Modulatory Contribution of Oxygenating Hydrogels and Polyhexamethylene Biguanide on the Antimicrobial Potency of Neutrophil-like Cells

Abstract

Neutrophils are a first line of host defense against infection and utilize a series of oxygen-dependent processes to eliminate pathogens. Accordingly, research suggests that oxygen availability can improve anti-infective mechanisms by promoting the formation of reactive oxygen species. Also, oxygen can synergistically upregulate the antibacterial properties of certain antibiotics against bacteria by altering their metabolism and causing an increase in antibiotic uptake of bacteria. Therefore, understanding the effects of oxygen availability, as provided via a biomaterial treatment alone or along with potent antibacterial agents, on neutrophil functions can lead us to the development of new anti-inflammatory and anti-infective approaches. However, studying neutrophil functions in vitro is often limited by their short life span and non-reproducibility, which suggests the need for cell-line-based models as a substitute for primary neutrophils. Here, we took advantage of the differentiated human leukemia-60 cell line (HL-60) as an in vitro neutrophil model to test the effects of local oxygen and antibacterial delivery by fluorinated methacrylamide chitosan hydrogels (MACF) incorporated with a polyhexamethylene biguanide (PHMB) antibacterial agent. Considering the natural modes of neutrophil actions to combat bacteria, we studied the impact of our dual functioning oxygenating-antibacterial platforms on neutrophil phagocytosis and antibacterial properties as well as the formation of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). Our results demonstrated that supplemental oxygen and antibacterial delivery from MACF-PHMB hydrogel platforms upregulated neutrophil antibacterial properties and ROS production. NET formation by neutrophils upon treatment with MACF and PHMB varied when chemical and biological stimuli were used. Overall, this study presents a model to study immune responses in vitro and lays the foundation for future studies to investigate if similar responses also occur in vivo.

Graphical Abstract

1. Introduction

Neutrophils are among the first and most prominent immune cells to accumulate at an injury site, mainly to protect the host against infection.¹ These cells contribute to bacterial elimination via multiple routes including production of reactive oxygen species (ROS), releasing cytotoxic proteases and antimicrobial peptides, generation of neutrophil extracellular traps (NETs), and phagocytosis.² In a normal wound healing cascade, towards the end of inflammatory phase the remaining neutrophils leave the site of injury, and their apoptotic debris is cleared from the wound site mainly by macrophages.³ This step is crucial to avoid prolonged wound inflammation caused by the presence of cytotoxic enzymes, ROS, and inflammatory signals,¹ which can lead to the formation of a chronic and non-healing wound, if not correctly progressed.

Hypoxia in the wound environment is known to promote persistence of neutrophils at the site and act as a secondary inflammatory signal to attract more neutrophils while upregulating pro-inflammatory cytokine levels at the site of injury.^{4, 5} To counteract the negative effects of hypoxia, delivering oxygen to injuries can alleviate inflammatory conditions by regulating the antibacterial functions of immune cells.⁶ Accordingly, we have focused on utilizing the excellent oxygen delivering properties of perfluorocarbon-based chitosan hydrogels in the form of our previously developed fluorinated methacrylamide chitosan (MACF) polymers to alleviate hypoxic conditions.^7–12 We have previously shown the application of MACF hydrogels as potent oxygenating platforms to improve wound healing responses in rodent and porcine models,^{7, 8, 11, 13} as well as to overcome oxygen transport limitations to enhance cell survival in vitro.^{9, 12, 14}

Clinically speaking, treating infection at a wound site is often achieved using a multimethod approach that most often includes using chemical antibacterial agents. Antibiotics are known to interact with leukocytes,¹⁵ stimulate their intracellular production of ROS,¹⁶ while altering overall oxidative mechanisms.¹⁷ However, there are still controversies as to whether the use of antibiotics promotes or inhibits endogenous antibacterial and inflammatory roles of immune cells. Moreover, the interaction and synergy between oxygen and chemical antibiotics in modulating immune cell functions are still not fully understood.

Direct in vitro application of primary neutrophils to study immune mechanisms is limited due to inadequate resources, donor-to-donor variations, extraction difficulties, and short life span.¹⁸ To address these limitations, the differentiated human leukemia cell line-60 (HL-60) has been used as a suitable and unlimited substitute for neutrophils to study phagocytic and migratory functions.^{18, 19}

In this study, we added additional function to MACF hydrogels by incorporating them with the potent chemical antibacterial agent, polyhexamethylene biguanide (PHMB), to create a dual oxygenating and antibacterial platform. The goal of this study was to understand how neutrophilic antibacterial functions in vitro are influenced by the presence of oxygen, as controlled via our novel MACF hydrogels and PHMB. Given the uncertainties and controversies over the distinct and synergistic roles of oxygen and antibacterial agents, we used differentiated HL-60 (dHL-60) cultures to test the hypothesis that oxygen supplemented via MACF hydrogels alone or along with a potent antibacterial agent, PHMB, could mediate neutrophil antimicrobial activity against Staphylococcus aureus (S. aureus), one of the most common human pathogens.²⁰ The antibacterial functions evaluated in this study include phagocytosis and antibacterial properties, as well as NET- and ROS-production.

2. Experimental Section

2.1. Materials and Methods

2.1.1. Preparation of fluorinated methacrylamide chitosan (MACF)

To make MACF, chitosan (ChitoClear 43010, DDA ~80% M_w = 200 KDa, Primex, Siglufjörður, Iceland) was dissolved in acetic acid (2% v/v) at a final concentration of 3 wt%. Methacrylation was achieved by mixing methacrylic anhydride (Sigma Aldrich, St. Louis, MO) with the chitosan solution at an appropriate ratio as previously described.^{7, 10, 11} The resulting methacrylamide chitosan was subjected to fluorination via a mixture of methanol and pentadecafluorooctanoyl chloride (PFOC, Sigma Aldrich) added dropwise. The reaction was allowed to proceed for 48 hours, followed by dialysis (12–14 KDa MWCO), and freeze-drying (Labconco, Kansas City, MO) to obtain MACF.

To confirm the degree of methacrylation as well as the concentration of fluorines in MACF, quantitative proton and fluorine nuclear magnetic resonance (¹H-NMR and ¹⁹F-NMR, Varian 500 MHz) was used on 3% (v/v) samples dissolved in deuterated acetic acid/D₂O at a final concentration of 1 wt%. Equation 1 was used to determine the degree of methacrylation using quantitative ¹H-NMR, as previously described.²¹

$$\text{DM}(\%)=3\times \left(\frac{{\text{I}}_{5.6}{+\text{I}}_{6.0}}{{\text{I}}_{2.8-4.0}}\right)\times 100$$ (1)

where I_x is the integral value for the corresponding peak in the NMR spectrum.

The concentration of fluorines per ml of MACF was also determined via quantitative ¹⁹F-NMR analysis with deuterated trifluoroacetic acid (d-TFA, Sigma Aldrich) as an internal standard (1 nM), according to equation 2.

$${\text{C}}_{\text{fluorine}}=\frac{{\text{I}}_{\text{fluorine}}}{{\text{I}}_{\text{internal}\text{standard}}}\times \frac{{\text{N}}_{\text{internal}\text{standard}}}{{\text{I}}_{\text{fluorine}}}{\times \text{C}}_{\text{internal}\text{standard}}$$ (2)

where C_x is the concentration of the compound x (μM), I_x is the sum of peak integral values related to the compound and N is the number of nuclei for the compound of interest,²² as we have previously reported.¹²

2.1.2. Preparation of MACF and MACF-PHMB hydrogels

MACF was dissolved in ultrapure water (18 MΩ, Millipore, Billerica, MA, USA) at a final concentration of 3 wt% and sterilized via autoclaving. 1-hydroxycyclohexyl phenyl ketone dissolved in 1-vinyl-2-pyrolidone (both Sigma Aldrich) was used as a photoinitiator at a ratio of (0.9 mg/g of polymer). The solutions were mixed and degassed using a speed mixer (DAC 150 FVZ, Hauschild Engineering, Hamm, Germany) at 3000 rpm for 2 minutes, followed by UV-crosslinking for 4 minutes at room temperature under sterile conditions. All procedures were the same for the MACF-PHMB hydrogels except that the polymer solutions were mixed with PHMB active agent (Musechem, Fairfield, NJ) before adding the photoinitiator. Sterile biopsy punches were used to cut the gels into cylinders (6 mm diameter, 4 mm height) to use for all experiments.

2.1.3. Release kinetics of PHMB from the MACF substrate

15 ml standard Franz diffusion cells (PermeGear, Hellertown, PA, USA) were used to conduct the release studies and to compare the release kinetics of PHMB inside MACF hydrogels. As previously described,²³ a single layer of a dialysis membrane (12–14 KDa MWCO, Spectrum Labs, Rancho Dominguez, CA) was placed in-between the donor and receptor chambers (filled with phosphate-buffered saline, PBS) and sealed. The hydrogels with a diameter of 6 mm and thickness of 4 mm were placed inside the donor chamber and sealed to avoid evaporation. The system was kept inside a 37 °C incubator while mixing with a stir bar at 80 rpm to maintain the sink conditions. At predetermined time intervals, the entire chamber media was discarded, sampled for measurement, and replaced with 15 ml of fresh PBS. Samples were then subjected to UV absorbance measurement at 235 nm and the concentration of PHMB was calculated using a PHMB standard curve at the same wavelength to evaluate the cumulative release behavior of PHMB. Furthermore, to investigate the dissolution mechanism through which PHMB is released from the MACF-PHMB hydrogel composite, various models were fit to the cumulative dissolution curves. The model parameters and coefficient of determination (R²) were determined using R software (R Core Team and the R Foundation for Statistical Computing, Vienna, Austria) as previously described.²⁴

2.1.4. Continuous oxygen partial pressure measurement from MACF hydrogels

Fiber optic oxygen sensor dots (OXF50 Firesting O2 (Pyroscience, Aachen, Germany) were used to measure the continuous oxygen partial pression in aqueous media with and without MACF hydrogels. MACF hydrogels were prepared as described in 2.1.2. Briefly, the sensor dots were attached to the bottom of a sterile 24 well cell culture plate as per the manufacturer’s protocol. The MACF hydrogels were then placed inside the wells containing 1 ml of sterile PBS. A group of PBS control was considered to compare with the readings from MACF hydrogels. The sensors were then connected to the oxygen meter (FireSting-O2, Pyroscience) and calibrated. Oxygen partial pressure was continuously recorded for 72 hours at 37°C in an enclosed incubator for each group.

2.1.5. HL-60s, culture, and differentiation

HL-60 cells (ATCC CCL-240, Manassas, VA) were grown in Roswell Park Memorial Institute medium (RPMI-1640, Thermofisher Scientific, Waltham, MA) supplemented 25 mM HEPES and L-glutamine, 20% heat-inactivated fetal bovine serum (FBS, Sigma Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO₂.²⁵ The cells were maintained at a concentration between 1 × 10⁵ and 10⁶ cells/ml with medium renewal every 2–3 days, as suggested by the supplier. All cells used throughout the studies were between passage numbers 10–25.

Differentiation to neutrophils was induced to HL-60s by the addition of 176 mM dimethyl sulfoxide (Sigma Aldrich) to the media for 5–7 days while monitoring the cell viability via trypan blue exclusion every 2 days to ensure that the cells have ceased proliferation to indicate the start of differentiation phase.²⁶ Upon differentiation to neutrophil-like cells (dHL-60s), HL-60 cells tend to shrink followed by a decrease in their nucleus to cytoplasm ratio. The nuclear morphology is expected to change from a normal round to a more segmented shape, indicative of granulocytic lineages. To confirm changes in nuclei morphology to a granulocytic lineage, dHL-60s were resuspended at a concentration of 2 × 10⁵ cells/ml in PBS. 250 μl of the cell suspension was placed into the Cytospin setup consisting of a glass slide, filter card, and funnel (Simport Scientific CytoSep Funnels for Shandon Cytospin, Fisher Scientific) and was spun down via a cytocentrifuge (Thermo Scientific) for 5 minutes at 125 ×g. The cells were then fixed on the slide using 4% freshly made paraformaldehyde solution (PFA) and stained using the Diff-Quik staining kit (Siemens Healthineers, Erlangen, Germany).

To further investigate the efficiency of the differentiation method via DMSO, flow cytometry was used to confirm the presence of CD11b, as a neutrophil-specific surface marker in the majority of the dHL-60 population. Two experimental groups were considered: HL-60s and dHL-60s differentiated for 5 days. Cells were resuspended in the flow staining buffer comprised of 2% FBS and 50 mg of sodium azide in 100 ml of PBS (5 × 10⁶ cells/ml). FITC-conjugated primary monoclonal anti-CD11b antibody (2.5 μg/ml, Sigma Aldrich) with the corresponding isotype control (igG2bk), as a negative control, were used to label the HL-60s both before and after differentiation. Samples were then rinsed twice with cold staining buffer to remove the unbound dye residues followed by centrifugation at 350 ×g for 5 minutes. An unstained control group was also included in this experiment as a control for the possible autofluorescence associated with HL-60s and dHL-60s. A flow cytometer (BD Accuri C6, Ann Arbor, MI) was used to detect the number of CD11b+ cells among the population with 50,000 events per session. BD Accuri C6 Plus software was used to analyze the data.

2.1.6. Impact of oxygenation on the phagocytic behavior of neutrophil-like cells

The effect of oxygenation by MACF-based hydrogels on the phagocytic activity of dHL-60s was studied via co-culturing dHL-60s with Staphylococcus aureus (Wood strain without protein A) BioParticles conjugated with Alexa Fluor^™ 488 (Invitrogen, Waltham, MA). Phagocytosis, as one of the antibacterial modes of action for neutrophils, is defined as the cellular uptake of small pathogens.²⁷ This mechanism can be assessed through the detection of the neutrophils that are bound to the bacteria in a direct-contact culture.²⁸ Briefly, sterile MACF hydrogels were used in both their non-oxygenated state (MACF-atm) and as supersaturated with medical-grade oxygen (MACF-O₂) under sterile conditions for 30 minutes, immediately before being in contact with dHL-60s. The non-oxygenated MACF hydrogels are still able to absorb oxygen from the atmosphere due to the presence of PFCs.⁹

A non-treated 24 well plate was used for all the experiments. To prime the neutrophils with oxygen, the gels were placed on well-inserts on top of the wells filled with 0.5 ml of dHL-60 cell suspension in PRIM medium supplemented with 2% FBS (heat-inactivated at 70 °C) and incubated on a rocker shaker at 50 rpm for 5 hours. This would ensure the direct contact between the hydrogels and cells to deliver higher concentrations of oxygen to the media containing cells.

Staphylococcus aureus (S. aureus) bioparticles were resuspended at a concentration of 2 × 10⁶ cell/ml in 2mM sodium azide solution in PBS and sonicated using an ultrasonic water bath, for 4 cycles of 20 seconds at 35 kHz as per manufacturer’s recommendation to ensure homogeneous dispersion right before use. After 5 hours of incubation with the hydrogels, dHL-60s and S. aureus bioparticles were co-incubated at a multiplicity of infection (MOI) of 3 for 30 minutes at 37 °C. The cell-bacteria suspension was then washed with ice-cold PBS twice to remove the unbound bioparticles via centrifugation at 125 ×g for 5 minutes and was subjected to flow cytometric measurements as previously described.²⁸ Oxygen-saturated and non-oxygenated MACF hydrogels with cells and bioparticles were used as treatment groups (in triplicate). A group with bioparticles-only and another group with dHL-60s-only served as our controls. After removing the signal associated with the remaining unbound bioparticles, the mean FITC fluorescent intensity in all groups was considered to be from the cells bound to bacteria and thus interpreted as phagocytosis.

2.1.7. Impact of oxygenation and antibiotic delivery on the antibacterial activity of neutrophil-like cells

To determine the antibacterial ability of the dHL-60s, cells were co-cultured with S. aureus (Staphylococcus aureus subsp. aureus Rosenbach (ATCC 25904)). Liquid cultures of S. aureus were prepared via inoculating a single colony of bacteria into sterile Todd-Hewitt broth (THB, Becton, Dickinson and Company, NJ) and allowed to cultivate overnight at 37 °C while shaking at 85 rpm. The optical density (OD) of the inoculum at 600 nm was adjusted to 0.5 which had a known number of colony-forming units (cfus) of approximately 10⁹ cfu/ml, as determined by dilution plate counts. The number of bacteria in the inoculum with the OD₆₀₀ of 0.5 was further adjusted to 2 × 10⁶ cfu/ml in RPMI medium to match that of dHL-60s. In all experiments, before co-culturing with bacteria, dHL-60s were pre-stimulated with phorbol 12-myristate 13-acetate (PMA, 160 nM, Sigma Aldrich) for 30 minutes before getting in contact with bacteria.¹⁹ PMA activates neutrophils via the protein kinase C (PKC) family of enzymes, that are directly responsible for the activation of NADPH oxidase and ROS production.²⁹ In the first set of studies, dHL-60s were primed with oxygen via MACF hydrogels for 5 hours in a plain RPMI medium to study the changes in neutrophil antibacterial behavior caused by oxygen. After the activation of dHL-60s with PMA, bacteria were added to dHL-60s, and the microplates were centrifuged to ensure full contact of cells with bacteria. The MOI and conditions used here were the same as in the phagocytosis studies (MOI = 3).

Next, to investigate the changes in the antibacterial behavior of neutrophils in response to a chemical antibiotic, experiments were performed in the presence of two different concentrations of PHMB in the media. To determine the minimum inhibitory concentration (MIC) of PHMB against S. aureus, known concentrations of PHMB were dissolved in broth when the inoculum was made. The cultures were allowed to grow overnight at 37°C while shaking at 60 rpm and serial dilutions were plated on the surface of agar plates. The smallest concentration in which no colony growth was observed was considered as the MIC value. For our experiments, concentrations were chosen that were lower than the minimum inhibitory concentration value of PHMB against S. aureus, to prevent total bacteria elimination caused by only PHMB. PHMB was dissolved directly in the plain RPMI media before adding the cells, and the PHMB incubation time with dHL-60s was set to 30 minutes at 37 °C while gently shaking on a 2D rocking shaker, after which the bacteria were added to the system. To capture the effects caused by both PHMB and dHL-60s, groups were included with PHMB-only (no cells) in both PHMB concentration as well as cell-only (no PHMB). A group with bacteria only was selected as the negative control. Finally, to investigate the synergy between PHMB and oxygen on neutrophil antibacterial activity, similar experiments were repeated with MACF-PHMB gels to introduce oxygenation and antibiotic delivery simultaneously.

All dHL-60-S. aureus cultures were incubated overnight at 37 °C while gently shaking at 60 rpm. After the incubation period, serial dilutions of all wells were plated on Todd-Hewitt agar plates for viable counts and comparison between the groups. All conditions were analyzed in triplicate and at least 3 dilutions per replicate were counted and averaged. Results were determined as the fold change in bacterial growth compared to the no-cell control (bacteria-only) per treatment group.

2.1.8. Effect of oxygenation and antibiotic delivery on the production of ROS by neutrophil-like cells

The production of ROS is among the most powerful weapons used by neutrophils to combat bacterial infections.¹ Therefore, the effect of oxygenation and antibiotic delivery via our MACF-based hydrogels on the capacity of neutrophils to produce ROS was studied via a cell-based dihydroethidium assay kit (DHE assay, Cayman, Ann Arbor, MI). Briefly, after the priming period of the dHL-60s (5 hours) with oxygenating MACF, soluble PHMB, and MACF-PHMB treatments, the treated dHL-60 suspension was added to a v-bottom well plate. Separate wells were designated to positive and negative assay controls, antimycin A and N-acetyl cysteine, respectively, as per the manufacturer’s protocol. Cells were pelleted down via centrifugation at 400 ×g for 5 minutes and the media was removed without disrupting the pellet. Then, the cells were washed with 150 μl of the cell-based assay buffer before re-centrifugation. Upon removal of the assay buffer, 130 μl of ROS staining buffer was added to each well. Next, N-acetyl cysteine reagent was added to the designated negative control assay wells and incubated at 37 °C for 30 minutes while protected from light, followed by an additional 1-hour incubation with antimycin A reagent to the positive control assay wells. The centrifugation was repeated, and the ROS staining buffer was switched with assay buffer. The cells were then transferred to a black well plate to measure their fluorescence intensity at the wavelengths indicated by the manufacturer via a top-read microplate reader (Tecan, Männedorf, Switzerland). Results were analyzed as the amount of DHE fluorescent corresponding to ROS formation in each group. All experiments were repeated three times to ensure reproducibility of the results.

2.1.9. Effect of oxygenation and antibiotic delivery on the production of neutrophil extracellular traps (NETs)

NETs are mainly composed of extracellular DNA fibers and antimicrobial proteins that entrap pathogens in their web-like structures and expose them to lethal concentrations of antimicrobial peptides.³⁰ Here, to investigate whether oxygenation from MACF alone and in conjugation with PHMB affects the NET formation capacity of dHL-60s, fluorescence-based Quant-iT^™ PicoGreen^™ assay (Invitrogen) was used to quantify the amount of extracellular DNA in each group both in the presence and absence of S. aureus pathogen. Briefly, cells were primed with oxygen from supersaturated MACF and MACF-PHMB hydrogels for 5 hours at the concentration of 2 × 10⁶ cells/ml. With the treatments still in contact with the cells, they were stimulated with 160 nM of PMA for 1.5 hours at 37 °C culture conditions. Following the oxygen priming and stimulation period, for the groups that did get the bacteria, the cells were co-incubated with S. aureus for 4 hours at an MOI of 3. The suspension was centrifuged at 472 ×g for 5 minutes to ensure uniform contact of the cells and bacteria immediately after the bacteria was introduced to the cell suspension. At the end of the co-incubation period, 150 μl of the supernatant was removed and subjected to PicoGreen assay (with a 10X dilution using the assay buffer) as per manufacturer’s protocol and the double stranded DNA (dsDNA) concentration was determined using the prepared standard curves. The results were shown as the amount of dsDNA per group as compared to the no-treatment controls. To confirm that the DNA detected was merely extracellular, the remaining samples were digested using cell lysis buffer (ThermoFisher) while mixing every 6 hours for 24 hours followed by a freeze-thaw cycle at 20 °C to ensure the full recovery of nuclear DNA content and quantified using PicoGreen assay.

2.1.10. Statistical analyses

All data were analyzed using GraphPad Prism 5.0 or JMP Pro 16.0 statistical software. Data are presented as mean ± standard deviation (S.D). p-values less than 0.05 were considered statistically significant and analyzed using one-way ANOVA with Tukey’s post-hoc analysis. In figures where letters are used, groups sharing the same letters are not significantly different. Groups with the highest mean values are indicated with the letter “A” and the rest of the groups are ranked alphabetically based on their mean values.

2.2. Results

2.2.1. MACF preparation and characterization

MACF possesses outstanding oxygen dissolving and carrying properties owing to its perfluorocarbons side chains, which provides an excellent platform for this study^{7, 8, 10, 11, 13} (Figure 1A). The degree of methacrylation and fluorination of MACF was characterized by quantitative proton and fluorine nuclear NMR, respectively, as shown in Figure 1B. Using these data, the degree of methacrylation was calculated to be 26.28 ± 1%, which confirms the successful substitution of the amine hydrogens from the chitosan backbone with methacrylic anhydride (Figure 1B). From ¹⁹F NMR, the concentration of fluorines incorporated within the MACF chains was calculated to be 87.2 μM which agrees with our previous reports to induce the required oxygen carrying properties (Figure 1).^{11, 12}

Figure 1.

(A) Synthesis process of MACF. (B) ¹H-NMR spectrum for MACF to determine degree of methacrylation (Left). ¹⁹F-NMR spectrum for MACF to determine degree of fluorination (Right) with d-TFA (1 nM) used as internal standard for quantifying concentration of fluorine. (C) In vitro release profile of PHMB from MACF hydrogel substrates. Data are shown as mean ± S.D. (n = 3). Average total mass of initially encapsulated PHMB is 9.14 mg. (D) Kinetic release variables derived from various mathematical models to describe PHMB release from MACF-PHMB hydrogels.

2.2.2. PHMB release kinetics from the MACF-PHMB hydrogels

Photo-crosslinked MACF hydrogels incorporated with PHMB were used as a dual-functioning system to simultaneously offer oxygen delivery and antibacterial properties to dHL-60s. To understand the release behavior of the MACF-PHMB hydrogels, a 7-day in vitro release study was carried out using Franz standard cells with PBS as the release media (Figure 1C). Within the first 30 minutes of the study, 20.2 ± 5.2% of the encapsulated PHMB was released in a burst, which was followed by a slow-release phase that further continued for 51 hours. From 51 to 171 hours (days 2–7 of the study) the changes in the amount of released PHMB were negligible (< 5%) and thus this phase was considered as the equilibrium. Figure 1D shows the results of fitting various dissolution kinetic models to the cumulative release data of PHMB from MACF-PHMB hydrogels. Based on these results the Weibull model with lag time (R² value of 99%) best describes the PHMB dissolution behavior from our MACF-PHMB substrates.

2.2.3. Measurement of oxygen partial pressure from MACF hydrogels

Oxygen tensions in PBS with or without MACF hydrogels were measured using a fiberoptic oxygen sensor inside cell culture plates incubated at 37 °C. Our results in Figure 2 showed the ability of MACF hydrogels to increase and then maintain an oxygen partial pressure (P_O2) of 174.6 ± 6 mm Hg after 24 h which was on average 23.4 mmHg higher as compared to PBS control (p < 0.0001), confirming the inherent ability of MACF hydrogels to attract and dissolve molecular oxygen to enhance the local oxygen levels in an aqueous buffer.

Figure 2.

(A) Illustration of the carbon-fluorine polarized bonding and electronegativity of fluorine atoms; (B) Oxygen dissolution capacity of PFCs vs. water³¹. (C) Chemical structure of MACF with PFC oxygen carrying units. (D) Hypothesized PFC agglomeration upon MACF hydrogel formation in aqueous conditions (~97–98% water) and the creation of oxygen-rich regions that dissolve/transfer oxygen mutually along with the atmospheric oxygen absorption (E) Continuous Oxygen partial pressure in PBS with and without MACF hydrogels measured over time (average of 3 measurements shown). Figure partially reproduced with permission from³².

2.2.4. Confirming HL-60 differentiation to neutrophil-like cells

To evaluate the DMSO-mediated granulocytic differentiation of HL-60s to neutrophil-like cells, a growth curve of dHL-60s was created and compared with control HL-60s (non-DMSO-treated) via counting the number of viable cells for 7 days. As shown in Figure 3A, the growth of dHL-60s was inhibited by the presence of DMSO starting from day 2 in culture and did not increase from day 3 onward. Conversely, HL-60s continued to proliferate until day 7 when they were ready for passage. Overall, on day 7, the mean number of viable cells in the dHL-60 group was 70.20 ± 4.03% less than that of the HL-60 population, which suggests the effective DMSO-mediated differentiation of dHL-60s.

Figure 3.

Successful differentiation of HL-60s to neutrophil-like cells (dHL-60s) upon exposure to DMSO. (A) Growth curve of HL-60s before and after differentiation for 7 days. Data mean ± S.D. (n = 3) (B) Diff-Quik staining of HL-60 cells before DMSO-mediated differentiation as well as days 5 and 7 after exposure to DMSO with changed nuclei morphology. Scale bars are 200 μm. (C) Flow cytometric analysis of CD11b+ population of HL-60s vs. dHL-60s.

The nuclear morphology changes of HL-60s when differentiated to neutrophils were assessed via Diff-Quik staining on days 5 and 7 after the addition of DMSO. As shown in Figure 3B, the ovoid nuclear morphology of HL-60s changed to lobulated and multilobed nuclei in dHL-60s. The DMSO-treated cells on day 5 showed a more uniform nuclear and membrane morphology as compared to day 7. Moreover, day 7 cells showed blebbing in cytoplasmic morphology, which could indicate that the cells are undergoing apoptosis.³³ Therefore, dHL-60s differentiated for 5 days with multilobed nuclei and more intact membrane were chosen for all other experiments moving forward.

Flow cytometry was next used to confirm granulocytic DMSO-mediated differentiation of HL-60 cells via measuring the number of the cells that were positive for the CD11b surface marker both before and after differentiation. Figure 3C shows the increased expression of CD11b by 4.03-fold in the DMSO-treated cells on day 5 compared to the vehicle control. In order to remove the effect of autofluorescence associated with HL-60s alone (both before and after differentiation), the signals from the unstained HL-60 and dHL-60 cell population were eliminated from the entire population as shown in Figure 3. The mean fluorescence values for the isotype controls were similar to that of the unstained HL-60s (data not shown).

2.2.5. Effect of oxygenation on the phagocytic activity of neutrophils

To test whether the presence of oxygen can affect neutrophil phagocytic behavior, Alexa Fluor 488-labeled S. aureus wood strain bioparticles were co-cultured with the cells at an MOI of 3. Two different levels of MACF treatment were used to introduce oxygen to the cells: 1. MACF equilibrated at atmospheric oxygen concentration (MACF-atm); 2. MACF supersaturated with pure medical-grade oxygen for 30 minutes before application (MACF + O₂). In both groups, MACF was in contact with dHL-60s using a culture well insert to prime the cells with oxygen for 5 hours after which time the bioparticles were introduced to the dHL-60s. After extensive washing steps to remove unbound bioparticles, FITC fluorescence was detected as a marker for phagocytosis using flow cytometry (Figure 4). Controls of dHL-60s-only and bioparticles-only were included to measure the autofluorescence from the dHL-60s and confirm the presence of FITC tags on the bioparticles (Figure 4A). Accordingly, to ensure that the collected signal is merely from the dHL-60s that are bound with bioparticles, the FITC autofluorescence from dHL-60 cells as well as the fluorescence associated with the bioparticles were subtracted from all the values via gating through BD Accuri software (Figure 4B).

Figure 4.

Effect of oxygenation via MACF-atm and MACF-O₂ hydrogels on the phagocytic activity of neutrophils. (A) Fluorescence histogram of dHL-60s autofluorescence vs. S. aureus bioparticles and the FITC-positive population of bioparticles and dHL-60s (B) Flow cytometric analysis of the number of phagocytic dHL-60s (negative control, MACF-treated and treated with MACF + O₂) (C) Mean fluorescent intensity as an index for neutrophil phagocytosis when primed with oxygen for 5 hours (MACF vs. MACF + O₂ compared to negative control). Data all mean ± S.D (n =3). One-way ANOVA with Tukey’s post-hoc analysis (ns: non-significant, p > 0.05).

Results from the mean FITC signals from the bioparticles conjugated with the dHL-60s in different groups, showed that no significant differences were detected in bacterial uptake when cells were in contact with oxygen from MACF-atm or MACF + O₂ (p > 0.05). Since no differences were detected in phagocytic capacity of neutrophils when MACF-atm or MACF + O₂ were used as treatments, the following experiments were carried out using the supersaturated MACF only (MACF + O₂) to ensure using the full oxygen dissolution capacity of MACF.

2.2.6. Impact of oxygenation and antibiotic delivery on the regulation of the antibacterial properties of dHL-60s

To investigate whether oxygen delivery from MACF and/or the PHMB antibacterial agent have discrete or synergistic effects on the capacity of neutrophils to kill bacteria, we co-incubated S. aureus Rosenbach with dHL-60s overnight in the presence of different oxygenating and antibacterial treatments (Figure 5). Before infection, dHL-60s were activated with PMA to induce phagocytosis (160 nM). The results are derived from the direct bacterial colony counts of the serial dilutions of each treatment group plated on the surface of Todd-Hewitt agar and shown as the fold change in the number of viable bacteria per treatment group as compared to the negative control (bacteria-only). Figure 5 shows the antibacterial capacity of dHL-60s in the presence of two different PHMB concentrations. As a result of the MIC determination experiment, all concentrations between 10 μM and 50 μM (namely 10, 20, 30 and 40 μM) showed visible bacterial colonies on agar surfaces, while PHMB concentrations equal to or above than 50 μM resulted in total bacterial elimination. Therefore, for this experiment the 10 μM PHMB concentration was selected to be less than the MIC of PHMB against S. aureus (i.e., 50 μM). Yet, to confirm the total killing capacity of PHMB at the MIC, the 50 μM PHMB concentration was also included in the study. As expected, the presence of dHL-60s and PHMB significantly reduced the bacterial load in all treatment groups. There was no growth detected in the groups where cells and soluble PHMB were used together (Figure 5). Moreover, oxygenated MACF hydrogels alone were able to significantly decrease the number of viable bacteria regardless of the presence of HL-60s (Figure 5). This effect was enhanced by the presence of dHL-60s in culture with oxygenating MACF hydrogels and S. aureus, where an even lower number of bacteria survived in the MACF + dHL-60s group (p < 0.05). To study the synergistic effects of oxygen and PHMB, MACF hydrogels were incorporated with 10 μM PHMB and used prime the cells with oxygen and PHMB similar to all previous treatments. As expected, MACF-PHMB hydrogels exhibited total killing capacity (zero growth on culture plates) against S. aureus when used with dHL-60s (Figure 5). MACF-PHMB hydrogels exhibited significantly reduced bacterial growth when used with dHL-60s when compared to the antibacterial properties of dHL-60s alone (p = 0.003).

Figure 5.

(A) Synergistic impacts of oxygenation and PHMB antibacterial agent on the antibacterial properties of neutrophils against S. aureus. Data all mean ± S.D. (n = 3). Letters denote significance by one-way ANOVA with Tukey’s post hoc analysis (p = 0.0001). Groups with same letters are not significantly different (p > 0.05). (B) Numerical data of each experimental group to highlight impacts of oxygenation and PHMB antibacterial agent on the antibacterial properties of neutrophils against S. aureus.

2.2.7. Effect of oxygen and antibiotic delivery on the production of ROS by neutrophils

The presence of an antibiotic agent is often known to promote the formation of ROS which plays a role in cell/pathogen death.³⁴ Oxygen is also involved in the regulation and formation of ROS by NADPH-oxidase.³⁵ Therefore, the discrete and synergistic effects of supplemental oxygen and PHMB were next quantified using the ROS-sensitive fluorescent dihydroethidium (DHE) dye after pre-stimulation of the cells with PMA. The results from Figure 6 show a slight trend of reduced ROS formation when the cells experienced oxygen delivery from MACF hydrogels, although this effect did not reach statistical significance (p = 0.405 dHL-60s vs. MACF + dHL-60s). The drug-induced oxidate stress was significantly increased in the groups with PHMB and caused more ROS formation in both soluble PHMB and MACF-PHMB groups as compared to dHL-60s alone (p = 0.042 and 0.01, respectively). Interestingly, MACF-PHMB hydrogels produced the highest amounts of oxygen radicals among all groups in good correlation with their distinctly high antibacterial activity. Even though the presence of MACF did not significantly change the ROS production patterns of dHL-60s, these results indicate that the presence of PHMB can significantly increase the ROS levels resulting in creating inflammatory responses, whereas the oxygen from MACF did not trigger such effects alone or in conjugation with PHMB.

Figure 6.

Effect of oxygenation and antibiotic presence on the production of ROS by neutrophils. Data all mean ± SD (n = 3). Statistical significance by one-way ANOVA (p = 0.001) with Tukey’s post hoc test (p = 0.001).

2.2.8. Effect of oxygen and antibiotic delivery on the production of NETs by neutrophils

The formation of extracellular traps are important mechanisms neutrophils use to entrap and deactivate pathogens and are mostly comprised of DNA, histones, and antimicrobial peptides.²⁸ Here, we quantified the formation of NETs by dHL-60s via measuring the dsDNA content using PicoGreen assay after the dHL-60s were stimulated with PMA both with and without the biological stimuli (S. aureus). To collect NETs, cells were co-incubated with PMA and bacteria after the oxygen priming period of 5 hours via MACF hydrogels. Sterile well inserts were used to hold the gels in place only to prime the cells with the oxygen from the MACF hydrogels and/or PHMB in the case of MACF-PHMB hydrogels. Upon introducing the bacteria to the dHL-60s culture, the hydrogels were removed to ensure no bacteria, cells, or NETs are absorbed into the hydrogel surfaces. Next, without permeabilizing the cells, the supernatant from each group was collected and subjected to PicoGreen assay.

Figure 7A shows the NET formation capacity of dHL-60s in response to the PMA chemical stimulation in the presence of oxygen from MACF hydrogels and the PHMB antibacterial agent. Consistent with the results for ROS formation, MACF-PHMB hydrogels triggered the highest amount of NET formation among the groups (p = 0.0001). However, oxygenation from MACF significantly reduced the amount of extracellular dsDNA by 42% (p = 0.0001) as compared to the negative control (dHL-60s).

Figure 7.

Assessment of NET production via PicoGreen assay in the co-presence of oxygen and antibacterial agent in the absence (A) and presence (B) of S. aureus pathogen. Data all mean ± S.D. (n = 3). Statistical significance by one-way ANOVA (p = 0.0001 for (A), p = 0.002 for (B)) with Tukey’s post hoc analysis (p < 0.05).

The observed trends were different when the neutrophils experienced biological stimulation from S. aureus (Figure 7B) in addition to the chemical stimulus. After 4 hours of incubation with S. aureus, the maximum NET formation was observed in the MACF-treated group (p = 0.001 compared to negative control). The results further revealed that with S. aureus present, MACF-PHMB hydrogels triggered less NET formation when compared to the MACF-treated group (p = 0.017), although still significantly higher than the negative control (p = 0.001). It is worth mentioning that since the NET formation from PHMB is mainly related to its ROS-inducing abilities, the NET formation from soluble PHMB would have most probably been the same as that of MACF-PHMB hydrogels. Therefore, we did not consider adding the soluble PHMB + dHL-60s group to the NET production experiment.

To ensure that the dsDNA is specific to NETs and not from intact dHL-60 nuclei, the remaining cells from all groups were digested using lysis buffer and subjected to PicoGreen assay. No significant differences were discovered in the DNA content of all, confirming similar cell numbers in each sample (p > 0.05). Importantly, these data verify that the DNA detected in Figure 7 was specifically from the NETs. Importantly, to ensure that trends are specific to NETs in Figure 7B, the total DNA content from S. aureus itself was measured using a similar PicoGreen assay, by considering a bacteria-only group with the same bacterial numbers as included in other experimental groups with bacteria. This DNA content was then subtracted from all groups containing bacteria to ensure that the values shown are only related to the DNA collected from the cells (i.e., NETs) and not the bacterial DNA.

2.3. Discussion

Neutrophils as the first line of innate immune defense, and pursue their anti-infective functions through several mechanisms including the production of toxic substances and ROS, phagocytosis, and formation of NETs.³⁶ These mechanisms are mediated by several interconnected pathways and signals, including oxygen abundance and antibiotic therapies, which facilitate the antibacterial functions of neutrophils.⁴ Accordingly, to simultaneously deliver both oxygen and an antibiotic, we designed a dual oxygenating and antibacterial hydrogel by incorporating PHMB inside MACF hydrogels. Obtaining direct measurements of oxygen sensed by cells faces several challenges such as a lack of accurate techniques/sensors to measure in vitro oxygen levels. Despite these challenges, our group has previously attempted to directly measure oxygen tensions resulting from our fluorinated chitosan materials in an aqueous buffers using 2D oxygen sensors integrating luminescent indicators. These data demonstrated that our hydrogels are able to attract and dissolve molecular oxygen to enhance the local oxygen levels in an aqueous buffered cell cultures^{9, 12}, which we again confirmed with our newest data (Fig, 2E). Additionally, due to the presence of oxygen-rich domains within MACF hydrogels, they can be seen as oxygen transport enhancers that are constantly re-absorbing and re-dissolving oxygen in the culture to benefit metabolically active cells, rather than simply serving as than a limited reservoir for oxygen.^{12, 14}

After confirming successful oxygenation from MACF treatment, studying the release profile of PHMB from MACF substrates was vital to understand the amount of antibiotic in contact with cells over time. Accordingly, the release kinetics of PHMB from the MACF substrates showed a burst phase profile during the first 30 followed by a slow-release phase (up to 51 hours in Figure 1C). Considering the short half-life of neutrophils (6–8 hours),³⁷ the relatively fast release of PHMB from the MACF substrate is ideal to evaluate the effects of PHMB on different antibacterial characteristics of dHL-60s. Although previous research has separately focused on creating oxygenating^38,39,40 and antibacterial hydrogels,^41,42 to the best of our knowledge, our system is the first to offer both benefits in a single platform to be useful in both in vitro and future in vivo applications. Three conventional release models including the Hixon-Crowell, Weibull, and Hill were fitted to the release data and analyses of regression was performed as previously described²⁴ (Figure 1D). Our regression data demonstrated the overall superiority of the Weibull model, with the highest R² value. The parameters of Weibull model are sensitive to ranges of release profile, where T_LAG is the delay time before the PHMB release from the MACF hydrogel substrate begins. The constant value a in the Weibull model is the time scale, or apparent rate constant. Constant b (shape parameter) is another determining factor in the Weibull model, which determines the shape of the release curve and is correlated with physiological behavior of the delivery system. When b = 1 the shape of the release profile is exponential, when b < 1 (such as in our case) a parabolic release behavior occurs and b > 1 predicts a sigmoidal form for the release profile.⁴³ For a parabolic release profile, such as for our data, the rate of drug release reduces as time increases.⁴⁴

Our dual functioning platform was used along with a differentiated human leukemia-60 cell line model to study the variations in neutrophil infection-fighting capacities, including phagocytosis, bacterial elimination, ROS, and NET production in response to oxygen abundance and antibiotic delivery. To induce antibacterial functions, HL-60s need to differentiate into phagocytic neutrophils through a series of chemical cues. For instance, Baxter et al.⁴⁵ used all-trans retinoic acid (ATRA, 1 μM, 5 days) to induce granulocytic differentiation to HL-60s. Other groups have used chemical clues, such as Bt₂cAMP,⁴⁶ phorbol 12-myristate 13-acetate,²⁶ TPA (12-O-tetradecanoylphorbol-13-acetate),⁴⁷ vitamin D₃,⁴⁸ and DMSO⁴⁹ to induce granulocytic differentiation to HL-60s. Gee et al., showed modulated rolling interactions of HL-60s when differentiated with DMSO. Improved cell survival was observed when HL-60s were treated with DMSO as compared to ATRA⁵⁰ and TPO.⁴⁷ Furthermore, DMSO-mediated HL-60s showed superior superoxide production compared to Bt₂cAMP.⁴⁶ Accordingly, our results indicated that differentiating HL-60s using 176 mM DMSO for 5–7 days can successfully induce the granulocytic differentiation to HL-60s. We showed an overall 68% reduction in cell numbers upon differentiation along with a decreased cytoplasm to nucleus ratio in the dHL-60s population both on day 5 and 7 after differentiation. The group of cells differentiated for five days demonstrated a more intact and uniform cytoplasmic morphology as compared to their conditions on day seven with obvious blebbing and degradation. The differentiation process was further confirmed by an approximately 4-times increase in the levels of neutrophil-specific surface marker, CD11b,⁵¹ in dHL-60s group compared to their non-differentiated state. Overall, we showed that using DMSO at a concentration of 176 mM for five days is optimal for granulocytic HL-60 differentiation (Figure 3).

Under normal biological conditions in vivo neutrophils experience oxygen partial pressures from 75–100 mmHg in main arteries to approximately 40 mmHg in capillaries.⁴ However, extremely low oxygen tensions increase the risk of delayed neutrophil apoptosis⁵²and tissue infections by disturbing the metabolic activity of neutrophils. Indeed, among all other monocytes⁵³, neutrophils consume the highest levels of molecular oxygen through the NADPH oxidase pathway.⁵⁴ We have previously shown that local application of MACF hydrogels for a period of 2 hours increases the oxygen partial pressure in the culture media up to 17 mmHg higher than media-only controls in vitro.⁹ Increasing oxygen partial pressures at the site of injury by 5–10 mmHg improves wound healing responses significantly in acute wound models,⁷ supporting the fact that MACF can be used as an efficient oxygenating platform. Our previous in vitro results also support the fact that additional oxygen from MACF can improve cellular migration and proliferation in cells with high oxygen demand.⁹ We have also shown that these hydrogels can be reloaded with oxygen as many times as needed with a maximum uptake capacity.²¹ Therefore, we aimed to study whether delivering additional oxygen via our MACF hydrogels to neutrophils impacts their antibacterial functions, including phagocytosis. Our results revealed that the phagocytic behavior of dHL-60s is not significantly influenced by oxygen availability as governed by MACF, even when it is used in its oxygen-supersaturated form (Figure 4). In accordance with our results, Reiss et al.⁵³ found that the oxygen consumption and uptake of zymosan particles by monocytes decreased to approximately zero upon the addition of Antimycin A, a mitochondrial respiratory chain inhibitor, as compared to no changes in neutrophil groups under the same conditions. It is also worth mentioning that PHMB-treated groups were not included in this part of the study assuming that antibiotics would not particularly impact the phagocytic behavior of dHL-60s, thus only the role of oxygenation via MACF was studied here.

Apart from phagocytosis, neutrophils often produce antibacterial cytokines and peptides to deactivate pathogens.⁵⁵ Therefore, it was important to probe the direct antibacterial capacities and see how this could vary with and without additional oxygen and PHMB. Based on our results, pre-treatment of the cells with PHMB significantly reduced the number of surviving bacteria, as compared to when dHL-60s and PHMB were used alone. This indicates that when PHMB is used in conjugation with dHL-60s, the antibacterial capacity of both can be upregulated, even at PHMB concentrations lower than the MIC value of 50 μM (Figure 5A).

Upon overnight contact with oxygenating MACF hydrogels, a lower number of bacteria survived both with and without dHL-60s (Figure 5). This observation could be explained by two different scenarios. The first possible explanation is the fact that MACF hydrogels are chitosan-based. The highly deacetylated chitosan carries a positive charge in biological buffers and can bind to the negatively charged bacterial membrane, alter their cell wall permeability, and result in bacterial death.⁵⁶ Chen et al.⁵⁷ showed a significant antibacterial activity against Escherichia coli (E. coli) associated with chitosan that increased with factors such as degree of deacetylation, molecular weight, and the density of positive charges. Using Gram-positive flora, Samar et al., also showed antioxidant and antibacterial properties of chitosan against S. aureus and Bacillus cereus. Moreover, oxygen can sustain the formation of antibacterial cytokines by neutrophils and facilitate their antibacterial processes. To support this explanation, Almzaiel et al., demonstrated increased antibacterial activity for dHL-60s induced by hyperbaric oxygen therapy (HBOT).²⁵ The fact that we did not see significantly reduced bacterial growth associated with MACF (Figure 5) might be because of the limitations in hydrogel size that can be put in contact with cells, and thus the amount of oxygen supplemented to them is smaller than hyperbaric oxygen pressures. Importantly, we observed total bacterial elimination when dHL-60s are treated with MACF-PHMB hydrogels and used against S. aureus (Figure 5). Although the neutrophil peripheral lifespan is a debated subject, some researchers⁵⁸ have reported that inflammatory signaling attracts more neutrophil populations to the injury site.^{59, 60} Therefore, with oxygen and PHMB acting as inflammatory signals,^{4, 5} this synergistic treatment might recruit more neutrophils to the site in favor of antibacterial characteristics and applications.

Exposure of PMA-activated dHL-60s to PHMB from MACF-PHMB hydrogels increased the formation of ROS as evidenced by an increase in DHE fluorescence signals (Figure 6). Antibiotic chemicals, specifically PHMB, can cause oxidative stress both in vitro and in vivo. To support this, Kim et al.⁶¹ have shown the important role of PHMB in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway. This pathway stimulates the secretion of the pro-inflammatory cytokines such as interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF)-α by neutrophils. These cytokines, in turn, recruit additional neutrophils to the site of inflammation and further amplify the oxidative stress. In agreement with our results, this previous study also showed a 3.32-fold increase in ROS production of adenocarcinomic human alveolar basal epithelial cells (A549) induced by PHMB in a dose-dependent manner. Since the presence of additional oxygen from MACF did not reveal significant effects on the ROS production patterns (p > 0.05), the majority of the observed response in the MACF-PHMB treated group is assumed to be associated with activation by PHMB (Figure 6).

Since DNA forms a major component of NETs, we used the PicoGreen double-stranded DNA quantification assay to quantify the amount of extracellular DNA as an indicator of neutrophilic NET formation. The PicoGreen assay is an accurate means of measuring DNA content, even at very low concentrations.⁶² Therefore, we used PicoGreen to demonstrate that the presence of oxygen and PHMB trigger different NET formation patterns when the cells are stimulated with PMA, a chemical stimulus, as compared to their biological stimulation with S. aureus (Figure 7). Correlating with our ROS production results, when PMA is used alone as the chemical stimulus, MACF-PHMB hydrogels produced the maximum amount of extracellular DNA (Figure 7A, p = 0.0001). Our results were in contrast with the observations of the Branitzki research group,⁶³ which showed that PMA-activated neutrophils produce significantly more NETs under normoxia as compared to hypoxic conditions. However, the evidence provided in a publication by the Kirchner group⁶⁴ aligns with our observations that the ROS derived from myeloperoxidase (MPO) enzyme activity, which is a hypochlorite (OCl⁻) superoxide, is mainly responsible for the formation of NETs. When hypochlorite reacts with hydrogen peroxide, singlet oxygen will be produced, which is essential for NET formation.⁶⁵ Therefore, as observed in our ROS production patterns, since the presence of PHMB triggers ROS production, it also induces improved NET formation to dHL-60s (Figure 7A).

Interestingly, contact with S. aureus, as a biological stimulant of NET formation, altered the described scenario (Figure 7B). Pilsczek²⁰ showed an approximately 10-fold increase in the amount of NETs produced by neutrophils upon contact with S. aureus, among a series of Gram-positive and Gram-negative bacteria. Therefore, they considered S. aureus as a unique NET inducer. In our case, we also observed the maximum amount of NET formation in the MACF-treated group without PHMB. This could be explained by the fact that MACF hydrogels provide additional oxygen to support aerobic S. aureus growth and thus facilitate the conditions that favor its growth to induce the maximum NET formation.⁶⁶ Conversely, since PHMB antibiotic inactivates some of those oxygen-derived proliferated bacteria, the biological NET stimulus is downregulated, explaining the lower NET formation in the MACF-PHMB groups. Overall, the results revealed that the capacity of dHL-60s to form NETs in the presence of supplemental oxygen and PHMB depends drastically on the type of stimulation. Chemically induced NET formation via PMA decreases in the presence of supplemental oxygen and increases when PHMB is added. This is while this trend is reversed with S. aureus as the biological NET inducer possibly due to the activation of different oxidative burst pathways.⁶⁷

3. Conclusions

The differentiated human leukemia cell line-60 was used in this study as a model to study the antibacterial properties of neutrophils as a response to a dual functioning treatment. Taken together, our data suggest that supplemental oxygen and antibacterial delivery from our MACF-PHMB hydrogel platforms, upregulate dHL-60s’ activity as shown by their enhanced antibacterial properties and production of reactive oxygen species against S. aureus. NET formation patterns in the presence of MACF and PHMB were shown to depend on the type of stimulus used. Namely, chemical, and biological stimuli, when used together or individually, triggered different NET formation responses to PHMB and additional oxygen. No substantial effects were discovered to be associated with supplemental oxygenation from MACF hydrogels on the phagocytic behavior of neutrophil-like cells. Besides creating a model platform to study immune responses in vitro, our dual functioning oxygenating-antibacterial hydrogels can potentially be used as a stand-alone treatment to improve inflammatory and wound healing processes in vivo.

Abbreviations

HL-60

human leukemia-60 cell line

MACF

fluorinated methacrylamide chitosan

A549 cells

adenocarcinomic human alveolar basal epithelial cells

AMP

antimicrobial peptide

ATCC

American type culture collection

ATRA

All-trans retinoic acid

cfu

colony-forming unit

D₂O

deuterium oxide

DHE

dihydroethidium

dHL-60s

differentiated HL-60s

DMSO

dimethyl sulfoxide

DNA

deoxyribonucleic acid

dsDNA

double stranded DNA

d-TFA

deuterated trifluoroacetic acid

E. coli

Escherichia coli

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

HBOT

hyperbaric oxygen therapy

Ig

immunoglobulin

IL

Interleukin

KDa

Kilodaltons

MIC

minimum inhibitory concentration

MOI

multiplicity of infection

MPO

myeloperoxidase

MWCO

molecular weight cutoff

NADPH

nicotinamide adenine dinucleotide phosphate

NETs

neutrophil extracellular trap

NF-kB

nuclear factor kappa-light chain-enhancer of activated B cells

NMR

nuclear magnetic resonance

OCl⁻

hypochlorite superoxide

OD

optical density

PBS

phosphate-buffered saline

PFA

paraformaldehyde

PFOC

pentadecafluorooctanoyl chloride

PHMB

polyhexamethylene biguanide

PKC

protein

C

kinase

PMA

phorbol 12-myristate 13-acetate

PMN

polymorphonuclear leukocyte

ROS

reactive oxygen species

RPMI

Roswell Park Memorial Institute medium

S. aureus

Staphylococcus aureus

S.D

standard deviation

THB

Todd-Hewitt broth

TNF

tumor necrosis factor

TPA

12-O-tetradecanoylphorbol-13-acetate

UV

ultraviolet