PLGA Nanoparticles Formulations Loaded With Antibiotics Induce Sustained and Controlled Antibiotics Release for Prolonged Antibacterial Action Against MRSA, and Pseudomonas aeruginosa FRD1 (2024)

ABSTRACT

INTRODUCTION

Antimicrobial resistance, driven by the overuse and misuse of antibiotics, has led to the rise of multidrug-resistant (MDR) bacteria, significantly impacting global health. MDR bacteria are a global crisis, increasing morbidity and mortality of infected individuals.¹ The most prevalent and challenging MDR infections are caused by methicillin-resistant Staphylococcus aureus (MRSA), representing up to 50% of all staphylococcal infections; Pseudomonas aeruginosa, the most common cause of nosocomial infections; and Acinetobacter baumannii, prevalent among immunocompromised individuals and now associated with hospital acquired infections.²

Many scientists have turned to nanotechnology because of their favorable physicochemical properties, drug targeting efficiency, enhanced uptake, biodistribution, and biocompatibility.^3–10 The use of nanoparticles (NPs) with antimicrobial properties offers a promising approach to combat MDR bacteria. These polymeric NPs are effective because of their targeted antibiotic delivery, controlled release, biocompatibility, biodegradability, and low toxicity to humans.¹¹ Copolymer poly(lactic-co-glycolic acid) (PLGA) is promising as a drug delivery system because of its biocompatibility, biodegradability, sustained drug release profile, and ability to be easily modified.¹² PLGA’s promising features led to its approval by the FDA and European Medicine Agency as a parental drug delivery system. Furthermore, modifying PLGA’s surface properties can enhance stealth and improve interaction with biological tissues.¹²

However, PLGA NPs may clump together, potentially impairing their effectiveness. Surfactants, which are amphiphilic, can stabilize these NPs by reducing surface tension.¹³ One common surfactant incorporated in the formulation of PLGA NPs is polyvinyl alcohol (PVA). PVA is a water soluble polymeric surfactant that can enable the formation of PLGA NPs with uniform and stable size distribution.¹³

By utilizing PLGA’s inherent properties as a nanocomposite drug delivery system combined with PVA’s stabilizing feature, a nanocomposite system was created. In this study, several different formulations of PLGA without and with PVA were created and incorporated with fluorescein to determine the dissolution rate of PLGA when release study experiments were conducted. These formulations consisted of varying ratios of PLGA without and with PVA (i.e., 25:75, 50:50, and 75:25). Then, a formulation with a steady biphasic drug release profile and a formulation with an increasing release profile were chosen to evaluate antibacterial capabilities of PLGA NP formulations containing the most effective antibiotics (i.e., ciprofloxacin and gentamicin) against pathogenic microbes MRSA, P. aeruginosa and A. baumannii, and measured the zone of inhibition produced by the PLGA NP complex. From a disc-diffusion susceptibility test, it was determined that ciprofloxacin and gentamicin were generally effective antibiotics against the pathogenic microbes tested. In this study, MRSA strain USA300 and P. aeruginosa strain FRD1 exhibited maximum inhibition.

Ultimately, antibiotic-loaded PLGA NP complexes can be incorporated into medical dressings for controlled, sustained release, preventing recurrent infections in chronic wounds. This approach ensures targeted delivery of antibiotics, enhances wound healing, reduces the need for frequent dressing changes, shortens hospital stays, advances point-of-care treatment, and improves patient’s quality of life.

MATERIALS AND METHODS

PLGA 502 H (poly[D, L-Lactide-co-glycolide] Molecular Weight [MW] 7,000-17,000, Sigma-Aldrich, Saint Louis, MO); pharmaceutical grade PVA (MW 31,000-50,000, 98-99% hydrolyzed Sigma-Aldrich, Saint Louis, MO); Tween 20 (purity ≥40% Sigma-Aldrich, Saint Louis, MO); Dichloromethane (anhydrous, purity ≥99.80% Sigma-Aldrich, Saint Louis, MO); 10× phosphate-buffered saline (10×-PBS, Research Products International, Mount Prospect, IL); polycarbonate (PC) membrane filter (Cyclepore Track Etched Membrane, pore size 1.0 µm Cytiva, Marlborough, MA); Dulbecco’s phosphate buffered saline (1×-PBS or DPBS, pH 7.1-7.5, Sigma-Aldrich, Saint Louis, MO); Dichloromethane (anhydrous, with a purity ≥99.80% was obtained from Sigma-Aldrich, Saint Louis, MO); 10×-PBS (was obtained from Research Products International, Mount Prospect, IL); PC membrane filter (Cyclepore Track Etched Membrane, pore size 1.0 µm was obtained from Cytiva, Marlborough, MA); Dulbecco’s phosphate buffered saline (1×-PBS or DPBS, pH 7.1-7.5, was obtained from Sigma-Aldrich, Saint Louis, MO); Rattler Plating Beads (4.5 mm, sterile, ready to use, Research Products International, Mount Prospect, IL); Oxoid Gentamicin Antimicrobial Susceptibility Discs (concentration = 10 µg ThermoFisher Scientific, Waltham, MA); Oxoid Ciprofloxacin Antimicrobial Susceptibility Discs (concentration = 5 µg ThermoFisher Scientific, Waltham, MA); Oxoid Vancomycin Antimicrobial Susceptibility Discs (concentration = 30 µg ThermoFisher Scientific, Waltham, MA); Gentamicin Sulfate powder (bioWORLD, Dublin, OH); Ciprofloxacin powder (purity ≥98.0% Tokyo Chemical Industry, Portland, OR); Water, sterile, nuclease-free (biotechnology grade VWR Life Science, Rad nor, PA); Acetic acid (purity ≥99% Sigma-Aldrich, Saint Louis, MO); Nanopure water (18 MΩ resistivity; ThermoFisher Scientific, Waltham, MA). Preparation of PLGA NPs with Fluorescein for Proxy Release Studies.

Preparation of PLGA NPs Containing Fluorescein

PLGA NPs loaded with fluorescein were prepared by using the double-emulsification solvent evaporation method (W/O/W), with PLGA 502 H as the polymeric system.⁸ In this method, the active component is dissolved in an aqueous phase (W) and emulsified in an organic solvent (O), which creates a primary W/O emulsion. The primary emulsion is then mixed in an emulsifier-containing aqueous solution (W) to create a W/O/W double emulsion.¹⁴ Supplementary Fig. S1A illustrates a schematic of this process.

Briefly, 120 mg of PLGA were dissolved in 4 mL of the organic solvent dichloromethane. About 0.6 mL of a stock fluorescein (0.05 mg/mL) was dissolved in 20 µL of the surfactant Tween 20. The fluorescein solution was added into the PLGA solution to form a primary W/O emulsion by stirring with an ultrasonicator for 10 seconds at an amplitude of 30%. The primary emulsion was slowly poured into a 100 mL beaker with 30 mL of 10×-PBS containing 0.4% w/v PVA, creating a W/O/W double emulsion, and stirred with a propeller in a water bath at 25°C for 30 min and then at 40°C for 1 h. This process synthesized PLGA NPs with PVA. For PLGA NPs without incorporation of PVA, the same procedure was followed, but instead of adding PLGA emulsion to 30 mL of 0.4% PVA solution, 0.14 g of PVA were added into the 100 mL beaker containing 40 mL of the total volume of 10×-PBS.

Using a 1.0 µm PC membrane filter, the solution was filtrated. The filter cake was washed with Nanopure water, and centrifuged 3× at 2,500 rpm for 10 min, dried at 25˚C and stored away from light.

Preparation of PLGA NPs Containing Antibiotics

The same double-emulsion method mentioned above in “Diameter of PLGA NP Formulations (Without PVA:With PVA)” was followed for the synthesis of PLGA NPs loaded with antibiotics (i.e., gentamicin and ciprofloxacin), with the exclusion of adding fluorescein. Instead, 100 µL of 100 mg/mL gentamicin stock in sterile water or 100 µL of 100 mg/mL ciprofloxacin stock in acetic acid were added into the PLGA solutions to form a primary W/O emulsion of PLGA-gentamicin and PLGA-ciprofloxacin, respectively.

Preparation of Varying Ratios of PLGA Formulations for Longitudinal Release Study

PLGA formulations, without and with PVA incorporated, in triplicates were transferred into centrifuge tubes and dispersed in 1.5 mL of DPBS and stored in the incubator at 37°C. The samples were centrifuged at 10,000 rpm for 10 min and at different intervals (1, 3, 7, 9, 24, 46, 71, 98, and 123 or 130 h), 0.5 mL of the supernatant of the samples were extracted and replaced by 0.5 mL of fresh DPBS. The mass formulations used in this experiment are shown in Table I.

Measurement of Absorbance and Calibration Curve

Using a plate reader, the absorbance of the extracted samples was determined at the maximum wavelength of the 0.05 mg/mL stock fluorescein solution in Nanopure water (i.e., 490 nm) and a calibration curve was constructed to determine concentration (mass/volume) of fluorescein recovered from the supernatant of release studies.

Characterization of PLGA NPs’ Size and Morphology

Hydrodynamic radius of PLGA NP formulations was determined by dynamic light scattering using a ZetasizerNanoZs (Malvern Instruments). Scanning electron microscopy (Prisma E-SEM or Helios FESEM) determined dry diameter of NPs. Ten microliters of each dilute PLGA NP solution were applied to a SEM sample stub and sputter coated with 5 nm of iridium before imaging. The samples were then imaged at a magnification of 100,000 times, and a high voltage of 20.00 kV. ImageJ analysis determined the diameter of the NPs formulations.

Zone of Inhibition of Antibiotic Discs

Three different bacteria (i.e., MRSA, P. aeruginosa FRD1, and A. baumannii) were streaked onto individual Luria-Bertani(LB)-agar plates (150 mm petri-dishes), and incubated at 37°C for 18 h with the plates stacked upside down to prevent condensation from interfering with bacteria culture. After 18 h, the streaked plates were removed from the incubator and stored in the refrigerator with the edges parafilmed and stacked upside down.

Then the inoculate was prepared by adding 6 mL of liquid lysogeny broth (LB) and one bacterial colony (from the streaked plates) onto a 10 mL centrifuge tube. This process was repeated for each of the three bacteria. This solution was mixed well and placed into a BT Lab Systems shaker with the lid of the centrifuge tube loosely tightened. The shaker was set to 37°C at a speed of 300 rpm for 12 h.

After 12 h, 50 µL of the respective bacterial inoculate was added to a fresh LB-agar plate, followed by the addition of 16 to 20 Rattler Plating Beads. With the lid closed, the LB-agar plates were agitated to ensure uniform distribution of the inoculate across the LB-agar plate and were left to dry for 2 minutes. After this, the antibiotic discs (i.e., ciprofloxacin, vancomycin, and gentamicin) were placed evenly apart on the LB-agar plates with sterilized tweezers. The plates were then inverted and incubated at 35°C for 24 h.

Following incubation, the diameter of the zone of inhibition, or the area where the bacteria did not grow, was measured in at least triplicate, with a Vernier-caliper for the different antibiotics. This process is known as the Kirby-Bauer disc diffusion susceptibility test, or the zone of inhibition test, which determines the sensitivity or resistance of pathogenic bacteria to various antimicrobial compounds (e.g., antibiotics).¹⁵ Supplementary Fig. S1C illustrates a schematic of this process.

Colony-Forming Units per Milliliter Assay

To count the number of viable cells that proliferate and form bacterial colonies, the three different bacteria used in this experiment (i.e., MRSA, P aeruginosa FRD1, and A baumannii) were plated for colony-forming units (CFU) per milliliter. The OD_600nm of the inoculates, prepared 12 h ago, were measured, followed by 10-fold serial dilution of inoculates and plating of dilutions on LB-agar plates. After incubating it between 12 and 17 h, the serial dilutions that had discrete spots were counted for colonies and used to determine the number of CFU/mL.

LB-Agar Well Diffusion Method

Well diffusion method was followed to determine antibiotic susceptibility.¹⁶ Bacterial inoculates (12 h old), were inoculated (50 µL) on LB-agar plates and spread evenly. The samples prepared included A baumannii, MRSA, and P aeruginosa FRD1. A 6 mm diameter sterile biopsy punches punched wells in LB-agar. These wells received different treatments: 100 µL of either PLGA25 or PLGA50 NP formulations loaded with ciprofloxacin or gentamicin. Additionally, wells containing 50 µL mixtures of both PLGA25 and PLGA50 formulations with either ciprofloxacin or gentamicin were prepared. After incubation at 37°C for 3 days, zones of inhibition were measured using a Vernier-caliper.

RESULTS AND DISCUSSION

Table IA shows the different PLGA NP formulations used in this experiment, namely the varied ratios of PLGA without PVA to PLGA with PVA incorporated. These different PLGA formulations were used throughout this study; first to determine the kinetics release, and later to determine the antibacterial activity when loaded with antibiotics.

Diameter of PLGA NP Formulations (Without PVA:With PVA)

DLS measurements of the PLGA (without PVA:with PVA) NP formulations of the ratios PLGA25, PLGA50, and PLGA75 demonstrate an average hydrodynamic diameter of 387 nm, 410 nm, and 339 nm, respectively (Table IB). SEM images of the PLGA25, PLGA50, and PLGA75 formulations of the PLGA NPs suggest that the particles have a spherical morphology with an average diameter of 100.1 nm, 311.5 nm, and 100.5 nm, respectively (Fig.1A–C, respectively).

Release Kinetics of PLGA NP Formulations (Without PVA:With PVA) Loaded With Fluorescein

The kinetics release profiles of the different PLGA NP formulations (i.e., PLGA25, PLGA50, and PLGA75) are shown in Fig.2. The PLGA25 shows an initial burst release of fluorescein from the PLGA matrix for hours, followed by a steadily increasing fluorescein release until the end of the release study (i.e., 123 h) at 37°C (Fig.2A). The PLGA50 ratio shows an initial rapid release of fluorescein from the PLGA matrix for 9 h, followed by a constant fluorescein release after the 9 h and until the end of the release study at 120 h at 37°C (Fig.2B). Similarly, the PLGA75 shows an initial burst release of fluorescein from the PLGA matrix for 9 h, followed by constant fluorescein thereafter (Fig.2C). However, at around 71 h, the fluorescein signal started to steadily increase and followed a constant release until the end of the release study.

Table IC tabulates the kinetics results for the different PLGA NP formulations. The PLGA25, PLGA50, and PLGA75 had zero order rate constants of 2 × 10⁻⁶, 6 × 10⁻⁷, and 5 × 10⁻⁷, respectively, for the first phase of release. The PLGA25, PLGA50, and PLGA75 had first-order rate constants of 0.18 h⁻¹, 0.16 h⁻¹, and 0.20 h⁻¹, respectively for the second phase of the release profile.

Zone of Inhibition Study for Antibiotic Discs

After 24-h incubation of the MRSA strain USA300, the P. aeruginosa strain FRD1, and A. baumannii BAA1605, at 35°C, with ciprofloxacin, gentamicin, and vancomycin antibiotic discs, zones of inhibition were measured using a Vernier-caliper (Supplementary Fig. S2). From triplicate experiments, MRSA produced zones of inhibition of ciprofloxacin, gentamicin, and vancomycin with average diameters of 10 ± 1.0 mm, 18 ± 0.6 mm, and 15 ± 1.2 mm, respectively. This indicated that MRSA was susceptible to both gentamicin and vancomycin, but resistant to ciprofloxacin. Pseudomonas aeruginosa FRD1 produced zones of inhibition of ciprofloxacin, gentamicin, and vancomycin with average diameters of 45 ± 2.0 mm, 21 ± 1.0 mm, and 0 mm, respectively. This indicated that P aeruginosa FRD1 was susceptible to both ciprofloxacin and gentamicin, but resistant to vancomycin. Acetobacter baumannii BAA1605 produced zones of inhibition of ciprofloxacin, gentamicin, and vancomycin with average diameters of 0 mm, 0 mm, and 9 ± 1.0 mm, respectively, indicating that A. baumannii BAA1605 was resistant to ciprofloxacin, and gentamicin, with minimal response to vancomycin.

A. baumannii was resistant to all three antibiotics; it was chosen as a negative control to demonstrate that the PLGA on its own is not antibacterial. The antibiotic discs of gentamicin and ciprofloxacin were susceptible to P. aeruginosa FRD1, demonstrating promising results to incorporate into the PLGA NP formulations. For MRSA, only gentamicin, and not ciprofloxacin, demonstrated to be susceptible. Nonetheless, both gentamicin and ciprofloxacin were chosen to be incorporated into the PLGA NP formulation to test the efficiency of the antibacterial activity of the PLGA NP formulations loaded with ciprofloxacin and gentamicin.

LB-Agar Well Diffusion Assay for the Determination of Antibacterial Activity of Selected PLGA NP Formulations

Antibacterial activity of PLGA25 and PLGA50 ratios against P aeruginosa FRD1

After the first 24 h, the PLGA25 and PLGA50 ratios of PLGA NP formulations loaded with ciprofloxacin produced zones of inhibition with average diameters of 26 mm and 22 mm, respectively, against P. aeruginosa FRD1 (Fig.3A(iii)). This indicated that P. aeruginosa FRD1 was susceptible to both the formulations. However, the zones of inhibition for the formulations loaded with gentamicin were very faint and not as distinguishable as those produced by ciprofloxacin. This indicated that P. aeruginosa FRD1 was resistant to the gentamicin-loaded formulations, which was contrary to what was expected given that the gentamicin antibiotic disc had shown to be capable of effectively killing P. aeruginosa FRD1. For the set of wells containing the 1:1 mass ratio of mixed PLGA25 and PLGA50, P. aeruginosa FRD1 produced a zone of inhibition for the mix ratio containing ciprofloxacin with an average diameter of approximately 25 mm (Fig.3A(iii)). Thus, demonstrating that P. aeruginosa FRD1 also to be susceptible to this mixed ratio containing ciprofloxacin. Similarly, for the first set of wells, the zone of inhibition of the mixed ratio loaded with gentamicin was very faint and not as distinguishable as that of the mixed ratio loaded with ciprofloxacin. The purpose of the mixed ratio was to demonstrate a controlled release of antibiotics from the PLGA matrix.

For the last day (i.e., day 3), P. aeruginosa FRD1 produced average zones of inhibition of the 25:75 and 50:50 ratio loaded with ciprofloxacin of 27 mm and approximately 23 mm, respectively (Fig.3A(ii, iii)). Thus, demonstrating that P. aeruginosa FRD1 is susceptible to both ciprofloxacin-loaded ratios over a 3-day period. The steady increase in the zones of inhibition of both formulations demonstrates promising results of antimicrobial activity for the treatment of chronic wounds susceptible to recurrent infections. Although the zones of inhibition produced by these formulations are less than those produced by the ciprofloxacin antibiotic discs (i.e., 45 mm), the fact that these PLGA NP formulations were able to inhibit the growth of bacteria shows their potential as drug delivery vehicles. For the 1:1 mass mixed ratio of PLGA25 and PLGA50, the ciprofloxacin-loaded formulation produced an average zone of inhibition of 25 mm (Fig.3A(i, iii)). This indicated that over a 3-day period, the mixed ratio formulation was able to steadily release ciprofloxacin, making P. aeruginosa FRD1 susceptible. Thus, this mixed formulation demonstrates the potential of a controlled release formulation that can effectively inhibit the growth of bacteria. The 1:1 PLGA25:PLGA50 mixture had a 14% increase in inhibition diameter compared to PLGA50 alone but it was still 4% less than the inhibition diameter of PLGA25. This seemed to indicate that a steady biphasic release profile that starts low is most effective against Gram (−) P. aeruginosa FRD1.

Antibacterial activity of PLGA25 and PLGA50 ratios against MRSA

After the first 24 h, MRSA produced zones of inhibition of the PLGA25 and PLGA50 NP formulation loaded with gentamicin with average diameters of 12 mm and 13 mm, respectively (Fig.3B(iii)). This indicated that MRSA was resistant to the PLGA25 ratio and intermediate to the PLGA50 ratio. As expected from the antibiotic disc studies, MRSA was resistant to the formulations loaded with ciprofloxacin. For the 1:1 mass ratio of PLGA25 and PLGA50, only the formulation loaded with gentamicin produced a zone of inhibition. The average diameter of the mixed ratio loaded with gentamicin was 13 mm, indicating that MRSA was intermediate to this formulation (Fig.3B(iii)).

For the last day (day 3), MRSA produced average zones of inhibition of the PLGA25 and PLGA50 ratios loaded with gentamicin of 13 mm and 13 mm, respectively (Fig.3B(ii, iii)). These results demonstrated that by day 3, MRSA was intermediate to both formulations. This was surprising, especially since MRSA had been resistant for the first 2 days to the PLGA25 formulation loaded with gentamicin but was intermediate at the end of the study. Although both ratios produced smaller zones of inhibition than the gentamicin antibiotic discs (i.e., 18 mm), the results obtained demonstrated that the PLGA NP formulations are capable drug delivery vehicles against MRSA. For the mixed formulation loaded with gentamicin, a 14 mm zone of inhibition was produced (Fig.3C(i, iii)). This again indicated that MRSA was intermediate to the mixed formulation loaded with gentamicin. In addition, the mixed formulation loaded with gentamicin slightly outperformed the individual 25:75 and 50:50 ratios loaded with gentamicin which indicated that a mixed formulation may be a more effective antimicrobial formulation against MRSA. Further tests will need to be repeated to obtain a more definitive conclusion. The 1:1 PLGA25:PLGA50 mixture had an ∼ 10% increase in inhibition diameter compared to PLGA50 and PLGA25. This seemed to indicate that a formulation that provided rapid release (PLGA50) mixed with a formulation that provides a steady biphasic release (PLGA25) is most effective against Gram (+) MRSA in stark contrast to Gram (−) P. aeruginosa FRD1.

Antibacterial activity of PLGA25 and PLGA50 ratios against A. baumannii

Over the 3 days, A. baumannii was resistant to all the PLGA NP formulations (i.e., the PLGA25, PLGA50, and 1:1 mass ratio of PLGA25:PLGA50). This is shown in Fig.3C(i, ii). This was expected given that the gentamicin and ciprofloxacin antibiotic discs had not produced effective zones of inhibition. As a result, the A. baumannii plates were used as a negative control group to compare the antibacterial efficacy of the antibiotic loaded PLGA NP formulations against the other two pathogenic bacteria.

CONCLUSIONS

PLGA NP formulations (i.e., PLGA25 and PLGA50) loaded with gentamicin and ciprofloxacin were successfully prepared by the double-emulsification evaporation method. The antibacterial efficacy of these PLGA NP formulations were tested against different MDR pathogens (i.e., the MRSA strain USA300, the P. aeruginosa strain FRD1, and A. baumannii BAA1605) by using the zone of inhibition test. At the end of a 3-day period, the PLGA25 and PLGA50 formulations loaded with ciprofloxacin produced zones of inhibition of 27 mm and 23 mm, respectively, against P. aeruginosa FRD1. Thus, indicating that P aeruginosa FRD1 was susceptible to both formulations. Similarly, for the 1:1 mass ratio of PLGA-25:PLGA50, only the formulation loaded with ciprofloxacin produced an effective zone of inhibition of 25 mm. This mixed ratio was 14% more effective than PLGA50, but 4% less effective than PLGA25 ratio. However, contrary to the results produced from using gentamicin antibiotic discs, P. aeruginosa FRD1 was resistant to both PLGA NP formulations loaded with gentamicin. This might be because gentamicin might need a very high starting diffusion concentration to be effective against Gram (−) P. aeruginosa FRD1, which might not have been possible with our PLGA formulation release kinetics.

The PLGA NP formulations tested against MRSA demonstrated that at the end of the 3-day period, only the gentamicin-loaded formulations produced intermediate results. The 1:1 mass ratio of PLGA-25:PLGA50 combination loaded with gentamicin produced intermediate results against MRSA (i.e., 14 mm) which was consistently 8% to 10% better than the inhibition zone of PLGA25 and the PLGA50 NP formulations loaded with gentamicin.

In conclusion, the results from the A. baumannii BAA1605 testing confirms that any antibacterial activity seen in MRSA and P. aeruginosa FRD1 is from the antibiotics and not the PLGA formulation on its own. A steady biphasic release profile with slow starting antibiotic concentration is most effective in overcoming Gram (−) P. aeruginosa FRD1. Conversely, a formulation that provided rapid release (PLGA50) mixed with a formulation that provides a steady biphasic release (PLGA25) is most effective against Gram (+) MRSA in stark contrast to Gram (−) P aeruginosa FRD1. This study aligns with the goals of the USAMRDC’s Combat Casualty Care Research Program, Military Infection Diseases Research Program, and Military Operational Medicine Research Program, and the U.S. Air Force School of Aerospace Medicine. If successful, this project will provide a unique pathogen agnostic antibacterial for immediate treatment to promote infection control of traumatic complex soft tissue, penetrating torso, and blast injury wounds through rapid hemostasis and proactive antibacterial activity. Despite higher survival rates in recent combat scenarios, infection rates, particularly from MDR bacteria, have risen, with 27% of evacuated injured personnel developing infections, increasing to 50% in ICU patients.¹⁷ Current prevention strategies include narrow-spectrum antibiotics, wound debridement, and irrigation. However, a shift from MDR Gram (−) bacteria to Staphylococcus spp., like S. aureus, has occurred in recurring infections, often resulting from nosocomial transmission, which pose significant health risks and long-term effects, highlighting the need for a broad-spectrum, resistance-preventing antibacterial agent that conforms to wounds. This study has demonstrated the importance of multi-release kinetics formulation in tackling MDR pathogens in a prolonged time period (3 days). This study also lays the foundation for sustained release dressings (e.g., topical creams, wound-conforming fillers, or bandages)^18–20 for prolonged wound care in the field with minimal bandage changes to keep bacterial load low while waiting for medevac. Although we have been able to fine-tune a PLGA NP formulation that gives us favorable release kinetics of the payload, ongoing efforts are being made to improve the loading efficiency of the payload as well.

ACKNOWLEDGMENTS

The Nallathamby lab would like to acknowledge the financial and material support of the Notre Dame Berthiaume Institute for Precision Health, and the Notre Dame Centre for Nano Science and Technology undergraduate summer research fellowships (NURF). We thank the staff of the Materials Characterization Facility, ASEND facility, Freimann Life Sciences Center, technician at the Berthiaume Institute for Precision Health, and the Notre Dame Integrated Imaging Facility for analytical services. Argerie Guevara, and Kevin Armknecht were undergraduate research associates. Argerie Guevara (NURF’21) and Kevin Armknecht (NURF’23) had Notre Dame Centre for Nano Science and Technology undergraduate summer research fellowships.

CLINICAL TRIAL REGISTRATION

Not applicable.

INSTITUTIONAL REVIEW BOARD (HUMAN SUBJECTS)

Not applicable.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

Not applicable.

INDIVIDUAL AUTHOR CONTRIBUTION STATEMENT

Conceptualization: Nallathamby, P.D.; Data curation: Guevara, A., and Nallathamby, P.D.; Formal analysis: Guevara, A., Armknecht, K., Kudary, C., and Nallathamby, P.D.; Funding acquisition: Nallathamby, P.D. Investigation: Guevara, A., Armknecht, K., and Kudary, C. Methodology: Guevara, A., and Nallathamby, P.D.; Project administration: Nallathamby, P.D.; Resources: Nallathamby, P.D.; Supervision: Nallathamby, P.D.; Validation: Nallathamby, P.D.; Visualization: Guevara, A., Armknecht, K., and Nallathamby, P.D.; Writing – original draft: Guevara, A.; Writing –review & editing: Armknecht, K., and Nallathamby, P.D. All authors read and approved the final manuscript.

INSTITUTIONAL CLEARANCE

Not applicable.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Military Medicine online.

FUNDING

The research was supported by a Project Development Team grant from the Indiana Clinical and Translational Sciences Institute (CTSI-PDT: 373037-31005-FY19CTSIK), Institutional Research Grants from the American Cancer Society (ACS IRG-17-182-04, ACS IRG-April 17, 2002, 0182) and seed grants from the Notre Dame Berthiaume Institute for Precision Health (372333-31025 and 372717-43310-FY17RFP).

SUPPLEMENT SPONSORSHIP

This article appears as part of the supplement “Proceedings of the 2023 Military Health System Research Symposium,” sponsored by Assistant Secretary of Defense for Health Affairs.

CONFLICT OF INTEREST STATEMENT

None declared.

DATA AVAILABILITY

The data that support the findings of this study are available on reasonable request from the corresponding author. All data are freely accessible.

REFERENCES

Author notes

Presented as a poster at the 2023 Military Health System Research Symposium, Kissimmee, FL, United States; MHSRS-23-10,092.

The data published in this material are those of the authors and do not reflect the official policy or position of the U.S. Government, the DoD, or the Department of the Army.

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