Nanoparticles Encapsulated with LL37 and Serpin A1 Promotes

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Nanoparticles encapsulated with LL37 and serpin A1 promotes wound healing and synergistically enhances antibacterial activity Miral D Fumakia, and Emmanuel Abraham Ho Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00099 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Molecular Pharmaceutics

Nanoparticles encapsulated with LL37 and serpin A1 promotes wound healing and synergistically enhances antibacterial activity Miral Fumakia and Emmanuel A. Ho*

Laboratory for Drug Delivery and Biomaterials, College of Pharmacy, Faculty of Health Sciences, University of Manitoba, 750 McDermot Ave, Winnipeg, Manitoba, Canada, R3E 0T5.

* Correspondence and Proofs to be sent to: Dr. Emmanuel A. Ho Laboratory for Drug Delivery and Biomaterials College of Pharmacy, Faculty of Health Sciences University of Manitoba 750 McDermot Ave, Room 329, Winnipeg, Manitoba, R3E 0T5, Canada Tel: 1-204-272-3180 Fax: 1-204-789-3744 Email: [email protected]

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 ABSTRACT Wound care is a serious healthcare concerns, often complicated by prolonged inflammation and bacterial infection, which contributes significantly to mortality and morbidity. Agents commonly used to treat chronic wound infections are limited due to toxicity of the therapy, multifactorial etiology of chronic wounds, deep skin infections, lack of sustained controlled delivery of drugs, and development of drug resistance. LL37 is an endogenous host defense peptide possessing antimicrobial activity and is involved in the modulation of wound healing. Serpin A1 (A1) is an elastase inhibitor and has been shown to demonstrate wound-healing properties. Hence, our goal was to develop a topical combination nanomedicine for the controlled sustained delivery of LL37 and A1 at precise synergistic ratio combinations that will significantly promote wound closure, reduce bacterial contamination, and enhance anti-inflammatory activity. We have successfully developed the first solid lipid nanoparticle (SLN) formulation that can simultaneously deliver LL37 and A1 at specific ratios resulting in accelerated wound healing by promoting wound closure in BJ fibroblast cells and keratinocytes as well as synergistically enhancing antibacterial activity against S. aureus and E. coli in comparison to LL37 or A1 alone.

KEYWORDS: Chronic wounds, wound healing; LL37; serpin A1; synergy; antibacterial; antiinflammatory; solid lipid nanoparticles; drug delivery; combination nanomedicine


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 INTRODUCTION Chronic wounds (CW) are one of the major health care concerns imposing profound clinical, economic, and personal implications 1. Acute wounds (AWs) are typically traumatic or surgical in origin that moves through the repair process predictably and repair as healing progresses in a timely manner. In contrast, CWs fail to resolve normally through the repair process and are characterized by an apparent conversion from a “resolving” to a “non-resolving” chronic inflammatory response 2. Persistent expression of high levels of unregulated proteinases at the wound site contributes significantly to the chronic wound conditions. It has been reported that non-healing chronic wound fluid in inflammatory phase have ten to forty fold higher levels of elastase (3 to 8 mU/mg protein) released from neutrophils in comparison to AW fluids (0.1 to 0.3 mU/mg protein)

3, 4

. Contaminated AW sites can successfully resist invasive infection. In

contrast, patients with non-healing CWs face unique healing challenges where the infection frequently ensues leading to excruciating pain and debilitation that can undermine the morbidity associated with the severely impaired healing process. Serpin A1 (A1) also known as α1 anti-trypsin/anti-proteinase is a major physiological elastase inhibitor and immuno-modulator that is found to be non-functional and degraded in CW fluids but is functional and active in AW fluids 5. Being an immunomodulator, A1 is capable of modifying and/or regulating one or more immune functions with broad physiological activities including its involvement in the coagulation, inflammatory and complement pathways affecting both wound healing and angiogenesis

6-8

. Thus, due to its ability to inhibit elastase and its

powerful anti-inflammatory property, it may help accelerate the CW healing process by reducing the over-zealous expression of inflammatory mediators.



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LL37 belonging to the cathelicidin family is a host defense peptide/antimicrobial peptide that has been described to play an important role in protecting the body against invading pathogens. LL37 has been identified to be present in humans and is overly expressed in keratinocytes and neutrophils of the inflamed skin 9-12. LL37 is an immunomodulator with broad physiological activities including: antiviral, antifungal, antimicrobial, endotoxin-binding properties, modulator of the inflammatory response, and is a promoter of wound healing by influencing cell proliferation and differentiation re-epithelialization process

14

13

. LL37 has been shown to be involved in the

. LL37 has previously been shown to induce the healing of

epithelial wounds in the airways by chemotaxis and by stimulating the proliferation of epithelial cells 15. Wound healing activity of LL37 has been attributed to its ability to induce the migration of keratinocytes via EGFR transactivation

16

. LL37 and LL37-derived peptides have

demonstrated potent activity in preventing biofilm formation and multidrug resistant bacterial infections, thereby maintaining the epithelial barrier integrity by resolving the infections 17-19. Current strategies for the management of CWs include topical antimicrobial agents, antiseptics,

antibiotics,

surgical

debridement,

antioxidant

inflammatory agents, and ischemic preconditioning

20, 21

complement

therapy,

anti-

. Unfortunately, with CWs being

multifactorial in etiology, single agent therapy may prove to be ineffective 22-24. For example, the prolonged use of antibiotics may result in the development of multidrug resistant bacteria. Furthermore, common agents such as cytokines/chemokines, growth factors, and hyaluronic acid/collagen, can result in adverse effects since they are not constrained to one specific cellular type

25

. Thus, combination therapy involving immuno-modulatory peptides may prove to be

effective in resetting the chronic inflamed phase back to an acute healing phase. Keeping the inflamed phase low, the wound will then be able to heal in a timely manner

26

. Providing 4

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continuous delivery of drugs to an active wound site over a prolonged time period is challenging, which results in patient non-compliance due to the need for repeated applications. The use nanoparticles (nano-sized drug carriers) can overcome these obstacles and provide a platform for controlled, sustained release and protect the peptides from degradation, resulting in rapid wound healing. In this paper, we investigated the potential therapeutic effects of combining LL37 and A1 into a single nanoparticle formulation to improve wound healing and synergistically enhance antibacterial activity in vitro, in comparison to individual drugs alone (Figure 1).

 EXPERIMENTAL SECTION HPLC method for LL37 and A1 quantitation HPLC separation was performed using a XBridge™ BEH300 C18 Column (300 Å, 5µm, 3.9 mm x 150 mm; Waters, MA, USA) at 60 ºC which was connected to the Symmetry C18 guard column (300 Å, 5 µm, 3.9 × 20 mm; Waters), fitted to a HPLC system (Waters® Alliance®) equipped with Separation module (Waters® 2690) and Photodiode Array detector (Waters® 996). Tuneable UV-Visible detector (Waters® 486) working at 210 nm was used for detection. The concentrations of LL37 (4.5 kDa, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) (custom synthesized by CPC Scientific Inc., CA, USA) and A1 (isolated from human plasma; cat# 16-16-011609; Athens Research and Technology, GA, USA) were determined using a gradient elution HPLC method with slight modifications

27

. Mobile phase A: 90% deionized

water (v/v), 10% acetonitrile (v/v) (HPLC grade; EMD Serono, ON, Canada); 0.01% trifluoroacetic acid (v/v) (EMD Serono, ON, Canada); mobile phase C: 90% Acetonitrile (v/v), 10% deionized water (v/v), 0.01% trifluoroacetic acid (v/v); the linear gradient from 85% Phase



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A to 99% was maintained within the first 8 min and then decreased to the initial conditions during the last 2 min. Total run was 12 min for each 200 µL injection. The flow rate was 2 mL/min maintained at 60 ℃ and detected at 210 nm. LL37 and A1 solutions of known concentrations (0.065-20 µg/mL) were used to plot the standard calibration curves (r2 > 0.99) and LL37 and A1 were quantitated using area under the curve (AUC).

Fabrication of LL37-A1 encapsulated SLNs SLNs were fabricated based on the emulsion solvent diffusion method as previously described with slight modifications whereby a water/oil/water (w/o/w) double emulsion was introduced into the method

28, 29

. Briefly, glyceryl monostearate (molecular weight 358.56 g/mol, Sigma-

Aldrich, MO, USA) (10 mg) and ∝ -L-phosphatidylcholine (Soy-95%) (molecular weight 770.123 g/mol; Avanti Lipids, AL, USA) (20 mg) were dissolved in equal volumes (1 mL) of acetone and ethanol (HPLC grade; EMD Serono, ON, Canada) at 55℃. LL37 and A1 were dissolved in 100 µL of deionised water and added to the lipid mixture under continuous sonication for 30 sec (QSonica, CT, USA) resulting in the formation of w/o primary emulsion. The resultant primary emulsion was then emulsified with 2% poly vinyl alcohol (PVA; 31-50 kDa; Sigma-Aldrich, MO, USA) aqueous phase solution (10 mL) for 50 sec under continuous sonication to form w/o/w secondary emulsion. The resultant dispersion was stirred overnight at 4℃ to evaporate the organic solvent. LL37 and A1 encapsulated SLNs (LL37-A1-SLNs) were collected by centrifugation (20,000g, 40 min, 4℃) (Sorvall RC6+; Thermo Fisher Scientific). The SLNs were then washed two times with autoclaved Milli-Q water. After taking into consideration the encapsulation efficiency (EE%), two SLN formulations loaded with varying


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concentrations of LL37 and A1 were prepared: prep 1 (final loading of 8.48 μg for LL37 and 43.5 μg for A1 per mg of SLNs) and prep 2 (final loading of 16.32 μg for LL37 and 62.47 μg for A1 per mg of SLNs). The SLNs were stored at -80°C for 5 h after re-suspending them in 25% trehalose solution which were further freeze-dried for 12 h (freeze-dry system; Labconco, MO, USA). Recovered SLN was calculated using the following equation:

Recovery =

Actual weight of SLN × 100 Theoretical weight of SLN

LL37-A1-SLN characterization Particle Sizing and Zeta Potential Measurements Dynamic light scattering (Brookhaven Instruments, NY, USA) was used to measure the particle size after re-suspending the lyophilized SLNs in autoclaved Milli Q water (25 μg/mL). Measurements were obtained at a 90° fixed-angle and a digital correlator was used to analyze the scattering intensity data. Measurements were made in triplicate at room temperature for three runs of 2 min each. The net surface charge of SLNs was analyzed by measuring the zeta potential (100 μg/mL; Smoluchowski mode) (ZetaPALS, Brookhaven Instruments, NY, USA) after resuspending the LL37-A1-SLNs and blank SLNs in autoclaved Milli-Q water (pH 7), in triplicate at room temperature for three runs of 2 min each.



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LL37 and A1 encapsulation efficiency (EE%) To determine the EE% of LL37 and A1 in LL37-A1-SLNs, the amount of un-encapsulated LL37 and A1 was quantified in the wash solutions using HPLC. The following equation was used to determine the encapsulation efficiency:

EE% =

[Amount of total drug (µg) − Amount of unentrapped drug(µg)] × 100 Amount of total drug (µg)

In vitro drug release profile The release profiles of LL37 and A1 were determined in artificial wound fluid (AWF; pH 7.4; consisting of 2% bovine serum albumin (BSA), 0.4 M sodium chloride, 0.02 M calcium chloride, and 0.08 M tris methylamine in deionized water)

30

. BSA, calcium chloride anhydrous, and tris

methylamine were purchased from Amresco, OH, USA. Sodium chloride was purchased from Fisher Scientific, ON, Canada. Briefly, 10 mg post-lyophilized LL37-A1-SLNs were resuspended in the AWF (5 mL, pH 7.4) as release media and maintained at 100 rpm speed using an orbital shaker (VWR, AB, Canada) at 37 ºC. Over a period of 15 days at specific time intervals, 200 μL of AWF was collected from the samples after centrifugation and replaced with same volume of AWF. After filtering the sample (0.2 μm filter; Pall, ON, Canada), the concentration of LL37 and A1 released was determined using HPLC.

Ex vivo skin permeation studies In accordance to the Canadian Council guidelines on Animal Welfare and Care, female New Zealand white rabbits (4-4.5 kg, 6-8 months old; Charles River) were sacrificed, ears were


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removed, and the inner ear skin (where the pinna is located) was peeled off from the underneath cartilage 31. Physical delipidization approach was used in order to carefully strip off the stratum corneum layer, which was then washed twice with saline solution (0.9% NaCl), stored at -80 ℃ (for no longer than 4 weeks), and used further as delipidized skin. Studies have shown that there were no differences in tissue integrity between fresh and frozen samples

32

. Before

initiating the permeation studies, the delipidized skin samples were equilibrated for at least 12 h with buffer solution. The skin was mounted onto a Franz diffusion cell (model # FDC-6 at 05 amplitude and 60 Hz; Logan Instruments, NJ, USA) and clamped between the donor and receptor compartments. AWF (5 mL, pH 7.4) was filled in the receiver compartment maintained at 37 ºC. The AWF in the receiver compartment was stirred continuously at 500 rpm/min. Prep 1 (10 mg/mL) and prep 2 (10 mg/mL) suspended in AWF (1 mL, pH 7.4) were added to the donor compartment and covered with Parafilm® (VWR, AB, Canada) to prevent evaporation. At various time points, 500 μL of AWF in the receptor compartment was withdrawn and replenished with fresh AWF. The concentrations of LL37 and A1 were analyzed by HPLC.

Degradation studies The influence of temperature and AWF on the stability of LL37 and A1 was evaluated over 5 days. Samples were incubated at 37 ºC in AWF (5 mL, pH 7.4) at 500 rpm/min. At specific time intervals, 500 μL of the medium containing the peptides was withdrawn and replaced with equal volume of AWF (pH 7.4). The concentrations of LL37 and A1 in the samples were determined using HPLC.



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Antibacterial studies Antibacterial activity was evaluated against gram-positive Staphylococcus aureus (S. aureus) (ATCC 25923, ON, Canada) and Escherichia coli (E. coli) (ATCC 25922, ON, Canada) cultured in tryptic soy broth no.2 (catalogue # 51228-500G-F) and stored on tryptic soy agar (catalogue # 22091-500G) at 4 ºC and mannitol salt phenol agar (catalogue # 63567-500G F) purchased from Sigma-Aldrich, MO, USA, respectively. The minimum inhibitory concentration (MIC) of LL37 and A1 were determined by the broth microtiter dilution method. Briefly, 100 μL of tryptic soy broth was added to each well of a 96-well plate. Treatment groups include prep 1 (2 mg/mL), prep 2 (2 mg/mL), LL37 only (5 μg/mL), and A1 only (20 μg/mL). To each well, 10 μL of bacteria (107 colony forming units (CFU)/mL) were added. Negative control were cells treated with 10 μL of phosphate buffered saline (PBS) (pH 7.4) (Lonza, NJ, USA) and positive control were cells treated with 10 μg/mL of ampicillin. The plates were incubated for 20 h at 37 °C and the optical density (OD) was measured at 620 nm by a spectrophotometer. Synergistic potential of the drug combination was assessed using a checkerboard method with a modified dilution scheme.

33

LL37 and A1 were titrated to determine the individual concentrations of these

peptides at which they exhibited no significant antibacterial activity. The same concentrations were then used in the combination experiments. Briefly, two-fold serial dilutions of each drug (LL37 and A1) up to at least double the MIC were prepared. 50 µL of tryptic soy broth was added into each well of a 96-well plate. The first drug (LL37) was serially diluted along the coordinate while the second drug (A1) was diluted along the abscissa. Each well was inoculated


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with 10 μL of 107 CFU/mL of bacterial inoculum and the plates were incubated for 20 h at 37 °C. After the addition of alamarBlue (DAL 1025; Thermo Fisher, ON, Canada) (10 μL) to each well, cells were incubated for another 4 h at 37ºC. The OD was read at 565 and 595 nm using a microplate reader. Synergy was calculated based on the degree of drug interaction for a given endpoint using the compusync Chou-Talalay combination index (CI) measurement as follows:

CI =

(D)1 (D)2 + (Dx )1 (Dx )2

Where in the numerators, (D)1 and (D)2 are the combination doses of drug 1 and drug 2 that inhibits x%. In the denominators, (Dx)1 and (Dx)2 are the doses of individual drug 1 and drug 2 that inhibits x%. Where, combination concentration ratios with CI less than 1 were considered synergistic, ratios with CI equal to 1 were considered additive, and ratios with CI more than 1 were considered antagonistic. Cellular Assays BJ fibroblast cells (ATCC CRL-2522, ON, Canada) were grown in eagle’s minimum essential medium (EMEM) (ATCC-30-2003, ON, Canada) containing 10% heat-inactivated FBS along with 1% penicillin-streptomycin (Lonza, NJ, USA). Primary human epidermal keratinocytes (ATCC PCS-200-011, ON, Canada) were cultured in dermal basal medium (ATCC-PCS-200030, ON, Canada) containing 1% v/v solution of antibiotics (10 units/mL of penicillin, 10 µg/mL of streptomycin, 25 ng/mL of amphotericin B) and keratinocyte growth kit (ATCC PCS-200040, ON, Canada).

BJ fibroblast cells and keratinocytes were cultured between 80-90% 11 ACS Paragon Plus Environment


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confluence at 5% CO2 and 37°C before passaging, and the medium was replaced every two days. BJ fibroblast cells and keratinocytes were used between passages four to six. Based on the synergistic combination ratios (Table 1), the concentration of prep 1 and prep 2 selected for studies will provide a 24 h release profile maintained between 2.5 – 5 µg/mL for LL37 and 10 – 20 µg/mL for A1. In order to compare prep 1 and prep 2 with the individual activities of LL37 and A1, the highest concentration of LL37 (5 µg/mL) and A1 (20 µg/mL) were selected for all cellular assays. In vitro cellular cytotoxicity studies Cytotoxicity was evaluated using MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay; Promega, WI, USA). BJ fibroblast cells and keratinocytes were grown in 96-well plates at a concentration of 5 x 105 cells/well. After 24 h, varying concentrations of prep 1 (0.156 mg/mL to 20 mg/mL), prep 2 (0.156 mg/mL – 20 mg/mL), blank SLNs (5mg/mL), LL37 only (0.781 µg/mL – 100 µg/mL), and A1 only (0.781 µg/mL – 100 µg/mL), in culture media were added to the cells in triplicate wells and were incubated in 5% CO2 for 24 h at 37°C. Blank cell media served as a negative control (NC) and 1% triton in cell media served as a positive control (PC).

After washing the cells once with PBS (pH 7.4), fresh cell media

containing 20 µL of MTS was added. Following incubation for a period of 4 h, the plates were analyzed using a spectrophotometer (BioTek) at 490 nm.

In vitro wound healing assay BJ fibroblast cells and keratinocytes were each seeded in 24-well plates and incubated in 5% CO2 for 24 h at 37°C until they were over 90% confluent. Cells were serum-starved for 12 h and 12 ACS Paragon Plus Environment

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then “wounded” using a 200 µL sterile pipette tip

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. Adhering cell debris were removed by

washing the wells twice with PBS (pH 7.4). Cells were cultured at 37°C in serum-starved medium containing 2 combination concentration ratios of LL37-A1-SLNs (3 mg/mL of prep 1 and 3 mg/mL of prep 2), blank SLNs (5 mg/mL), LL37 only (5 µg /mL), A1 only (20 µg/mL) and untreated cells in serum-starved medium as well as untreated cells in serum-rich medium (with growth stimulating factors) as negative and positive controls, respectively. The final volume for each of the wells was 1 mL. For cell membrane staining, DiO dye (3,3′Dioctadecyloxacarbocyanine perchlorate) (catalogue # D4292, Sigma-Aldrich, MO, USA) was used at 2 µM final concentration for 1 x 107/mL cells in fluorescence activated cell sorting solution. The adherent cells were incubated with the dye solution for 30-45 min. The dye solution was then aspirated. After washing the wells twice with PBS (pH 7.4), fluorescent images of the in vitro wounds were taken at 0 h, 12 h, and 24 h with a fluorescent microscope (Nikon TE2000) and the average extent of “healing” was determined by measuring the width of wound closure (the distance migrated by the cells into the wounded area) and quantified using Image J software. Cellular monolayer integrity Integrity of the BJ fibroblast cells and keratinocytes monolayers grown on cell culture inserts (0.4 μm membrane pore size, 12 mm in diameter; Millipore, MA, USA) were evaluated by measuring transepithelial electrical resistance (TEER) using a epithelial volt-ohm meter (millicell ERS-2, Millipore) attached to a STX01 electrode (Millipore). The cells were grown on the cell culture inserts at a density of 2 x 105 cells/well containing 400 µL of growth medium, while 800 μL of growth medium was added to the culture plates. The cells were grown to form a confluent monolayer on the inserts. The cells were then treated with LL37-A1-SLNs (3 mg/mL 13 ACS Paragon Plus Environment


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of prep1 and 3 mg/mL of prep 2), Blank SLNs (5 mg/mL), LL37 only (5 µg/mL), and A1 only (20 µg /mL) for 24 h. Controls include untreated naïve cells (negative control) and lipopolysaccharide (LPS) treated cells (positive control) (10 μg/mL; pH 7.4). The electrical resistance of the insert without skin (blank resistance) was subtracted from the experimental values and TEER was calculated using the following formula: TEER = resistance x area of the insert (Ω x cm2) Measurement of cytokine and collagen-I production in LPS-activated BJ fibroblast cells and keratinocytes Briefly, BJ fibroblast cells and keratinocytes were each plated in 24-well plates at a density of 5 x 105 cells/well and incubated in 5% CO2 for 24 h at 37°C until over 90% confluent. The cells were then treated for 24 h with LL37-A1-SLNs (3 mg/mL of prep 1 and 3 mg/mL of prep 2) as well as LL37 only (5 µg/mL) and A1 only (20 µg/mL). Controls include untreated naïve cells (negative control) and LPS treated cells (positive control; 10 μg/mL). After 24 h incubation, supernatant from cell cultures were collected and evaluated for IL-6, IL-1β, TNF-α, and collagen-I concentrations using enzyme linked immunosorbent assay (ELISA; R&D Systems, MN, USA).

Statistical Analysis The values are expressed as the mean ± standard deviation. Significant differences (P80% EE% and exhibited a net negative surface charge.

In vitro release study of LL37 and A1 from LL37-A1-SLNs The release of LL37 and A1 from 10 mg of LL37-A1-SLNs occurred in a controlled and sustained manner throughout the study period. LL37 and A1 release profile followed a biphasic pattern with a release of 13.6 ± 2.24% (1.025 ± 1.68 µg/mg of SLNs) and 14.1 ± 1.4% (5.30 ± 5.2 µg/mg of SLNs) of LL37 and A1, respectively, for prep 1 (combination SLNs loaded with 8.48 μg of LL37 and 43.5 μg of A1 per mg of SLNs) (Figure 2A) in the first 24 h. Prep 2 (combination SLNs loaded with 16.32 μg of LL37 and 62.47 μg of A1 per mg of SLNs) released around 10.4 ± 1.20% (1.565 ± 1.81 µg/mg of SLNs) and 12 ± 1.22% (6.80 ± 6.8 µg/mg of SLNs) of LL37 and A1, respectively (Figure 2B) in the first 24 h. This was followed by a slow and sustained release over the entire study period of 15 days. 15 ACS Paragon Plus Environment


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Ex vivo skin permeation studies The cumulative amount of LL37 and A1 that permeated across the skin (per diffusion area as a function of time) from LL37-A1-SLNs is shown in Figure 3. Approximately, 14 ± 1.73% (1.053 ± 1.29 µg/mg of SLNs) and 13.4 ± 1.83% (5.048 ± 6.86 µg/mg of SLNs) of LL37 and A1, respectively, from prep 1 permeated across the skin (Figure 3A) while 7.3 ± 0.73% (1.09 ± 1.10 µg/mg of SLNs) and 11.1 ± 1.13% (6.24 ± 6.3 µg/mg of SLNs) of LL37 and A1, respectively, from prep 2 permeated across the skin (Figure 3B) in the first 24 h.

Degradation studies Since both release and ex vivo permeation studies were performed in AWF (pH 7.4) at 37 ºC, we evaluated whether direct exposure of the peptides to AWF at 37 ºC had any impact on the degradation of the peptides. According to the HPLC chromatographs (data not shown), no changes were observed in the concentration of either peptide up to 8 h. However, prolonged exposure (> 8 h) resulted in a decrease and/or absence of chromatographic peaks for both LL37 and A1 (Supplementary Figure S3). Furthermore, peptide aggregation was visually observed in the AWF after 3 days of incubation.

Synergistic antibacterial effects of LL37 and A1 on S. aureus and E. coli We evaluated the synergistic effects of various combination concentration ratios of LL37 and A1 against S. aureus and E. coli by using sub-lethal doses of LL37 and A1. When LL37 and A1 16 ACS Paragon Plus Environment

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were combined (Table 1), we observed that several combination ratios resulted in synergistic enhancement of antibacterial activity against S. aureus and E. coli in comparison to single drug therapy (Supplementary Figure S4). Furthermore, certain combination ratios can impart additive and antagonistic effects.

As per Chou Talalay’s definition of combination index (CI),

combination concentration ratios with CI less than 1 were considered synergistic, ratios with CI equal to 1 were considered additive, and ratios with CI more than 1 were considered antagonistic 35, 36

.

Antibacterial activity of LL37-A1-SLNs The antibacterial activity of LL37-A1-SLNs, blank SLNs, LL37 only, and A1 only were evaluated against 107 CFU/mL of S. aureus or E. coli. Prep 1 provided a release of 2 µg/mL for LL37 and 10.6 µg/mL for A1 while, prep 2 provided a release of 3 µg/mL for LL37 and 13.6 µg/mL for A1. As a result 5 µg/mL of LL37 only and 20 µg/mL of A1 only were used as controls for comparison. As shown in Figure 4, LL37-A1-SLNs exhibited 42.3 ± 0.56% (prep 1) and 46.93 ± 0.52% (prep 2) inhibition against S. aureus and 65 ± 0.34% (prep 1) and 72.3 ± 0.27% (prep 2) inhibition against E. coli. LL37 only and A1 only inhibited the growth of S. aureus by 34 ± 0.64% and 28.3% ± 0.70%, respectively, and inhibited the growth of E. coli by 50 ± 0.48% and 45.8 ± 0.53%, respectively (Figure 4).

In vitro cytotoxicity studies The cytotoxic effects of LL37-A1-SLNs on on BJ fibroblast cells and keratinocytes was evaluated by an MTS assay. Our studies showed that prep 1 and prep 2 (up to a concentration of 17 ACS Paragon Plus Environment


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5 mg/mL), and blank SLNs (5 mg/mL) demonstrated no significant reduction on cell viability (Figure 5A). Treating the cells with LL37 only (higher than 25 µg/mL) and A1 only (higher than 50 µg/mL) resulted in a significant reduction to cell viability (Figure 5B). In vitro wound healing assay The effects of LL37-A1-SLNs, LL37 only, and A1 only on the migratory capacities of BJ fibroblast cells and keratinocytes were determined by their abilities to induce in vitro wound closure. Both BJ fibroblast cells and keratinocytes were successfully stained with DiO dye, imparting the cell membrane with green fluorescence (Figure 6). Prep 1 provided a release of 3 µg/mL for LL37 and 15.9 µg/mL for A1 while, prep 2 provided a release of 4.5 µg/mL for LL37 and 20.4 µg/mL for A1. As a result 5 µg/mL of LL37 only and 20 µg/mL of A1 only were used as controls for comparison. Cells treated with LL37-A1-SLNs (prep 1 and prep 2) showed faster migration whereas LL37 only and A1 only treated cells showed lower migratory effects. By 24 h post-wounding, LL37-A1-SLNs significantly improved closure of in vitro wounds by 79.38 ± 7.5% (prep 1) and 103 ± 4.04% (prep 2) for BJ fibroblast cells (Figure 6A and 6C) and by 65 ± 19.5% (prep 1) and 107 ± 4% (prep 2) for keratinocytes (Figure 6B and 6D), respectively. In comparison, LL37 only and A1 only treatment groups showed 60.31 ± 10.14% and 70.62 ± 5.85% of wound closure, respectively, in BJ fibroblast cells. In keratinocytes, LL37 only and A1 only treatment improved wound closure by 47.85 ± 6.9% and 61.26 ± 16.5%, respectively.


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Impact of LL37-A1-SLNs on transepithelial electrical resistance (TEER) of LPS-treated BJ fibroblast cells and keratinocytes LPS treatment resulted in a significant reduction in TEER values of the cells (P 1 indicates antagonism. A LL37 (μg/mL)

A1 (μg/mL)

Combination index (CI) for S. aureus

2.5

10

0.576

5

10

0.666

2.5

20

0.889

5

20

0.696

10

20

3.709

2.5

40

3.896

5

40

1.680

2.5

80

1.369

10

80

0.968

20

80

3.369

LL37 (μg/mL)

A1 (μg/mL)

Combination index (CI) for E. coli

2.5

10

1.541

5

10

0.697

2.5

20

0.950

5

20

0.631

10

20

1.073

2.5

40

0.505

5

40

1.593

2.5

80

0.855

10

80

0.357

20

80

0.695

B


Page 33 of 46

120

S. aureus

100

80

% Inhibition

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***

***

60

**

40

E. coli *** ***

**

**

**

*

*

20 0 NC -20

Prep 1

Prep 2

LL37-A1-SLNs

5

20

LL37 only

A1 only

PC

Concentration (µg/mL)

Figure 4. Antibacterial activity of LL37-A1-SLNs (prep 1 and prep 2), LL37 only, and A1 only in PBS (pH 7.4) against S. aureus and E. coli. Studies were performed using bacteria at a concentration of 1 x 107 CFU/mL incubated at 37°C for 6 h. Cells were treated with prep 1 (2 mg/mL), prep 2 (2 mg/mL), LL37 only (5 µg/mL) and A1 only (20 µg/mL). After treating with 10% alamarBlue, cells were further incubated at 37°C for 4 h. Negative control (NC) = cells treated with 10 μL of PBS (pH 7.4); positive control (PC) = cells treated with ampicillin (10 μg/mL). Values represent mean±S.D; n=3. * P

Nanoparticles Encapsulated with LL37 and Serpin A1 Promotes

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