Antibiofilm Potential of Silver Sulfadiazine-Loaded Nanoparticle

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Anti-biofilm potential of silver sulfadiazine loaded nanoparticle formulations: a study on the role of DNase-I in microbial biofilm and wound healing activity Krishna Kumar Patel, D. Bhavya Surekha, Muktanand Tripathi, Md. Meraj Anjum, M.S. Muthu, Ragini Tilak, Ashish Kumar Agrawal, and Sanjay Singh Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00527 • Publication Date (Web): 18 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Molecular Pharmaceutics

Anti-biofilm potential of silver sulfadiazine loaded nanoparticle formulations: a study on the role of DNase-I in microbial biofilm and wound healing activity Krishna Kumar Patel1, D. Bhavya Surekha1, Muktanand Tripathi2, Md. Meraj Anjum1, M. S. Muthu1, Ragini Tilak2, Ashish Kumar Agrawal1* and Sanjay Singh1*

1Department

of Pharmaceutical Engineering and Technology, Indian Institute of Technology (IIT-BHU), Varanasi, India. 2Department

of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

* Corresponding Authors: Prof. Sanjay Singh & Dr. Ashish Kumar Agrawal Department of Pharmaceutical Engineering and Technology, IIT (BHU), Varanasi, India Email: [email protected]; [email protected]

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Highlights 

Process and formulation variables were statistically optimized using Box-Behnken Design to develop silver sulfadiazine loaded solid lipid nanoparticles (SSD-SLNs) with desired characteristics



Developed SSD-SLNs demonstrated sustained activity up to 48h on minimum inhibitory concentration (MIC) as compared to pure SSD



SSD-SLNs in combination with DNase-I further facilitated the biofilm disruption as compared to SSD-SLNs alone



Developed SSD-SLNs were able to reduce the fibroblast toxicity associated with SSD as confirmed by MTT assay



Final formulation of chitosan gel having SSD-SLNs and DNase-I accelerated the wound healing process and showed the normal re-epithelialization in animal models


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Abstract Biofilm resistance is one of the severe complication associated with chronic wound infections which impose the extreme microbial tolerance against antibiotic therapy. Interestingly, DNase-I have empirically proved efficacy to improve the antibiotics susceptibility against biofilmassociated infections. DNase-I hydrolyzes the extracellular DNA, a key component of the biofilm responsible for the cell adhesion and strength. Moreover, Silver sulfadiazine, a frontline therapy in burn wound infections, exhibit delayed wound healing due to fibroblast toxicity. In this study, solid lipid nanoparticles of silver sulfadiazine (SSD-SLNs) laden chitosan gel supplemented with DNase-I have been developed to reduce the fibroblast cytotoxicity and overcome the biofilm imposed resistance. The extensive optimization by using Box-Behnken Design (BBD) resulted into the formation of SSD-SLNs with smooth surface as confirmed by scanning electron microscopy and controlled release (83%) for up to 24h. The compatibility between the SSD and other formulation excipients was confirmed by FTIR, differential scanning calorimetry and powder X-ray diffraction studies. Developed SSD-SLNs demonstrated improved cell viability (90.3±3.8%) as compared to SSD alone (76.9±4.2%) and combination of SSDSLNs with DNase-I, inhibited around 96.8% of biofilm of Pseudomonas aeruginosa as compared to SSD with DNase-I (82.9%). In line with our hypothesis, SSD-SLNs were found less toxic (cell viability 90.3±3.8% at 100 µg/mL) in comparison with SSD (Cell viability 76.9±4.2 %) against human dermal fibroblasts cell line. Eventually, the results of in-vivo wound healing study showed complete wound healing after 21 days’ treatment with SSD-SLNs along with DNase-I, whereas, marketed formulation, SSD and SSD-LSNs showed incomplete healing after 21 days. Data in hand suggest, SSD-SLNs with DNase-I as an effective treatment strategy against the biofilm-associated wound infections and to accelerate the wound healing. Keywords: Silver sulfadiazine, burn wounds, biofilm, SLNs, eDNA, DNase-I.



1.

Introduction

Burns, covering ≥40% of total body surface area (TBSA), are classified as severe burns leading to 75% of total deaths due to septic wound infections [1]. Burn injury is a severe traumatic condition which damages the skin frontline external protective barrier and provides open access to microbes viz. S. aureus, E. coli, and P. aeruginosa and requires the immediate hospitalization and critical care. [2]. Metabolically inactive or less active form within relatively stable and dense communities of mixed microbial species embedded in the extracellular polymeric matrix (ECP), popularly known as biofilm, makes the condition more critical [3]. The biofilm is comprised of self-secreted dense polysaccharides, proteins, nucleic acid and extracellular DNA molecules which makes the antimicrobial therapy ineffective or less effective due to the poor antimicrobial penetration in the biofilm [4, 5]. Due to the biofilm resistance, higher concentration of antimicrobials is required which ultimately results in to the toxic effect on fibroblast and delayed wound healing. A biofilm can further promote the chronic wound formation and may also lead to the systemic infection of the microbes [4]. In addition to the other components, eDNA is the principal component of the biofilm matrix responsible for initiating the microbial cell attachment to the tissue surface, aggregation and conservation of biofilm by binding all the components of EPS together [6] and hence can be a potential target for biofilm disaggregation. Silver Sulfadiazine (SSD) is a non-ionized, water-insoluble, fluffy pale yellow color powder with poor permeability across the skin. SSD is considered as gold standard and most preferred drug for the treatment of burn wound infections [7]. Though SSD efficiently acts against burn wound infections, yet burning sensation, fibroblast and keratinocyte toxicity and delayed wound healing are the major complications with SSD therapy [8, 9]. Additionally, biofilm occurrence in the wounds exaggerates the complication and hampers the wound healing. Although, a variety of nanoformulation based approaches viz. aloe vera gel containing SSD nanosuspension [10], SSD loaded silk fibroin nanofibers [11] were reported to reduce the fibroblast toxicity and accelerate the wound healing, yet a much more efficient approach is needed to combat the biofilm-associated wound infection and enhance antimicrobial therapy against biofilm. In the current report we have developed SLNs laden chitosan gel in combination with DNase-I to reduce the fibroblast toxicity and improve the therapeutic efficacy by overcoming the biofilm mediated resistance. The selection of the SLNs as carrier is based upon the earlier reports in


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which SLNs have shown superiority over conventional formulation in terms of enhanced drug solubility, protection from physiological drug degradation (for instances by pH, Enzyme, etc.), controlled drug release, attenuated drugs toxicity, thin film formation over skin, avoiding the direct exposure of tissue with drug [12, 13]. Nano size and higher surface area of SLNs are supposed to facilitate the penetration into the biofilm and promote the close interaction of drug with microbial colonies. The selection of chitosan gel is based upon the earlier reports in which chitosan has been reported to have antimicrobial efficacy, hemostatic property and wound healing potential [14]. Deoxyribonuclease-I (DNase-I) has been reported to reduce the viscosity of sputum carrying microbial biofilm and proved clinically safe when administered in cystic fibrosis (CF) patients. Consequently, the rhDNase administration along with medication improved the pulmonary function in CF patients [15]. DNase-I breaks the phosphodiester bonds next to pyrimidine nucleotides in DNA strands and degrades it leading to the dispersal of biofilm matrix. Hence, the combination of SLNs, chitosan gel along with DNase-I was hypothesized to improve the therapeutic efficacy of SSD against biofilm resistance. Along with the optimization and usual tests for nanoformulations, the developed formulation was evaluated for its therapeutic efficacy against P. aeruginosa biofilm by testing for MIC, antibiofilm activity, toxicity and in vivo wound healing potential in animals. 2. Materials and Methods 2.1.

Materials

Silver sulfadiazine was procured from India Platinum Pvt. Ltd., Mumbai. Compritol 888 ATO, and Lutrol F 68 were obtained as genuine gift sample for research purpose from Gattefosse Sas, France and BASF, India, respectively. Moreover, the LIVE/DEAD cell imaging kit was purchased from Thermo Fisher scientific, Mumbai India and the DNase-I was purchased from Sigma Aldrich, Bangalore, India. All other analytical grade chemicals, solvents, and reagents were purchased from the different vendors. 2.2.

Preparation of SSD-SLNs

SSD-SLNs were fabricated by the double emulsification-sonication method. In short, 100 µL span 80, a w/o surfactant, was added drop wise in a lipid melt (at 70°C). Afterward, adequate



SSD dissolved in ammonia (25% v/v) solution was added drop wise in the lipid melt to prepare primary w/o emulsion with concurrent application of ultrasonication. Subsequently, the aqueous surfactant (Lutrol F 68) solution maintained above the melting point temperature of lipid (75°C) was added to primary emulsion (w/o) with continuous ultrasonication. Eventually, the prepared hot double emulsion (w/o/w) was dispersed in cold distilled water (2±3°C) under slow stirring (200 rpm) for 10 min to precipitate the fine lipid droplets in the emulsion and produce the SLNs. 2.3.

Design of formulation experimentation

The Box-Behnken response surface design created the SSD-SLNs experiment matrix by using the Design-Expert software (7.0.0, Minneapolis, USA). Factors viz. lipid to drug ratio, sonication time and surfactant concentration were selected as independent variables while the particle size (PS) and entrapment efficiency (EE) were the critical quality attributes or dependent variables. Levels with constraint set are depicted in Table 1. 2.3.1. Optimization of SSD-SLNs The response of dependent variable against each set of combination (different batches) of independent variables analyzed by lack of fit test and model statistic data to find a suitable model. A polynomial equation obtained from model specifically explains the effect of variables with the help of response surface graph generated by Design Expert. Furthermore, applying the desirability index optimized levels of independent variables with optimized PS and EE were predicted by the numerical method. Finally, the optimized SSD-SLNs were prepared based on the predicted dependent variables and used for further characterization. 2.4.

Characterization of SSD-SLNs

2.4.1. Particle size, polydispersity index and Zeta potential Particle size, polydispersity index (PDI) and Zeta potential (ZP) of SSD-SLNs were measured using Delsa Nano C particle size analyzer (Beckman Coulter, USA) at 25°C temperature. Fundamentally, the dynamic light scattering and electrophoretic movement of charged particle under the applied electric field is the basic principle for measurement of PS and ZP, respectively [16].


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2.4.2. Entrapment efficiency The % EE of SSD-SLNs was determined by direct method described in the previous study using UV analysis [17] as follows; %𝑬𝑬 =

𝑨𝒎𝒐𝒖𝒏𝒕 𝒐𝒇 𝒅𝒓𝒖𝒈 𝒊𝒏 𝒕𝒉𝒆 𝑺𝑺𝑫 ― 𝑺𝑳𝑵𝒔 𝑿 𝟏𝟎𝟎 𝐓𝐨𝐭𝐚𝐥 𝐚𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 𝐝𝐫𝐮𝐠 𝐢𝐧𝐭𝐢𝐚𝐥𝐥𝐲 𝐮𝐬𝐞𝐝

2.4.3. Scanning electron microscopy (SEM) The shape and surface characteristics of the optimized SSD-SLNs were observed by scanning electron microscope (ZEISS, EVO18). Briefly, a drop of SSD-SLNs dispersion was placed on the glass slide with the help of micropipette and kept at room temperature for air drying to form the thin film. Later, the dried thin film of SSD-SLNs was coated with gold using a gold sputter coater QUORUM (Q150R ES) in a high vacuum evaporator to make the surface conductive. Finally, the gold coated SSD-SLNs were observed under scanning electron microscope [18, 19]. 2.4.4. In-vitro release study The modified dialysis bag diffusion technique was used for in-vitro drug release study of SSDSLNs [20, 21]. Briefly, 3 mL of SSD-SLNs dispersion (equivalent to 5 mg of SSD) was filled in a pre-soaked dialysis bag. Tightly tied bag was immersed in 500 mL beaker containing 250 mL of pH 6.8 phosphate buffers (supplemented with 0.5%w/v Tween 80) and maintained at 37±0.5°C under continuous stirring (50 rpm). Adequate samples from the release medium were pipetted out at predefined time points and replaced by fresh medium to maintain the sink condition. The diluted samples were analyzed at λmax 256 nm using UV spectroscopy to find out the drug concentration and total drug released in release medium. The regression coefficients of the release profile were calculated for different release kinetics to understand the exact release mechanism. 2.5.

Drug-excipient compatibility study

2.5.1. Fourier transformation infrared spectroscopy (FT-IR) Spectra of pure SSD, compritol 888 ATO, Lutrol F 68, and SSD-SLNs, were taken by KBr pellet method using Infrared spectrophotometer (SHIMADZU, Model 8400S, Tokyo, Japan). Samples



were scanned over the wavenumber ranging from 4000 cm-1 to 400 cm-1 to obtain the FT-IR spectra [22, 23]. 2.5.2. Differential scanning calorimetric (DSC) study Approximately 5 mg of samples were hermetically sealed into an aluminum crimp cell and scanned from 0°C-400°C temperature at the rate of 10°C/min under the constant nitrogen atmosphere (purging rate of 25 ml/min) against an empty aluminum pan as a reference. At the end, properties like melting enthalpy and melting points of the sample were determined for further analysis [24]. 2.5.3. Powder X-ray diffraction (PXRD) study Pure SSD, physical mixture of SSD with other excipients and SSD-SLNs samples were mounted on 0.2 mm thick standard quartz sample holder and scanned from 5°-80° diffraction angle (2Ɵ) to record X-ray diffraction pattern using X-ray diffractometer (Rigaku, MiniFlex 600). The detector DTEX Ultra had high resolution with scanning speed of 10°/min [25]. 2.6.

Bacterial strain and growth conditions

Pseudomonas aeruginosa PA01 (ATCC 15692) was collected from the Institute of Medical Sciences, Banaras Hindu University, Varanasi. Later, the P. aeruginosa PA01 was grown overnight in Luria Bertani (LB) (Pronadisa, Spain) liquid medium at 37°C. Finally, cells were centrifuged at 8000 ×g for 10 min and the absorbance at A550 was recorded to confirm the growth. 2.7.

Minimal inhibitory concentration (MIC) assays

MIC values of different groups (pure SSD, SSD+DNase-I, SSD-SLNs and SSD-SLNs+DNase-I) were determined with reference to Clinical and Laboratory Standards Institute guidelines on planktonic culture by two fold broth dilution method [26, 27]. In brief, 96-well microplates (Polystyrene, transparent, sterile, BRAND plates) containing 100 μL of different test samples with concentration varying from 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.7812μg/mL (equivalent to SSD) were inoculated with 100 μL of P. aeruginosa culture at a density of 5 × 105 CFU/mL in LB culture medium. Afterwards, the plates were incubated at 37°C and absorbance


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recorded for each well using SYNERGY/HTX Multi-mode reader (Biotek, USA) at 550 nm after 24, 36 and 48 h. The motive of observing absorbance at different time points was to evaluate the time-dependent effect of SLNs on MIC. 2.8.

Anti-Biofilm activity of SSD-SLNs

The anti-biofilm activity of various test groups (pure SSD, DNase-I, SSD+DNase-I, SSD-SLNs and SSD-SLNs+DNase-I) was evaluated on P. aeruginosa established biofilms and the biofilm was quantified by the method described previously [28, 29]. Concisely, the peg lids were immersed in the 96 well plates containing 5 × 105 CFU/mL P. aeruginosa PAO1 suspension (200 μL) in LB medium. Inoculated microplates were kept at 37°C for 48 h in the static condition. After that, peg lids were withdrawn from microplate and cleaned thrice with phosphate buffer saline pH 7.4 to remove lightly attached planktonic cells from the biofilm. Subsequently, the peg lids with biofilm were immersed into fresh 96-well plates filled with 200 μL LB enriched with different test groups (SSD concentration equivalent to18.75 µg/mL) to evaluate the biofilm dispersal potential of SSD-SLNs against 48 h established biofilm of P. aeruginosa PA01. In this study, biofilm was treated with three subsequent dosing on 24, 48, and 72 h to evaluate the % biofilm inhibition after incubation at different time points. Each time the new 96 well microplate with fresh LB medium containing the SSD-SLNs and other test samples were used. After that, biofilm was sonicated to remove the viable cells from peg lid in 200 μL LB and then the extracted biofilm centrifuged at 3000 rpm in an Eppendorf Microcentrifuge tube to get the remaining cells. Extracted cells were transferred in LB agar plates after proper serial dilution to count the CFU and calculate the % biofilm residue. Similarly, the biofilm was grown on cover slips in 12 well culture plate following the different treatments to determine the change in the thickness of biofilm. The untreated and treated biofilms were stained by SYTO 9 (6 μM) and propidium iodide (30 μM) of the LIVE/DEAD BacLight Bacterial Viability kit (Thermo Fisher). Finally, the stained biofilms sections were scanned at excitation wavelengths of 560 and 488 nm using confocal scanning laser microscopy (ZEISS, Germany). The thickness of biofilms was determined by acquiring Z-stack of scanned section at z step size of 2.03μm. Acquired microscopic images were further processed with Zen lite software (Zen lite 2012).



2.9.

Determination of toxicity on Human Dermal Fibroblast

Cell viability was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay (Sigma-Aldrich, India). Briefly, human dermal fibroblast (HDF) cells were plated in 96 well tissue culture plates at density 2 x 105 in medium and incubated with varying sample’s concentration (SSD equivalent to 100, 50, 25, 12.5, and 6.25 μg/mL). The culture supernatant separated after 24 h incubation of cell line with varying concentration of test sample was treated with 10% of MTT for 3 h at 37°C. Subsequently, the blue formazan obtained was solubilized in DMSO and absorbance at 550 nm was recorded using a microplate reader SYNERGY/HTX Multi-mode reader (Biotek) to calculate the % cell viability [30]. 2.10. Preparation of SSD-SLNs loaded chitosan gel Chitosan gel (1% w/v) was formulated by incorporating the weighed amount of chitosan into 0.5% v/v acetic acid and kept at stirring for 3h at room temperature. Subsequently, the pH was adjusted to 7 using the 1N NaOH. Later the SSD-SLNs were added to previously prepared chitosan gel and mixed homogeneously with the glass rod for 20 min followed by bath sonication for 10 min to expel out the entrapped air bubbles. Finally, to obtain the SSDSLN+DNase-I gel, DNase-I was added and mixed thoroughly. 2.11. Characterization of SSD-SLN loaded chitosan gel 2.11.1. Texture profile analysis (TPA) TPA of prepared hydrogel was performed as per the previously reported study using the Texture Profile Analyzer (TA.XT plus Texture Profile Analyzer, Stable Micro Systems, UK) [31]. Mechanical properties such as hardness, adhesiveness and elasticity were calculated from the resultant force–time plots. 2.11.2. In-vivo wound healing studies on rat model In-vivo burn wound healing study was carried out on male Wistar albino rats (200-250 g). Animals were divided into four groups (n=6) as follows; Group 1: untreated (diseased control), Group 2: treated with 1% w/w marketed cream of SSD, Group 3: treated with SSD-SLNs gel and Group 4: treated with SSD-SLNs gel containing DNase-I. Initially, the hairs of the rats on the


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lateral abdominal region were shaved off to ensure the formation of the even burn wound. After removing the hairs, rats were given 24 h rest to recover any possible skin injury due to shaving. Thereafter, full thickness burn wounds were inflicted on animals under ether anesthesia using the hot cylindrical iron rod on the lateral abdominal region. Briefly, cylindrical iron rod (20 mm diameter) was heated on flame for 5 min and placed in contact with the rat for 10 sec to create the wound. Successively, the treatment was initiated from the next day of burn induction. The different formulations were applied once a day for 21 days and the results of in-vivo wound healing study were attained in terms of % wound retraction as compared to original burn area. Eventually, on day 21 the rats were sacrificed and the histopathology of healed area was performed to check the epithelialization [32, 33]. Formula used for % retraction is as follows; % wound retraction on day x =

(𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎 𝑜𝑛 𝑑𝑎𝑦 𝑧𝑒𝑟𝑜 ― 𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎 𝑜𝑛 𝑑𝑎𝑦 𝑥) 𝑋 100 (𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎 𝑜𝑛 𝑑𝑎𝑦 0)

2.12. Statistical analysis. All graphs and its statistical evaluations were carried out using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). 3. Results 3.1.

Optimization of SSD-SLNs and analysis of variables

Quality attributes of the optimized batch of SSD-SLNs are shown in Table 1. Based on the analysis of variable and responses, the quadratic model was selected to further illustrate the effect of different independent variables on PS and EE. 3.1.1. Effect of various factors on PS The particle size of all the experimental SSD-SLNs batches varied from 256.5±8.8 to 461.1±11.3 nm. The final polynomial equation obtained from the mathematical modeling depicting the effect of variables on PS is as follow: 𝑷𝒂𝒓𝒕𝒊𝒄𝒍𝒆 𝑺𝒊𝒛𝒆 = +𝟐𝟗𝟗.𝟓𝟒 ―𝟒𝟓.𝟏𝟏 ∗ 𝑨 ―𝟑𝟎.𝟑𝟓 ∗ 𝑩 +𝟐𝟓.𝟒𝟒 ∗ 𝑪 + 𝟏𝟎.𝟑𝟓 ∗ 𝑨 ∗ ―𝟏𝟎.𝟎𝟖 ∗ 𝑨 ∗ 𝑪 +𝟐𝟕.𝟑𝟒 ∗ 𝑨𝟐 +𝟐𝟓.𝟐𝟐 ∗ 𝑩𝟐 ―𝟑.𝟒𝟔 ∗ 𝑪𝟐 ………………………………….(1)



The negative and positive sign indicates the decrease and increase in the PS on increasing the level of variable. As shown in Figure 1A-1C and equation 1, all the independent factors including sonication time, surfactant concentration prominently influenced the PS. Sonication time with highest magnitude (-45.11) and surfactant concentration with magnitude -30.35 reduced the size once these factors were elevated however, on increasing the lipid: drug ratio (+25.44), PS was increased. 3.1.2. Effect of variables on EE Entrapment efficiency of SSD-SLNs batches varied from 70.3±5.1 to 86.7±6.5% (Figure 1D-1F). The polynomial equation 2 mentioned below illustrates the effect of different variable: %𝑬𝑬 = + 𝟕𝟕.𝟕𝟖 ―𝟐.𝟖𝟓 ∗ 𝑨 +𝟐.𝟏𝟎 ∗ 𝑩 +𝟒.𝟏𝟓 ∗ 𝑪 +𝟏.𝟒𝟖 ∗ 𝑨 ∗ 𝑩 +𝟎.𝟑𝟐 ∗ 𝑨 ∗ 𝑪 +𝟏.𝟏𝟖 ∗ 𝑩 ∗ 𝑪 +𝟐.𝟐𝟎 ∗ 𝑨𝟐 ―𝟎.𝟗𝟎 ∗ 𝑩𝟐 ―𝟏.𝟐𝟓 ∗ 𝑪𝟐………………………………………(𝟐) Similar to our observation in PS, all the factors had the major role and affected the EE accordingly. The equation 2 depicting the effect of various factors on response has magnitude equal to -2.85, +2.10 and +4.15 for sonication time, surfactant concentration and lipid: drug ratio, respectively. The magnitude indicated that increase in sonication time resulted in the reduction in EE however, surfactant concentration and lipid: drug ratio had favorable influence on the EE and on increasing these factors the EE was also improved. 3.2.

Scanning electron microscopy (SEM)

The SEM image of SSD-SLNs (Figure 2) revealed spherical shape and uniform size distribution of the developed SLNs. Moreover, the particle size obtained by SEM (~ 300 nm) was approximately similar to the particle size measured by differential light scattering. 3.3.

In-vitro drug release study

SSD-SLNs showed initial burst release up to 35.3±3.89% in 3 h followed by sustained release up to 83.1±2.64% of SSD in 24 h (Figure 3A). The kinetic evaluation of release data suggested that SSD-SLNs exhibited diffusion controlled release mechanism (followed Higuchi model; R2 0.9686). 3.4.

FT-IR


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The physicochemical compatibility of SSD and excipients was studied using FT-IR analysis. The FT-IR spectra of SSD (Figure 3B) depicted the specific and intense peaks at 3390.97, 3344.68 cm-1 (N-H stretching), 3074.63 cm-1 (aromatic =C-H stretching), 1654.98 cm-1 (N-H bending), 1599.04, 1581.68, 1552.75, 1500.67 cm-1 (aromatic C=C stretching), 1232.55 cm-1 (aromatic CN stretching), 1356.00 cm-1 (S=O asymmetric stretching), and 1118.39 cm-1 (S=O symmetric stretching). Similarly, all these peaks were intact and present with minor or no transformation in intensity and position in the spectra obtained for SSD-SLNs. 3.5.

Differential scanning calorimetric study

The DSC thermogram (Figure 3C) corresponding to pure SSD had a sharp endothermic peak at 286.56°C suggesting the crystalline nature of SSD. The crystalline behavior of SSD was well maintained in the physical mixture with insignificant shift in endothermic peak (282.90°C). Precisely, the minor shift in melting point peak indicated the better compatibility and no interaction of SSD with excipients. However, thermogram for SSD-SLNs possessed endothermic peak at same melting point 286.56°C as SSD but with reduced and broadened peak intensity. 3.6.

PXRD study

The diffraction pattern of SSD (Figure 3D) demonstrated the complete crystalline behavior with many peculiar, intense and sharp peaks noticed at 2θ of 8.77, 10.32, 16.21, 19.93, 24.5, 27.97, 33.13, 37.46, 38.65 and 40.82 and many more minor peaks up to 60°. On contrary, most of these diffraction peaks were absent, deformed and broadened in the diffraction pattern of SSD-SLNs. 3.7.

MIC determination

The results of in-vitro MIC studies (Table 2) elucidate that pure SSD and SSD-SLNs both had the same MIC (18.75 µg/mL) after 24 h. But after 24 h, MIC of pure SSD shifted to higher side from 18.75 to 25 µg/mL in a temporal manner during incubation. Besides that, SSD-SLNs had a constant MIC of 18.75 µg/mL at all-time point during incubation (24, 36 and 48 h). 3.8.

Anti-biofilm properties using repeated doses of treatment

This study was performed to evaluate the anti-biofilm potential of SSD-SLNs with or without DNase-I on 48 h grown P. aeruginosa biofilm (Figure 4A). Three subsequent doses of SSD and



SSD-SLNs without DNase-I could remove only 58.1 and 78.7% of biofilm after 72 h treatment, respectively. The antibacterial property of formulation enhanced significantly (P

Antibiofilm Potential of Silver Sulfadiazine-Loaded Nanoparticle

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