experimental

A solution of Etoposide in methanol was gradually added to equimolar concentration of EPS, β-CD, EPS with β-CD in water and agitated at 50 °C for 30 min and toward the end of addition turbidity developed in the mixture. At the end of this period the solution was filtered, and the moist solid was kept in oven 50 °C for removal of last trace of solvent. The mass was then pulverized and passed through sieve number 100.

2.1. Materials

2.4. Phase solubility studies

Etoposide was generous gift from Macleodes Ltd., Mumbai. βCyclodextrin was received from Hi-media chemical company Mumbai India. Methanol, potassium dihydrogen phosphate and sodium hydroxide was purchasing from SRL (Mumbai, India). Distilled water was used throughout the experiment.

The phase solubility studies were carried per the method reported by (Higuchi and Connors). An excess of Etoposide (10 mg) was added to 10 ml of distilled water (pH 7.4) containing various concentrations of βCD (0 to 0.012 M) the flask were sealed and were shaken for 7 days at room temperature (25 ± 0.5 °C). After 7 days of shaking to achieve equilibrium, 5 ml of aliquots were withdrawn and filtered immediately through Whatman Qualitative Filter Paper. The filtered samples after suitable dilution were assayed spectrophotometrically for EPS at 288 nm. The phase solubility studies were conducted in triplicate. From the phase solubility curve, the stability constant Ks can be calculated by assuming 1:1 stoichiometric ratio in the following equation

2.2. Characterization FT-IR spectra of EPS, EPS:β-CD solid inclusion complexes is recorded with the Nicolet.380 Thermo electron Spectrophotometer (Tokyo, Japan) using KBr pelleting method. The spectra were measured 97

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Ks =

Slope S0 (1 Slope)

(Fig. S1) at 283 nm. Upon increasing the concentration of β-CD, the absorption maxima are regularly increased the absorbance at 283 nm. However, the absorption intensities of pH 7.0 solutions are regularly increases with increasing concentration of β-CD, no clear isosbestic point is observed in the absorption spectra. The absorption spectra show in absorption maxima even in the presence of the highest concentration of β-CD used (1.2 × 10−2 mol dm−3) and there is no change in the absorbance by further addition of β-CD (Inset Fig. S1). Here, Increasing the concentration of β-CD from 0 to12 × 10−3 M increased the absorbance of anticancer drug EPS without any wavelength shifts of λmax. The observed λmax may be attributed to the perturbation of chromophore electrons of anticancer drug EPS into the cyclodextrin cavity. Therefore the neutral form of EPS drug can form an inclusion complex with β-CD in aqueous solution. In this regard, the phase solubility analysis allows the estimation of the attraction between β-Cyclodextrin (β-CD) and anticancer drug etoposide (EPS) in water. The phase solubility diagram for the inclusion complex formation between EPS drug and β-CD was represented in Fig. S1 and Table 1. From this plot/figure suggested that the aqueous solubility of drug increases linearly as a function of β-CD concentration. As per the Higuchi and Connors [44] classification of the phase solubility diagram for EPS:β-CD inclusion complex follows AL type which clearly indicating the formation of water soluble complex. The linear straight line with a slope value 0.078 (R2 = 0.999) of less than unity clearly recommended the formation of 1:1 complex in solution. The obvious stability constant KS obtained from the slope and intercept of the linear straight line was found to be 133.4 mol−1 [45].

(1)

where, S0 is the solubility of EPS, in the absence of β-CD. 2.5. In-vitro dissolution studies The dissolution studies were performed using dissolution test apparatus with a paddle stirrer. Per USP reference standard 11, dissolution studies were performed using 900 ml of 0.1 N HCl as dissolution medium at 37 °C by applying 100 rpm. 50 mg of pure drug (or) its equivalent quantity of solid EPS:β-CD inclusion complexes is dispersing over the dissolution medium. At different time intervals 10, 20, 30, 40, 50, 60 min samples of dissolution medium (5 ml) were withdrawn and replaced with a fresh dissolution medium suitably diluted, and assayed for etoposide by measuring absorbance at 286 nm. The dissolution experiments were conducted in triplicate (n = 3). 2.6. In vitro anti-microbial studies The antibacterial activity of the Etoposide drug and its solid inclusion complexes were examined against two different microorganism Bacillus licheniformis (B.lich), a gram positive and Vibrio parahaemolyticus (V.para) a gram negative bacteria in agar broth medium. The 24 h bacterial cultures were inoculated into agar broth supplemented with various concentrations (25 μl, 50 μl and 75 μl). The plates were incubated at 37 °C for 24 h. After incubation, plates were observed for antimicrobial activities by determining the diameters of the zone of inhibition developed in and around of each sample.

3.2. Fourier transform infrared spectroscopy (FT-IR) analysis

2.7. In vitro antibiofilm studies

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis is a highly sensitive method to characterize the formation of solid inclusion complexes because generally included part of the guest molecule is shifted (or) their intensities are altered. Fig. 1a is the FT-IR spectrum of β-CD,

The effect of β-CD inclusion complex on biofilm formation was done in 24-polypropylene micro titre plates. The microorganisms used for this study was Bacillus lucheniformis (B.lich), a gram positive and Vibrio parahaemolyticus (V.para) a gram negative bacteria. Briefly overnight cultures of the test organisms were incubated at 37 °C for 24 h until a visible biofilm formation was established. After incubation, the plates stained with 0.01% acridine orange dye solution for 20 min. The obtained biofilms monitored under a confocal laser scanning microscopy (CLSM- Carl Zeiss, Germany). 3. Results and discussion 3.1. Phase solubility studies The absorbance of neutral form of anticancer drug EPS is remarkably altered by the addition of β-CD, which may have been caused by the conjugation effect of the host and guest molecules. Evident absorbance changes of anticancer drug EPS have been observed with βCD, and are shown in the inset Fig. S1. The UV–vis absorption spectral data of EPS molecule in different concentrations of β-CD are compiled in Table 1. The absorption peaks of EPS molecule in pH 7.0 appears Table 1 Absorption maxima and absorbance values of Etoposide with β-CD in phase solubility studies. S·No.

Conc. of β-CD(mmol)

λ max (nm)

Absorbance

1 2 3 4 5 6 7

0 0.002 0.004 0.006 0.008 0.010 0.012

283 283 283 283 283 283 283

0.630 0.799 0.944 1.123 1.251 1.433 1.578

Fig. 1. FT-IR spectra of EPS:β-CD System (a) β-CD (b) EPS Drug(c)Inclusion complex by Physical Mixture (d) Inclusion complex by Kneading Method (e) Inclusion Complex by Solvent évaporation Méthod. 98

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(c)

β-Cyclodextrin

Etoposide

(b) Etoposide

(a) β-Cyclodextrin

Fig. 2. 1H NMR spectra (500 MHz) at 25°C of (a) β-cyclodextrin, (b) Etoposide and (c) Etoposide:β-CD complex.

groups connected by the intramolecular hydrogen bonds (the C– 2, eOH group of one glucopyranose unit and C–3, eOH group of the adjacent glucopyranose unit) [46,47]. Also, in the FT-IR spectrum of βCD, the absorption band with maximum at 2926 cm−1 has been observed. This is belonging to the stretching vibrations of the CeH bonds in the CH and CH2 groups. Absorption bands of the stretching vibrations of the С-О bonds in the ether and hydroxyl groups of β-СD has been gave at 1080 cm−1 and 1027 cm−1 respectively. FT-IR spectrum of anticancer drug EPS Fig. 1b showed the stretching vibrations of ether (O-CH3) and aromatic ketones for the region at 1354 cm−1 and 1648 cm−1 respectively. The strong absorption band maxima indicating the presence of lactone ring in anticancer drug EPS molecule, it nearly observed at 1763.5 cm−1. A prominent absorption band at 1426 cm−1 is due to the CeH bending vibration. The broad peak obtained at 2916.8 cm−1 and 3402.78 cm−1 is due to the –C-H sp3 hybridization and aromatic –OH stretching vibration respectively. Further, analysis of FT-IR spectra of the solid inclusion complexes prepared by physical mixture and kneading method (Fig. 1c and d) the components exposed the most frequent changes to the ranges 950–1500 cm−1, which were interpreted as the intensity and shape of these bands dramatically changes for the solid inclusion complexes as compared to the pure EPS drug. The intensities of the characteristic peak for eOH bending vibration and ether group are reduced to lower intensities at 941.06 cm−1 and 1331 cm−1 respectively. Additionally, the peak intensities corresponding to CH-SP3 hybridization and

Table 2 (a) 1H NMR chemical shifts of β-CD in free and complexed state determined in D2O at 303 K. (b) 1H NMR chemical shifts of Etoposide in free and complexed state determined in D2O at 303 K. (a) Proton

β-CD δ (ppm)

EPS:β-CD δ (ppm)

Δδ (ppm)

H1 H2 H3 H4 H5 H6

4.985 3.545 3.877 3.579 3.774 3.790

4.889 3.541 3.700 3.571 3.698 3.780

−0.006 −0.004 −0.055 −0.008 −0.076 −0.010

Proton

EPS δ (ppm)

EPS:β-CD δ (ppm)

Δδ(ppm)

Ha Hb Hc

3.477 5.277 6.174

3.507 5.288 6.146

−0.030 −0.044 −0.028

(b)

in this spectrum the broad band has been observed with the absorption maximum at 3320 cm−1, which is caused by the stretching vibrations of the OeH bonds in the primary hydroxyl groups (C–6, eOH) connected by the intermolecular hydrogen bonds or in the secondary hydroxyl 99

100

EPS:β-CD

β-CD

EPS

Fig. 3. The 2D H NMR (ROESY) spectra of inclusion complex (EPS:β-CD) in DMSO-d6 at 300 K, β-Cyclodextrin(β-CD) structure and etoposide(EPS) drug molecular structure.

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aromatic eOH stretching vibrations significantly reduced to 2922 cm−1 and 3379 cm−1. Further looking the FT-IR spectra of EPS:β-CD solid inclusion complex prepared by solvent evaporation (Fig.1e) product is the most important characteristic peaks of eOH bending vibration, ether group are completely disappeared, this is clearly suggested that the aromatic ring containing ether and eOH groups of anticancer drug EPS molecule is included to the β-CD cavity (Scheme S2), which is clearly indicates the anticancer drug EPS is encapsulated with β-CD by solvent evaporation method. 3.3. Proton nuclear magnetic resonance spectral analysis Proton nuclear magnetic resonance (1H NMR) spectra of β-CD, anticancer drug EPS and solid complexes of anticancer drug EPS with βCD are given in Fig. 2. The values of chemical shift, δ for different protons of β-CD, β-CD:EPS complex and pure EPS were listed in Tables 2a and b. The 1H NMR spectra of the inclusion complex, a variation of β-CD chemical shift as well as EPS of protons are clearly mentioned in the Tables. The up-field signal of H3 and H5 protons of β-CD is lies on inner side of cavity. On the other hand, as shown in Fig. 2b, when anticancer drug EPS form the complex with β-CD, the change in microenvironment leaded the phenyl ring proton signals to split. The chemical shift and their changes upon complexation for the EPS protons where Δδ = δcomplex − δ free EPS. The 1H resonance of β-CD was assigned as per the reported procedure [48]. The changes of the chemical shift (Δδ) of H3 and H5 (Scheme S3) suggested that the EPS drug entered the β-CD cavity. Phenyl ring containing hydroxyl and ether group protons of EPS shifts the signals of β-CD protons (H3 and H5) into up-field shift. The chemical shift of Ha, Hb and Hc protons of EPS are in to the inner nano hydrophobic cavity of β-CD which clearly indicates the existence of strong interaction between anticancer drug EPS and β-CD molecule. On the contrary, the chemical shifts of H1, H2, H4 and H6 which are on the outer surface of β-CD (Scheme S3) were only affected by the guest molecule which due to the conformational rearrangement in the host molecule. The significant information obtained from the 1H NMR spectra strongly confirmed the formation of solid inclusion complexes. In addition, it is well identified that Nuclear Overhauser effect (NOE) cross correlation through the 2D ROESY NMR reflects significant information about the spatial proximity between host and guest molecules [49,50]. To put on further information about the interaction between anticancer drug EPS and β-CD, 2D NMR of EPS:β-CD inclusion complex was recorded and shown in Fig. 3. The ROESY spectrum showed appreciable cross peaks between the Hc, Hd protons of anticancer drug EPS with the H5 protons of β-CD, indicating that the A ring of EPS was included into the β-CD cavity. In addition to other peak were assigned to the interaction of H3 protons of β-CD with ortho positioned Hb protons of the EPS drug (Fig. 3). In each case the interaction of anticancer drug EPS with only internal protons of the β-CD were observed. In addition the H6 protons of the β-CD were not affected by the inclusion process. Further, the inclusion of etoposide into the β-CD cavity is evidenced by the chemical shift changes observed in the 1H NMR spectra of both β-CD and drug molecule. The 1H NMR spectra of etoposide inclusion complex displayed upfield shift changes compared to pure drug. On the other hand, changes in shape and size of chemical shift were observed for the aromatic proton of etoposide and the downfield shift was relatively small. These observations confirmed the complex formation. Here, 3.35–3.63 ppm for the Methylene and Methine proton in the backbone of cyclodextrin (6), 4.8 ppm is the Methine group close to oxygen (1), 4.5 ppm for the proton peak of OeH adjacent to methylene (CH2) was appeared (7), and 5.7 ppm for the OeH proton attached to the methane group on the backbone of the CD (8). We confirmed that the EPS drug included into the β-CD cavity via wider rim [51,52], it's confirmed by Fig. 3. Based on these results of 1H and 2D NMR, the possible mode of the inclusion complex of anticancer drug EPS with β-CD was recognized.

Fig. 4. X-ray diffraction pattern of EPS:β-CD system (a) β-CD (b) EPS Drug (c) Inclusion complex by Physical Mixture (d) Inclusion complex by Kneading Method (e) Inclusion Complex by Solvent évaporation méthod.

3.4. Powder X-ray diffraction pattern analysis The formation of the solid inclusion complex can be confirmed by Powder X-ray diffractometry (XRD) pattern. Here, Fig. 4 is the powder X-ray diffraction pattern of β-CD, anticancer drug EPS and their solid inclusion complexes. The X-ray diffraction pattern of the solid inclusion complex shown in Fig. 4c,d and e was evidently different from that of βCD host molecule itself shown in Fig. 4a. The difference between different diffraction patterns indicated that the solid inclusion complex is due to the interaction of β-CD with anticancer drug EPS molecule. β-CD powder X-ray diffraction pattern exhibits the diffused diffraction pattern typical of an amorphous state. Fig. 4b and the 4c, d and e exhibit the sharp intense peak indicative of its crystalline character. From the results of XRD patterns strong evidence of the solid inclusion complex of EPS: β-CD was formed.

Fig. 5. DSC Thermogram of EPS:β-CD system (a) β-CD (b) EPS Drug (c) Inclusion complex by Physical Mixture (d) Inclusion complex by Kneading Method (e) Inclusion Complex by Solvent évaporation method. 101

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Fig. 6. SEM images of (a)β-CD, (b) EPS Drug, (c)Inclusion complex by Physical Mixture (d) Inclusion complex by Kneading Method (e) Inclusion complex by Solvent evaporation method.

3.5. Differential scanning calorimetry analysis

temperature observed due to melting point depression of drug molecule inclusion nature into the β-CD cavity [54]. While turning to the kneading method solid inclusion complex (Fig. 5d) and solvent evaporation method solid complex (Fig. 5e), there was only a dehydrated peak of β-CD, the endothermic peak (243.5 °C) responsible for EPS drug molecule completely disappeared, it's suggesting that EPS drug encapsulated into inner nano cavity of β-CD.

Differential scanning calorimetry (DSC) can be used for the identification of the solid inclusion complexes. When guest molecules were encapsulated into β-CD cavity, their melting, boiling or sublimating points generally shifted to different temperatures or disappeared [53]. The thermograms of β-CD, EPS and their solid inclusion complex systems were shown in Fig. 5. The DSC thermogram of β-CD (Fig. 5a) showed a very broad endothermic peak at 110 °C which corresponds to their dehydration process, followed by another sharp peak around 300 °C is due to its dehydration process. EPS DSC thermogram (Fig. 5b) exhibited a sharp endothermic peak at 243.5 °C indicating their melting point. The solid inclusion complexes such as physical mixture of EPS: βCD, (Fig. 5c) the drug endothermic peak shifted from 243.5 °C to 200 °C and the peak of β-CD 300 °C peak shifted to 280 °C. The lower

3.6. Scanning electron microscopic analysis Scanning electron microscopic (SEM) images of β-CD, anticancer drug EPS and their solid inclusion complexes are shown in Fig. 6. The βCD topography image (Fig. 6a) was found in irregular rectangular broken bricks shaped crystal structure and pure EPS drug (Fig. 6b) appeared as flower petals structure. The solid inclusion complex prepared by physical mixture (Fig. 6c) revealed flower petals and

Table 3 Absorption maxima and absorbance values of pure Etoposide drug and its inclusion complex through in vitro studies. S.No

1 2 3 4 5 6

Time (min)

10 20 30 40 50 60

Pure drug

EPS:βCD(PM)

EPS:βCD(KM)

EPS:βCD(CP)

λ max (nm)

Absorbance

λmax (nm)

Absorbance

λ max (nm)

Absorbance

λmax (nm)

Absorbance (SE)

286 286 286 286 286 286

0.172 0.240 0.366 0.428 1.173 1.234

286 286 286 286 286 286

0.261 0.229 0.375 0.861 1.336 1.398

286 286 286 286 286 286

0.342 0.388 0.559 0.976 1.417 1.885

286 286 286 286 286 286

0.530 0.598 0.827 1.065 1.696 2.253

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rectangular broken bricks mixed topography image was appeared. Likewise, kneading method (Fig. 6d) complex image appeared as like a broken rock structure. The solvent evaporation method solid complex image (Fig. 6e) appeared as some broken smallest bricks crystals with different shapes. This SEM topography images clearly elucidates the formation of the solid inclusion complex and shows the difference of each other the materials [46,47].

45 ± 3.2%, while the percentage of anticancer drug EPS dissolved from the physical mixture was higher, 50.9 ± 4.7% (p < 0.001). The enhancement in the drug dissolution rate from physical mixture could be due to the surfactant-like properties of β-CD, thus improving the wettability and dissolution properties of the drug [55,56]. The kneading method solid product gives higher dissolution percentage (68.7 ± 0.1%) when compared to both physical mixture and free anticancer drug EPS (p < 0.001), EPS dissolved after 60 min. This enhancement has been recognized to the formation of stable soluble complexes in the solid state, improving the hydrophilicity of the molecule and reducing its crystallinity, as designated by DSC and XRD studies [54]. Interestingly, the solvent evaporation method is greater performances to the dissolution percentage (81.9%), it's may be attributed to a few factors such as formation of soluble complexes, superior wettability, amorphisation, reduction of particle size. In addition, the increase in dissolution of solvent evaporation method can be surfactant

3.7. Dissolution studies Dissolution profile of 50 mg of pure anticancer drug EPS or its equivalent amount in physical mixture, kneading method and solvent evaporation products were carried out in 0.1 N HCl at 37 ± 0.5 °C. The results expressed percentage of drug release/dissolved versus time (Fig. S2). The percentage of drug releases (Table S2) and the corresponding absorption maxima values are given in Table 3. Table S2 gives the information about the percentage of free drug dissolved after 60 min is

Fig. 7. (A) Confocal Laser Scanning Microscopy images of gram positive microorganism B.lich treated with (a) EPS drug (b) Inclusion Complex by Physical Mixture (c) Inclusion complex by Kneading Method (d) Solvent evaporation method. (B) (A) CLSM images of gram negative microorganism V.para treated with (a) EPS drug (b) Inclusion Complex by Physical Mixture (c) Inclusion complex by Kneading Method (d) Inclusion complex by Solvent evaporation method. 103

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like properties of β-CD, which can be reduce the interfacial tension between EPS and the dissolution medium. Finally, the high energetic state of solvent evaporation method might be another reason in the enhanced dissolution from the complex further increase in solubility of the drug [57].

of the anticancer drug EPS. Finally, the high energetic state of solvent evaporation method might be better enhanced dissolution from the complex further increase in solubility of the anticancer drug.

3.8. In vitro antimicrobial activity studies

Dr. Stalin is grateful to the UGC, New Delhi, India for the award of Raman Fellowship No. F: 5-107/2016 (IC), and heartfelt thanks to Prof. V. Ramamurthy, Department of Chemistry, University of Miami, USA for giving me a great opportunity to work in his laboratory as a part of Raman Fellowship-Postdoctoral Research programme (January 2017January 2018).

Acknowledgments

Antimicrobial activity of anticancer drug EPS and their solid inclusion complexes were examined by disk diffusion agar method, and the relevant experimental results are shown in Fig. S3. The corresponding zone of inhibition values for all the systems are given in Table S3. It was clearly obvious that compared to pure anticancer drug EPS, all the tested solid inclusion complexes showed greater inhibitory effect on both gram negative or gram positive microorganisms. Whereas comparing three different solid complexes, solvent evaporated complex only exhibits superior inhibitory against both bacterial pathogens (B.lich and V.para). Because, the cell wall materials of both B.lich and V.para made up of high hydrophobic materials of mucopeptide. The anticancer drug EPS molecule (i.e) absence of β-CD is less interaction and efficiency with the same hydrophobic materials present on the surface of the cell wall due to its hydrophobic–hydrophobic repulsion. While in the case of EPS: β-CD inclusion complex systems, the hydrophobic part of the anticancer drug EPS is included into the nanohydrophobic β-CD cavity, here there is a possibility of hydrogen bonds and Vander wall interactions between hydroxyl oxygen atoms of β-CD with drug molecule occurs. The resulting hydrophobic exterior experiences a good interaction between hydrophobic components of cell wall materials, which is control the growth inhibition and finally causes cell death [58].

Conflict of interest Author stated that there is no conflict of interest for publishing this manuscript in your reputed journal. Appendix A. Supplementary data Supplementary data (characterization data). Supplementary data to this article can be found online at doi:https://doi.org/10.1016/j.msec. 2019.04.033. References [1] The American Society of Health-System Pharmacists. Archived from the Original on 31st March-2016 and Retrieved 8th December 2016. [2] Cancer, International Agency for Research on Cancer, World Health Organization, vol. 223, (2013), p. 101. [3] K.R. Hande, Etoposide: four decades of development of a topoisomerase II inhibitor, Eur. J. Cancer 34 (10) (1998) (1514–152). [4] G. Su Kim, J. Kang, S. Woong Bang, D. Su Hwang, Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner, Biochem. Biophys. Res. Commun. 456 (3) (2015) 763–767. [5] I.A. Najar, R.K. Johri, Pharmaceutical and pharmacological approaches for bioavailability enhancement of etoposide, J. Biosci. 39 (1) (2014) 139–144. [6] T. Wang, J.J. Wei, D.M. Sabatini, E.S. Lander, Genetic screens in human cells using the CRISPR-Cas9 system, Science 343 (2014) 80–84. [7] N. Akhtar, S. Talegaonkar, R.K. Khar, et al., A validated stability-indicating LC method for estimation of etoposide in bulk and optimized self-nano emulsifying formulation: kinetics and stability effects, Saudi Pharm. J. 21 (1) (2013) 103–111. [8] H. Chen, Y. Gu, Y. Hu, Comparison of two polymeric carrier formulations for controlled release of hydrophilic and hydrophobic drugs, J. Mater. Sci. Mater. Med. 19 (2008) 651–658. [9] H. Peng, B. Cui, G. Li, Yingsai Wang, Nini Li, Zhuguo Chang, Yaoyu Wang, A multifunctional β-CD-modified Fe3O4@ZnO:Er3+,Yb3+ nanocarrier for antitumor drug delivery and microwave-triggered drug release, Mater. Sci. Eng. C 46 (2015) 253–263. [10] Y. Aso, S. Yoshioka, Y. Takeda, Epimerization and racemization of some chiral drugs in the presence of cyclodextrin and liposomes, Chem. Pharm. Bull.(Tokyo) 37 (10) (1989) 2786–2789. [11] P. Vinayak Sant, Mangal S. Nagarsenker, S.G. Anand Rao, Rajiv P. Gude, Sterically stabilized etoposide liposomes: evaluation of antimetastatic activity and its potentiation by combination with sterically stabilized pentoxifylline liposomes in mice, Cancer Biother. Radiopharm. 18 (5) (2003) 811–817. [12] C. Alvarez, J. Calero, J.C. Menendez, et al., Effects of hydroxypropyl-beta-cyclodextrin on the chemical stability and the aqueous solubility of thalidomide enantiomers, Pharmazie 63 (2008) 511–513. [13] Y.H. Zhang, N.P. Wang, Q.P. Zhang, X.Q. Shi, The interaction of beta-cyclodextrin with etoposide studied by fluorescence spectroscopy, Yao Xue Xue Bao 44 (12) (2009) 1416–1420. [14] SUN, He-wen; HAN, Jing; ZHANG, Yue; GUO, Xiao-ran, Analysis of complex forming ability of medium substituted degree hydroxypropyl-β-cyclodextrins on etoposide, Chin. J. Pharm. Anal., 31(11) (2011) 2103–2107(5). [15] B. Siefert, U. Pleyer, M. Müller, C. Hartmann, S. Keipert, Influence of cyclodextrins on the in vitro corneal permeability and in vivo ocular distribution of thalidomide, J. Ocul. Pharmacol. Ther. 15 (5) (2009) 429–438. [16] R. Kale, P. Tayade, M. Saraf, A. Juvekar, Molecular encapsulation of thalidomide with sulfobutyl ether-7 beta-cyclodextrin for immediate release property: enhanced in vivo antitumor and antiangiogenesis efficacy in mice, Drug Dev. Ind. Pharm. 34 (2) (2008) 149–156. [17] E.M. Martin Del Valle, Cyclodextrins and their uses: a review, Process Biochem. 39 (9) (2004) 1033–1046. [18] Q. Hu, Gu-Ping Tang, Paul K. Chu, Cyclodextrin-based host−guest supramolecular nanoparticles for delivery: from design to applications, Acc. Chem. Res. 47 (2014) 2017−2025. [19] L. Liua, X. Fengb, Yueting Peia, J. Wanga, J. Dingb, Li Chena, α-Cyclodextrin

3.9. Antibiofilm studies by confocal laser scanning microscopy Confocal Laser Scanning Microscopy (CLSM) images of gram positive and negative microorganism (B.lich and V.para) [59] treated with anticancer drug EPS and their β-CD inclusion complex shows in Fig. 7. The inhibitory effect of EPS drug and EPS:β-CD inclusion complex systems against B.lich, and V.para microorganisms after 24 h incubation was studied. The difference between the thicknesses of the biofilm formation was examined for both pure anticancer drug EPS and EPS:βCD inclusion complex systems. The current study, the β-CD inclusion complex reduces the biofilm formation to noticeable extent compared to the pure drug. Hydrophobic nature of anticancer drug EPS interacts with the same hydrophobic materials present in the cell wall of both pathogens. Hence, this hydrophobic repulsive interaction could not cause any significant change in the biofilm formation compared to the control system. In the case of EPS: β-CD inclusion complex systems the efficient exterior hydrophilic interactions with lipophillic cell wall materials which disturb the architecture of biofilm by loosening the microcolonies. This inhibition and dispersion effect of inclusion complex system on biofilm provides an attractive antibiofilm strategy [60] when compared with pure anticancer drug EPS. 4. Conclusions Inclusion complexation is a viable strategy was established for in vitro dissolution rate, antimicrobial and antibiofilm studies of anticancer drug EPS. The strategy is based on three types of preparation procedure, such as physical mixture, kneading and solvent evaporation methods and the solid prepared complexes were characterized by FT-IR, 1 HNMR XRD, DSC and SEM techniques. Indeed, this is the case observed with the stability constant (Ks) is 133.4 mol−1 and 1:1 stoichiometric ratio of the inclusion complex was proposed from the phase solubility studies. Finally, we conclude in this part, the solvent evaporation solid complex exhibits superior activity and promising strategy for improving the solubility, dissolution rate, and antimicrobial activity 104

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