Anti biofilm effect of dihydromyricetin-loaded nanocapsules on urinary catheter infected by Pseudomonas aeruginosa

Colloids and Surfaces B: Biointerfaces 156 (2017) 282–291

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Anti biofilm effect of dihydromyricetin-loaded nanocapsules on urinary catheter infected by Pseudomonas aeruginosa A.J.F. Dalcin a,b,∗ , C.G. Santos a,b , S.S. Gündel a , I. Roggia a,b , R.P. Raffin b , A.F. Ourique b , R.C.V. Santos b,c , P. Gomes b a

Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, Brazil Post Graduate Program in Nanosciences, Centro Universitário Franciscano, Santa Maria, Brazil c Laboratory of Oral Microbiology Research, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil b

a r t i c l e

i n f o

Article history: Received 3 February 2017 Received in revised form 29 April 2017 Accepted 10 May 2017 Available online 15 May 2017 Keywords: Antimicrobial activity ® Eudragit RS 100 Polymeric nanoparticles Stability study DMY

a b s t r a c t Nosocomial infections associated with biofilm formation on urinary catheters are among the leading causes of complications due to biofilm characteristics and high antimicrobial resistance. An interesting alternative are natural products, such as Dihydromyricetin (DMY), a flavonoid which presents several pharmacological properties, including strong antimicrobial activity against various microorganisms. However, DMY, has low aqueous solubility and consequently low bioavailability. Nanoencapsulation can contribute to the improvement of characteristics of some drugs, by increasing the apparent solubility and sustained release has been reported among other advantages. The aim of this study was to evaluate, for the first time, the feasibility of DMY nanoencapsulation, and to look at its influence on nanoencapsulation of DMY as well as verify its influence on antimicrobial and antibiofilm activity on urinary catheters infected by Pseudomonas aeruginosa. The physicochemical characterization showed an average diameter less than 170 nm, low polydispersity index, positive zeta potential (between +11 and +14 mV), slightly acidic pH. The values of the stability study results showed that the best condition for suspension storage without losing physical and chemical characteristics was under refrigeration (4 ± 2 ◦ C). The antibiofilm activity of the formulations resulted in the eradication of biofilms both in free DMY formulations and in nanocapsules of DMY during those periods. However, within 96 h the results of the inhibition of biofilm by DMY nanocapsules were more effective compared with free DMY. Thus, the nanocapsule formulation containing DMY can potentially be used as an innovative approach to urinary catheter biofilm treatment or prevention. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Urinary tract infections associated with catheters are the most commonly acquired infections in the hospital environment [1]. These infections result in prolonged hospitalizations and high mortality rates [2]. The surface of the catheter acts as a platform for bacterial proliferation and biofilm formation because it provides ideal conditions for the development of biofilm populations. The deposition of urine into the catheter forms a protein film which increases the adhesion of microorganisms and facilitates the formation of biofilms [3–5]. Biofilm associated with the urinary catheter may become a

∗ Corresponding author at: Centro Universitário Franciscano, Laboratory of Nanotechnology, Rua dos Andradas 1614, Santa Maria, RS, 97010-032, Brazil. E-mail address: [email protected] (A.J.F. Dalcin). http://dx.doi.org/10.1016/j.colsurfb.2017.05.029 0927-7765/© 2017 Elsevier B.V. All rights reserved.

potential risk for systemic infection and major complications such as pyelonephritis and sepsis [5–8]. Among the most frequently encountered pathogens when using a urinary catheter is Pseudomonas aeruginosa. These infections are usually hospital-acquired and may act as a reservoir in most hospital equipment and materials, especially with liquid components, which, combined with their natural resistance, can facilitate their distribution in the environment [9–12]. P. aeruginosa is associated with biofilm formation, which has a worse and is difficult to treat due to its intrinsic resistance to several drugs and therefore it is an important pathogen [13]. The high tolerance of P. aeruginosa biofilms to antimicrobial agents has been attributed to a combination of factors that contribute to the protection of the bacterial cells by exopolysaccharide matrix present in the biofilm [14]. The appearance of multiresistance strains is a matter of concern, and new therapeutic approaches must be found [15].

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It is extremely important to search for new therapeutic options, and natural products with antimicrobial activity have received increased attention due to their wide use, potential therapeutic effect and low incidence of adverse effects [5,16,17]. Dihydromyricetin (DMY), a flavonoid compound, is mainly extracted from Ampelopsis grossedentata, a wild plant from South China. It has low toxicity and different pharmacological properties, such as antioxidant, anti-tumor, lowering blood pressure, and is hypoglycemic, anti-thrombotic, anti-inflammatory and immunostimulatory [18–21]. Furthermore, DMY showed potential antimicrobial activity with significant inhibitory effects on Staphylococcus aureus, Bacillus substillis, Aspergillus flavus, Penicillium sp. and Vibrio parahaemolyticus [22,23]. However, the disadvantage of DMY is its low aqueous solubility, which contributes to poor tissue penetration and, consequently, low bioavailability [24]. Nanoencapsulation is an attempt at overcoming the obstacles of DMY. Nanotechnology is one of the most important areas with the potential to solve problems caused by microbial infections [25]. Drug delivery by polymeric nanoparticles is considered a promising strategy to overcome the resistance of biofilms of P. aeruginosa [14,26]. Several studies have used nanotechnology to try to potentiate the antimicrobial activity of conventional drugs [1,13,26–32]. Nanoparticles can direct antimicrobial agents to the site of infection so that lower doses of the drug can be administered at the site of infection overcoming antimicrobial resistance without exceeding the systemic toxicity of the drug and preventing possible side effects [5,7,26,31–34]. The delivery of active compounds of polymeric nanocapsules is considered a promising strategy to overcome biofilm resistance. These nanocapsules are constituted by polymers which due to their structural characteristics allow a sustained release of the encapsulated drug maintaining plasma concentrations at therapeutic levels during certain periods of time, and are very important to control drug release [35–37]. These particles can improve antimicrobial delivery in the bacterial cell, increasing treatment efficacy [26,28,29,31,32]. Reports in the literature demonstrate the biosynthesis of gold nanoparticles using DMY, which resulted in gold nanoparticles with special shapes and different sizes that led to an important role in reduction during the synthesis process [67]. The result of this biosynthesis demonstrated significant antioxidant and antibacterial properties (for Escherichia coli and Staphylococcus aureus) in vitro [68]. However it should be emphasized that ours is the first work that allows the insertion of DMY in polymeric nanocapsules. Thus, this study aims to verify for the first time the feasibility of encapsulating DMY in nanocapsules and evaluating the influence of nanoencapsulation in antimicrobial and antibiofilm activities on P. aeruginosa infected urinary catheters.

2. Materials and methods 2.1. Materials DMY (98% w/w) and acetonitrile grade-High performance liquid chromatography (HPLC) were obtained from Sigma-Aldrich (St. Louis, USA), ethanol grade-HPLC was acquired from Panreac ® (Barcelon, Spain). Eudragit RS100 was kindly donated by Evonik (Germany), A medium chain triglycerides mixture was obtained ® from Alpha Química (São Paulo, Brazil), Polysorbate 80 (Tween 80 ) and acetone were provided by Synth (Diadema, Brazil). All chemicals and solvents were analytical grade and were used as received.

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2.2. Pre-formulation studies Initially, formulations were prepared at different concentrations of DMY. In order to choose the concentration of study for this DMY nanocapsules were prepared at concentrations 1.0 mg mL−1 (NC-1), 2.0 mg mL−1 (NC-2) and 5.0 mg mL−1 (NC-5). The formulations were evaluated for their physicochemical characteristics after 60 days of preparation in terms of particle size, polydispersity index, pH, value zeta potential and drug content. The samples were stored at room temperature (25◦ C ± 2◦ C) in amber glass flasks. After choosing the best formulation considering stability, cost, and performance in biological pilot tests, nanocapsule suspensions were prepared at a final concentration of 1 mg mL−1 of DMY for further studies on characterization, stability and biological activity. 2.3. Preparation of nanoparticle suspensions Nanocapsule suspensions were prepared by interfacial deposition of a preformed polymer, according to the method described ® by Fessi [38]. Eudragit RS100 (0.25 g) was solubilized in acetone (68 mL), DMY (0.025 or 0.050 or 0.125 g) and medium chain triglycerides (0.825 ␮L). After 20 min under moderate magnetic stirring at room temperature this organic phase was added to an aqueous phase (132 mL) containing polysorbate 80 (0.19 g). Magnetic stirring was maintained for 10 min at room temperature and then the organic solvent was eliminated by evaporation under reduced pressure to achieve a final volume of 25 mL. Blank nanocapsule suspensions (NC-B) were similarly prepared, omitting the presence of DMY. 2.4. Physicochemical characterization of nanocapsule suspensions and evaluation of stability 2.4.1. Determination of pH The pH values of the suspensions were determined directly from the formulations using a previously calibrated potentiome® ter (Digimed DM − 20) at room temperature. The results were expressed based on three different readings of the suspensions. 2.4.2. Particle size and zeta potential analysis The mean particle sizes and polydispersity index (size distribution) were measured (n = 3) by photon correlation spectroscopy after dilution of an aliquot of nanoparticle suspension in ultrapurified water (1:500 v/v) employing a Zetasizer instrument ® (Zetasizer Nano-ZS model ZEN 3600, Malvern Instruments, UK). Zeta potential analyses were measured (n = 3) by electrophoretic mobility then performed in the same equipment after diluting the samples in a 10 mM NaCl solution (1:500 v/v). 2.4.3. Determination of drug content DMY content was determined (n = 3) by High Performance Liquid Chromatography (HPLC) using a previously validated method. The nanocapsule suspensions that were used for the evaluation of all parameters were prepared at a concentration of 1 mg mL−1 . Nanocapsule suspensions (1.0 mL) were diluted with acetonitrile to a concentration of 100.0 ␮g mL−1 and subjected to ultrasonication ® (Unique , Brazil) for 30 min. Subsequently, a portion of this solution (2.0 mL) was diluted with mobile-phase and again ultrasonicated for 30 min to yield a final concentration of 20.0 ␮g mL−1 . The result® ing solution was filtered through a 0.45-␮m membrane (Millipore , Brazil) and injected into the HPLC system (n = 3). Chromatographic instruments and conditions were the following: Shimadzu HPLC system (Kyoto, Japan) was used and equipped with an LC-20AT pump, an SPD-M20A photodiode array (PDA) detector, a CBM-20A system controller, a C18 Phenomenex (4 × 3.0 mm) precolumn and

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RP-18 Phenomenex column (150 mm × 4.0 mm, 5 ␮m particle size, 100 Å pore diameter). The mobile phase was acetonitrile–water (20:80 v/v) at pH 4.0 (adjusted with acetic acid) at an isocratic flow rate (0.6 mL min−1 ). Each run was 10.0 min at room temperature, retention time: 5.6 min. The injection volume was 20 ␮L. Detection was performed at 290 nm. 2.4.4. Evaluation of encapsulation efficiency Nanocapsules were submitted to ultrafiltration/centrifugation ® using centrifugal devices (Amicon 10 kDa, Millipore) at 7000×g for 10 min. Free DMY was determined in the ultrafiltrate. The encapsulation efficiency (%) was calculated as the difference between the total (drug content) and the free drug concentrations using the HPLC method described [30]. 2.4.5. Morphology The nanocapsule morphology was determined by means of transmission electron microscopy (JEM 1200 Exll operating at 80 Kw). The aqueous suspension sample was deposited on a Formvar/Carbon grid and negatively stained with uranyl acetate solution (2% w/v). 2.4.6. Stability studies The nanocapsules DMY (1 mg mL−1 ) (NC-DMY) and blank nanocapsules (NC-B) were monitored after preparation for 90 days under different storage conditions. These conditions were: refrigeration (4 ± 2 ◦ C), room temperature (25 ± 2 ◦ C), and climatic chamber (40 ± 2 ◦ C). Samples were stored in amber glass flasks. The formulations were prepared in triplicate. 2.4.7. In vitro drug release study The in vitro release of DMY from nanocapsules and Free-DMY was performed using the dialysis bag diffusion technique. Formulations of the DMY ethanolic solutions and DMY nanocapsules at 1 mg mL−1 were placed in a dialysis bag (Cellulose acetate membranes − 10,000 Da, 25 mm, Sigma Aldrich), and this system was immersed in 50 mL of pH 4.5 acetate buffer. The medium was kept at 37 ◦ C under continuous magnetic stirring at 50 rpm. At predetermined intervals (0; 0.25; 0.5; 0.75; 1; 2; 3; 6; 9; 12 and 24 h), 1 mL aliquots of dissolution medium were withdrawn and replaced by the same volume of medium. The percentage of drug released was determined using the HPLC conditions mentioned previously. The release profiles were mathematically modeled to fit mono and/or biexponential equations (Eqs. (1) and (2)) and the selection of the best model considered the correlation coefficient (r), the model selection criteria (MSC) and graphics adjustment (Micromath Scientist, USA). C = 100(1-e−kt )

(1)

C = 100[1-(Ae−˛t +Be−ˇt )]

(2)

Where C is the concentration at time t; k, ␣ and ␤ are dissolution rate constants; A and B are portions of the initial concentrations of drug that contributed to the burst and sustained phases, respectively. The drug release mechanism was predicted by fitted experimental points to Korsmeyer-Peppas model (Eq. (3)) [62,63]. The experiments were performed in triplicate and under sink conditions.

2.5. Evaluation of antimicrobial activity 2.5.1. Microorganisms The P. aeruginosa PA01 strain was used for this test. The strain was plated on Brain Broth Heart Infusion agar (BHI) and incubated in a bacteriological heater for 24 h at 37 ◦ C. After the incubation time, part of the colony was suspended in sterile saline and turbidity was adjusted to 0.5 in a spectrophotometer McFarland scale (OD600 = 0.08 to 0.1). 2.5.2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (CBM) The broth microdilution technique was performed in a sterile 96-well plate [64]. 100 ␮L of the BHI culture medium were added to all wells. In the first well 100 ␮L of the treatment (FreeDMY, DMY-loaded nanocapsules and blank nanocapsules) were added, and serial dilution was performed. For inoculum formation, the microorganism was placed in sterile saline and adjusted to the 0.5 McFarland scale, then 10 ␮L were added to each well. The positive control was composed of the culture medium and the inoculum. The negative control was composed only by the culture medium. After 24 h of incubation at 37 ◦ C, the result of the Minimum Inhibitory Concentration (MIC) was verified using the 2,3,5-triphenyltetrazolium chloride dye indicating bacterial growth. CIM was considered the lowest concentration capable of inhibiting microbial growth. For the determination of Minimum Bactericidal Concentration (CBM), each well where no microbial growth was observed was seeded in Petri dishes containing nutrient agar that was incubated for 24 h at 37 ◦ C. CBM was considered the lowest concentration at which there was no microbial growth in the plates. The assay was performed in triplicate. 2.5.3. Time-kill studies This assay was performed using the method of Thomas, Lulves and Kraft [39] with some modifications. For this assay tubes were used containing the liquid culture medium (BHI) in order to verify the effect of the formulations on microbial growth. Five groups were used for this test: negative control (only BHI), positive control (culture medium BHI broth and inoculum), DMY-loaded nanocapsules 1 mg mL−1 (culture medium BHI broth, inoculum and suspension of nanocapsules of DMY), blank nanocapsules (culture medium BHI broth, inoculum and nanocapsules blank suspension) and free DMY culture medium BHI broth, inoculum and suspension free DMY. The free DMY was diluted with water containing suspension/dimethylsulfoxide at a proportion of 3:1. The tubes were incubated in a shaker (90 rotations per minute) at 37 ◦ C. At 0, 6, 12, 24, 48, 72 and 96 h aliquots were withdrawn from each tube, diluted in saline and plated on BHI agar. The plates were incubated for 24 h at 37 ◦ C. After incubation time, the colonies were visually counted to compare the efficacy of the nanocapsules of formulation DMY as opposed to the free compound, blank nanocapsules and the positive control. This assay was performed in triplicate.

(3)

2.5.4. Antibiofilm activity in urinary catheter 2.5.4.1. Urinary catheter. This study consisted of an in vitro experimental study using the catheter that is the standard model manufactured and most widely used at public hospitals in Brazil. The Foley type bladder catheter was purchased commercially. It was made of silicone latex, size 16 (French), two-way and the brand was Solidor Brazil (Barueri, Brazil), registered at the Ministry of Health under Nr. 10237580014, Lot number BE14A1630/14C06.

Where ft is the drug dissolved at time t; n is the release exponent indicative of the mechanism of drug release and a is the constant structural and geometric characteristics of the drug form.

2.5.4.2. Biofilm formation and evaluation. A method by Jones and Versalovic [40] was adapted to assess biofilm formation with some modifications in order to produce the biofilm on a urinary catheter.

ft = atn

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The catheter was cut into 6.0 cm portions and added to test tubes containing BHI, the inoculum of P. aeruginosa (PA01) and aerobically incubated at 37 ◦ C for 24 h. After the incubation time, treatments (negative and positive controls, DMY-loaded nanocapsule at 1 mg mL−1 (NC-DMY), blank nanocapsule (NC-B) solution free DMY (F-DMY), and other constituents of the nanocapsules in ® their production concentrations, Eudragit RS 100 , Polysorbate 80 and mixtures of capric and caprylic acid triglycerides (MCT)) were added to tubes containing the catheter and incubated for 72 and 96 h in a shaker agitator. After 72 and 96 h of incubation, the catheter portions were washed twice with 0.9% saline (NaCl), and dried at 60 ◦ C for 60 min. Then, the portions were stained with 0.1% crystal violet (w/v) for 10 min. They were then washed (five times) with 0.9% saline and added to 95% ethanol for 15 min. The absorbance was performed in microplate reader (570 nm). The negative control was represented by catheter fragments added in sterile BHI. All assays were evaluated in five replications. 2.6. Statistical analysis Formulations were prepared and analysed in triplicate and results are expressed as mean ± standard deviation. Statistical analysis was performed to study pre-formulation stability using Student’s t-test for two groups, comparing the conditions evaluated to the initial one. The results of time-kill studies and biofilm formation were analysed using the t-test, comparing it with the positive control treatments, and nanocapsules with free DMY. The GraphPad Prism program, version 5.0, was used for this analysis. 3. Results 3.1. Physicochemical characterization 3.1.1. Pre-formulation study The results of the pre-formulation study can be demonstrated in Table 1. As to the particle diameter value obtained by photon correlation spectroscopy, during the trial period all samples were between 148 and 163 nm. There was no significant effect of drug concentration by referring to this parameter, likewise, no statistical differences between the mean values of zeta potential and pH of the formulations were evidenced. Formulations containing 1 mg mL−1 and 2 mg mL−1 showed a low polydispersion index, indicating the homogeneity of the system during the 60 days of analysis. However, the DMY nanocapsules at a concentration containing 5 mg mL−1 showed a significant increase in polydispersity index after 14 days and a phase separation and the presence of precipitated DMY were observed, therefore this formulation was discarded. The presence of the cationic polymer resulted in a zeta potential between 11 and 14 mV, both positive, as expected due to the ® characteristics of the Eudragit RS100 polymer. A slightly acid pH was observed in all formulations. The higher the concentrations of DMY in the formulation, the more acid was the pH. Regarding the drug content, it was significantly decreased for samples containing 1 mg mL−1 , and maintained for the formulation containing 2 mg mL−1 DMY. Although the formulation with 1 mg mL−1 showed a statistical reduction in content compared to a formulation with 2 mg mL−1 , these are preliminary results from a single production held under only one storage condition. Furthermore, results of the pilot study were performed for antibiofilm activity in a urinary catheter. The incubation time of the treatment (NC-1 and NC-2) was 48 h and there was no significant difference (t-test) in the biofilm reduction effect at concentrations of 1 and 2 mg mL−1 (p = 0.3967). Thus, the

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formulation chosen in order to conduct stability studies and activity was the formulation containing 1 mg mL−1 because of its cost and performance in microbiological assays.

3.1.2. Stability study After the Pre-formulation study step the formulations were prepared in 3 batches and their morphology was analysed. The morphology of the nanocapsules was evaluated by transmission electron microscopy (TEM) and can be seen in Fig. 1. In the images, it can be observed that the nanocapsules have a spherical and irregular distribution, with no difference between the DMY-loaded nanocapsules and blank nanocapsules. While performing the stability study, the physicochemical characteristics could be evaluated during the 90-day analysis and there was no phase separation and presence of precipitations under all storage conditions (refrigeration, room temperature and climatic chamber). The results in Fig. 2A show that the technique has enabled the formation of nanometric particles of the evaluated formulations in the 90-day period and, independent of the storage condition, there was no significant difference in particle size, affording a mean diameter of around 170 nm and polydispersity index of less than 0.140 (Fig. 2B). The polydispersity index (Fig. 2B) and zeta potential (Fig. 2C) also showed no significant difference in the 90 day test under different storage conditions analysed. For pH values (Fig. 2D) there was a significant decrease under the climate chamber condition within 90 days of analysis by changing the initial pH of 4.84 ± 0.03 to 4.00 ± 0.03. Possibly this decrease in value can be attributed to the degradation of some components of the nanocapsules that could have provided these pH changes [30]. The decrease in drug content (Fig. 2E) proved to be significant from 30 days, compared to the initial periods under the climatic chamber condition. There was a decrease of approximately 35% of the drug content in the nanocapsules placed in a climatic chamber for 7 days. This value further decreases with time, and at the end of the experiment, it was possible to determine about 14% of the drug. For the samples there was no significant difference in drug content when compared to the original conditions at room temperature, but in 30 days it was observed to decrease by approximately 25% content increasing gradually and obtaining a 45% decrease of the content in 90 days analysis. The decrease in drug content can also be viewed in Fig. 3 looking at the chromatograms obtained after 90 days of stability under different storage. It has been verified that when the drug content is stored under refrigeration, the nanocapsules show less decay until the end of the experiment (90 days) quantifying approximately 90% of the drug. This decrease in drug content at storage conditions of 25 and 40 ◦ C can be attributed to a possible degradation of the drug, suggesting the best storage condition at 4◦ C in order to maintain the drug content during the 90 days of storage. The initial encapsulation efficiency was 80.88% ± 2.88 and was stable during the 90 days analysed (80.80 ± 3:57). Although there was a decrease of the content under room temperature conditions, the encapsulation efficiency remained the same, this may have occurred due to degradation of the drug that was adhered to the external phase of the wall of the nanocapsule and not encapsulated in the core of the nanocapsule. The results of physicochemical characterization of blank nanocapsules (results not shown) showed no significant difference in the particle size, polydispersity index, pH and zeta potential parameters, during the 90 days of storage under different conditions evaluated.

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Table 1 Pre-formulation study results for the DMY nanocapsules at concentrations of 1, 2 and 5 mg mL−1 (NC-1, NC-2, NC-5). The samples were stored for 60 days at 25 ◦ C. Analysis time

Formulation

Particle Size (nm)

Polydispersity index

Zeta potential (mV)

pH

Content DMY (%)

Initial

NC-1 NC-2 NC-5 NC-1 NC-2 NC-5

161 ± 2.50 151 ± 0.79 123 ± 1.01 163 ± 1.01 148 ± 0.96 ND

0.079 ± 0.01 0.098 ±0.01 0.134 ± 0.02 0.125 ± 0.02 0.095 ± 0.02 ND

11.4 ± 0.60 12.7 ± 0.44 13.4 ± 1.09 12.7 ± 0.55 13.6 ± 1.27 ND

5.63 ± 0.04 4.26 ± 0.01 3.82 ± 0.01 5.67 ± 0.02 4.27 ± 0.01 ND

104.5 109.5 111.1 67.6* 92.1 ND

60 days

* ND: Not determined. *±: Standard Deviation.

Fig. 1. Photomicrographs obtained by transmission electron microscopy (TEM) (A) DMY-loaded nanocapsule and (B) Blank nanocapsule.

3.1.3. In vitro drug release study Release of the drug from the formulations tested (DMY-loaded nanocapsules and free DMY) were best described by the biexponential model (Fig. 4). The DMY-loaded nanocapsules obtained r = 0.998 and MSC = 3.85, for free DMY, r = 0.997 and MSC = 3.49, that is, two release phases were detected, where part of the drug is released rapidly in an effect called burst and part of the drug is released progressively over time. The nanocapsules released 44% of DMY with k = 0.756 h−1 and t½ of 0.91 h, followed by a sustained release, where the remaining 56% of the drug contained in the nanocapsules presented a rate constant = −0.011 h−1 and ½ of 63.01 h. The drug in its free form showed a burst release of 70% with k = 1.045 and t½ of 0.66 h, the remaining 30% of the drug was released at k = −0.03 and t½ of 23 h. 3.2. Evaluation of the antimicrobial activity 3.2.1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (CBM) The MIC results for Free-DMY, DMY-loaded nanocapsules and blank nanocapsules were respectively 2.27 mg mL−1 , 2.27 mg mL−1 and 4.76 mg mL−1. For CBM, there was bacterial growth at all the concentrations of the analysed treatments. Indicating that in 24 h there was no bactericidal activity inr the samples tested. 3.2.2. Time-Kill studies The statistical analysis showed a significant difference between free DMY and DMY-loaded nanocapsules, and between free DMY and blank nanocapsule. The blank nanocapsule did not show a significant difference when compared to the positive control, indicating that no constituent of the formulation has a significant effect on the organism tested in their planktonic state. In the positive control there is a tendency to increase the microbial population

while the control without the microorganism showed no growth of microorganisms, showing absence of test contamination. The results shown in Fig. 5 demonstrate that the compound in free form eliminated the microbial population in 12 h while the DMY-loaded nanocapsules took 24 h to demonstrate significant removal of a microbial population that extended to 96 h of analysis, suggesting a sustained antimicrobial effect of nanocapsules. In this case there was a decrease in the number of microorganisms, but the population is not eliminated.

3.2.3. Evaluation of anti-biofilm activity on urinary catheters In 72 h of treatment (Fig. 6A), the DMY-loaded nanocapsules eradicated 78.6% of the biofilm population, and free DMY eradicated 70.5%, already in 96 h of treatment (Fig. 6B), the formulation of DMY-loaded nanocapsules eradicated 67.% of the biofilm against 41.2% treated with the drug in free form.The blank nanocapsules eradicated 82.8% of the population of the biofilm in 72 h of treatment (Fig. 6A) and 44.8% in 96 h. (Fig. 6B). Because of the biofilm eradication activity by the blank nanocapsules, constituents of the formulation of the nanocapsule were evaluated in order to verify their individual activity on the biofilm of P. aeruginosa. Polysorbate 80 was used as a surfactant in order to stabilize the formulation. The results did not show a significant effect on the biofilm population treated at 72 and 96 h. Although an apparent difference was displayed in biofilm populations when treated with a medium chain triglycerides (MCT) mixture of capric/caprylic acid, this difference was not significant compared to the positive control. ® The cationic polymer Eudragit RS 100 demonstrated significant results in the eradication of the biofilm in 72 h, 78.3% reduction of the biofilm population. At 96 h eradication was 51.1% of the population, but in 96 h that eradication was not significant compared to the positive control.

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Fig. 2. Stability study results of DMY-loaded nanocapsules at 1 mg mL−1 under different storage conditions. (A) Particle size, (B) Polydispersity index, (C) Zeta potential, (D) pH, (E) Drug content. Statistically significant values when ** to P < 0.01 and * to value P < 0.05.

4. Discussion The method employed to produce the nanocapsules was nanoprecipitation or interfacial deposition of preformed polymer, as described by Fessi et al., [38], most often used due to its easy handling and reproducibility [36]. DMY has a complex molecular and lipophilic character and is difficult to dissolve in aqueous solvents. Thus, acetone was used in this formulation, a polar solvent with rapid diffusion in the aqueous phase with low viscosity and characteristically conducting smaller particle sizes [41] corresponding to 33.74% of the formulation.

®

The polymer chosen for the formulation was Eudragit RS100 , which has a positive surface charge due to the presence of a quaternary ammonium grouping and appropriate physicochemical characteristics. This polymer was chosen because of its surface properties which can provide higher adhesion to the negatively charged microorganisms of the type of bacteria with the positive charge of the polymer [46,57,42]. After preparation, the formulations exhibited a macroscopically homogeneous milky appearance and showed a bluish opalescent reflection due to the tyndall effect resulting from the brownian

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Fig. 3. Chromatograms obtained after 90 days of stability of DMY-loaded nanocapsules under different storage conditions.

motion of nanoparticles in suspension, characteristic of suspensions with submicron particles [43,44]. The concentrations of the formulation constituents used in this study are usual for the production of polymeric nanocapsules by preformed polymer deposition technique which showed stability, confirming the results found in the literature [30,45,46]. The phase separation and presence of precipitate formulation of nanocapsules containing 5 mg mL−1 possibly occurred due to a high concentration of drug that destabilized the formulation. Pohlmann et al., [47] reported that the saturation of the colloidal phase can form a nanocrystal drug that increases and precipitates and thus destabilizes the formulation. The zeta potential values found (between 11 and 14 mV) corroborate other results found [30,41,46], and indicate that positive results demonstrate that polymer is effectively added to the oilwater interface as a polymer wall around the oil core by preventing particle aggregation due to the repulsion of small particles. The slightly acidic pH values found in the nanocapsule formulations containing DMY were expected in accordance with the drug characteristics and corroborate results obtained of a pH around 5.0 using different drugs and the same polymer [41,46]. After stability studies, it was possible to determine the refrigeration condition as the best form of storage of the DMY-loaded nanocapsules. This condition has ensured the preservation of the physicochemical characteristics up to the time of analysis at 90 days. The results obtained in the in vitro release study showed an important release phase where the nanocapsules released the drug about 3 times slower than the release of the drug in the free form, demonstrating sustained effect for a period of up to 63 h, thus prolonging the release of the drug. This more sustained liberalization by nanocapsules occurs due to the formation of the polymeric wall around the drug [65]. The initial release of the drug (burst effect) may have occurred by the drug absorbed to the surface [66]. Initially, the antimicrobial activity of the formulations: DMYloaded nanocapsules, blank nanocapsules and free DMY was evaluated against the P. aeruginosa PA01 strain in their planktonic phase (not adhered to the biofilm). In both the evaluation of antimicrobial activity and antibiofilm activity in urinary catheter a control pH = 5.0 (adjusted with HCl) was performed and this variation did not interfere in the growth of the microorganisms.

The evaluation of antimicrobial activity showed results consistent with previous studies that demonstrated the action of DMY against Gram negative and positive bacteria and fungi [22,48]. But this is the first study that shows the antimicrobial activity of free DMY and nanostructured DMY against P. aeruginosa. It is believed that the DMY-loaded nanocapsules are undergoing sustained release in comparison with DMY formulation in a free form, a typical characteristic of a nanostructured system, similar to results found in the literature [27,49]. At the same time, sustained release formulations can enhance the therapeutic index of the drug and facilitate patient compliance with antimicrobial treatment [32] and may have the effect of treating infections caused by P. aeruginosa. Catheter-related urinary tract infections cause great concern due to the high morbidity and mortality among hospitalized patients [50]. Contributing to this scenario is the formation of microbial communities in biofilms that form inside and outside these devices when inserted into the urinary tract. The results of the evaluation of anti-biofilm activity on urinary catheters demonstrate a tendency of further reduction compared to nanoencapsulated drug and the drug in free form. It is also clear that the longer the treatment (96 h) the greater the difference between the eradication of the biofilm by treatment with nanostructured and free drug, suggesting a potentiation of anti-biofilm activity of DMY during the time of analysis, there by confirming a sustained release on the part of the nanocapsules when compared to conventional formulation. This study shows for the first time the action of nanoparticles, especially cationic nanocapsules containing DMY in biofilms formed by Pseudomonas species in urinary catheters. This study demonstrated that both the free DMY formulation and nanocapsules led to a significant reduction of biofilm formation when compared to positive control (Fig. 6). This sustained release of the drug is very important due to biofilm formation which occurs gradually over time in urinary catheters. According to Maki and Tambyah [51] the risk of developing urinary tract infections associated with catheters increases by 5% for each day of catheterization and virtually all the patients are colonized by 30 days. In this context, DYM can be used for the treatment or prevention of biofilms related to urinary catheters. The results presented here may agree with those reported by Frank et al., [36] who showed that the nanocapsules may increase the interaction between the drug and the tissue, leading to increased penetration of the drug, or modified drug biodistribution.

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Fig. 5. Time kill curves of DMY-loaded nanocapsules (NC-DMY), free DMY (F-DMY) and blank nanocapsules (NC-B). Data presented as mean ± standard deviation. Fig. 4. In vitro DMY release profiles from DMY-loaded nanocapsules and Free DMY.

The activity found by blank nanocapsules may be due to the positive charge of the affinity of the nanocapsule, which may have adhered to the negative bacterial cell membrane leading to eradication of the biofilm. Positively charged nanocapsules with a negative charge of the epithelium and other tissues cause strong adhesion and greater penetration may act as an effective stragegy to prolong residence time in the mucosa [32,36]. Blank nanocapsules showed activity in the eradication of Pseudomonas biofilm on urinary catheters. Hadinoto and Cheow [14] suggest that the formulation constituents have a significant influence on anti-Pseudomonas activity, but these factors are not well understood. The constituents were tested at the same concentrations used in the formulation of nanocapsules. Although no reduction was found in the biofilm population when treated with a medium chain triglycerides (MCT) mixture of capric/caprylic acid, there are reports of antimicrobial activity of capric acid and caprylic in isolation against strains of S. aureus, Chlamydia trachomatis, Helicobacter pylor and Campylobacter jejuni [52]. The results of the eradication of biofilm by polymer Eudragit RS ® 100 in isolation can be clarified because the contact properties of the cationic surface feature that inhibits microbial colonization tend to remain, by electrostatic attraction, the adhered bacteria [53,54]. However, no results were found in the literature relating ® Eudragit RS 100 polymer to antimicrobial and antibiofilm activity, but studies using this polymer in nanocapsules formulation obtained higher antimicrobial and antifungal results when compared to the free drug (no polymer) [27,30,46]. However, cationic polymers with quaternary ammonium groups exhibited antimicrobial activity against B. subtilis, S. aureus, E. coli, Enterobacter aerogenes and P. aeruginosa [53,55]. The antimi-

crobial activity of this type of polymer has been interpreted as the polymer type conducts a positive charge to negative charge of the bacterial cell membranes. After binding to the negatively charged phospholipid, the hydrophobic polymer portion interacts with the hydrophobic core of the bacterial inner membrane leading to a disruption of the cytoplasmic membrane, releasing potassium and other components that cause cell death [55,56]. This effect on the eradication of the biofilm positive charge type of the polymer with the negative charge of P. aeruginosa membrane can allow a long residence time of the nanoparticles on the surface of the microorganism [27,57,58]. Another possibility involves ion exchange and the release of multivalent cations into the bacterial membrane in the region where the membrane of the bacterial cell comes into contact with the cationic surface [59]. ® Importantly, although Eudragit RS 100 showed considerable reduction activity in the biofilm, the DMY- loaded nanocapsule showed the largest reduction in P. aeruginosa biofilm population on urinary catheters compared to the other treatments evaluated. The most satisfactory results (which occurred at higher inhibition) were obtained during the treatment in 72 h treatment. It was possible to verify the effects of free DMY and DMY-loaded nanocapsule, which showed similar results: free DHM reduced biofilm formation by 70.5% and 78.6% in nanocapsules. However, within 96 h the results of the inhibition of biofilm by DMY-loaded nanocapsule were significantly higher (p = 0.0024) compared to the elimination of free DMY. The results of this paper agree with results found by Roe et al., [49], where the antimicrobial and antibiofilm evaluation was investigated in urinary catheters coated with silver nanoparticles. They viewed inhibition in cell growth and biofilm formation in 72 h against several test microorganisms, resulting in 67% inhibition of the formation of P. aeruginosabiofilm in 72 h. These results were

Fig. 6. Effect of the formulations evaluated on the P. aeruginosa biofilm formation (A) after 72 h of treatment and (B) after 96 h of treatment Statistically significant comparing positive control with*** to p < 0.001 and ** to p < 0.01.

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similar to those found in our study (78.6% inhibition DMY-loaded nanocapsule in 72 h). However, studies show that silver nanoparticles have high toxicity and a systemic distribution of silver and accumulation in tissues such as the kidneys, spleen, liver, lungs, stomach and brain, which may be harmful to human health [60]. It is also important to note that there are few reports in the literature using nanotechnology to coat urinary catheters, and studies found, nanoparticle formulation in the absence of the drug in question (blank nanocapsules) and other components used in the formulation are not shown. The use of nanoparticles for successful drug delivery by herbal extracts was shown in this study and confirmed by literature reports [16,17], demonstrating increased pharmacological activity, stability, sustained release and protection from degradation. Increased antibiofilm activity of the nanocapsules can be explained by some characteristics: reduced particle diameter can penetrate into the bacterial cell and the biofilm, release the drug into cells via endocytosis and then release the drug to treat intracellular infections, causing an interference membrane leading to loss of cellular viability and thus greater antimicrobial effect [32,61]. In addition to the nanometric size, the nanocapsule formulations presented prolonged drug action that can be shown both in timekill studies and in antibiofilm activity. This feature is an advantage for long-term implementation of catheters, which often happens in hospital practice. 5. Conclusions This study demonstrated for the first time the association of DMY with nanocapsules. DMY-loaded nanocapsules at a concentration of 1 mg mL−1 demonstrated adequate physicochemical characteristics and the best condition to preserve their characteristics was under refrigeration. DMY incorporated the nanocapsule demonstrating sustained release as compared to free DMY. DMYloaded nanocapsules reduced 67% of the biofilm population in urinary catheters in 96 h of treatment, while free DMY eliminated 41%. The results found were satisfactory for the eradication of biofilms in urinary catheters and it may be used as a promising antimicrobial and antibiofilm formulation. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements: The authors would like to thank FAPERGS, CNPq (Conselho Nacional de desenvolvimento Científico e Tecnológico), CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior) ® for financial support and Evonik relented Eudragit RS 100 . References [1] L.E. Fisher, A.L. Gancho, W. Ashraf, A. Yousef, D.A. Barret, D.J. Scurr, X. Chen, E.F. Smith, M. Fay, C. Parmenter, R. Parkinson, R. Bayston, Biomaterial modification of urinary catheters with antimicrobials to give long-term broadspectrum antibiofilm activity, J. Controlled Release 202 (2015) 57–64. [2] R.D. Scott II, The Direct Medical Costs of Healthcare Associated Infections in US Hospitals and the Benefits of Their Prevention, 2017 (Disponível em: https://www.cdc.gov/HAI/pdfs/hai/Scott CostPaper.pdf. Acessado em: 03 de janeiro de 2017). [3] L. Conterno, J. Lobo, W. Masson, Uso excessivo do cateter vesical em pacientes internados em enfermarias de hospital universitário, Revista da escola de enfermagem 45 (5) (2011) 89–96. [4] D. Kowalczuk, G. Ginalska, T. Piersiak, M. Miazga-Karska, Prevention of biofilm formation urinary catheters: comparison of the sparfloxacin-treated long-term antimicrobial catheters with silver-coatedones, J. Biomed. Mater. Res. B: Appl. Biomater. 100 (7) (2012) 1874–1882.

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