Erin
Myles
,
Raechelle. A.
D'Sa
and
Jenny
Aveyard
*
School of Engineering, University of Liverpool, The Quadrangle, Brownlow Hill, L69 3GH, UK. E-mail: zippy78@liverpool.ac.uk
First published on 8th April 2025
Antimicrobial resistance (AMR) represents a significant global health challenge, contributing to increased mortality rates and substantial economic burdens. The development of new antimicrobial agents with dual antimicrobial and antibiofilm capabilities is crucial to mitigate AMR. Nitric oxide (NO) is a broad-spectrum antimicrobial agent which has shown promise in treating infections due to its multiple antimicrobial mechanisms. However, the high reactivity of NO poses a challenge for effective delivery to infection sites. We investigated the antimicrobial and antibiofilm capabilities, and the shelf life, of NO-releasing gelatin nanoparticles (GNP/NO) against three common hospital-acquired pathogens: Staphylococcus aureus, Escherichia coli, and Candida albicans. The synthesised GNP/NO were found to be cytocompatible and exhibited significant antimicrobial and antibiofilm efficacies against the tested pathogens in both nutrient-rich and nutrient-poor conditions. Furthermore, we found that the antimicrobial capabilities of GNP/NO were maintained for up to 6 months post synthesis, against Staphylococcus aureus (2.4log), Escherichia coli (1.2
log) and Candida albicans (3
log) under nutrient-poor conditions. Our study demonstrates the use of a novel broad-spectrum antimicrobial with a prolonged shelf life for the treatment of infections. These findings offer an effective alternative to traditional antibiotics which would contribute to mitigating the current global AMR threat resulting from antibiotic overuse.
Nitric oxide (NO) is a broad-spectrum antimicrobial agent which is endogenously produced by macrophages within the immune system, in response to pathogens. The antimicrobial capabilities of NO are attributed to its ability to react readily with oxygen, superoxide (O2−) and hydrogen peroxide (H2O2) to form highly reactive nitrogen and oxygen species (RNS and ROS), such as peroxynitrite, nitrogen dioxide and dinitrogen tetroxide. The presence of RNS and ROS over time leads to intracellular nitrosative and oxidative stress within bacterial cells, causing DNA alterations, lipid peroxidation and enzyme inactivation, making NO an effective antimicrobial agent.4,5 Owing to the multiple antimicrobial mechanisms by which NO can inactivate microorganisms, there has been intense interest in the use of NO-releasing delivery systems as a potential therapy for treating infections.6,7 Moreover, studies by Privett8 and Grayton9 have demonstrated that exposure to sublethal dosages of exogenous NO is unlikely to induce resistance in bacteria or fungi, likely due to the multifaceted antimicrobial mechanisms of NO.5,8,9
Despite its antimicrobial properties, the radical nature of NO makes it highly reactive, resulting in a short shelf life (<10 s). Therefore, targeted NO delivery to the site of infection is challenging.10 To address this issue NO donors, such as N-diazeniumdiolates have been developed to improve the storage capabilities of NO and enhance delivery of therapeutic doses to target sites. N-diazeniumdiolate compounds, characterised by their diolate [N–(O)NO] functional group, are formed by the reaction of amines with NO under high pressure in the absence of oxygen. This reaction forms a diolate group bound to a nucleophile adduct to form on a nitrogen atom within the amine.11 The rate of NO release from N-diazeniumdiolates is highly dependent on the pKa of the amine group to which they are attached. Primary amines typically produce less stable N-diazeniumdiolates with rapid release rates.12 Consequently, secondary amines and polyamines are more commonly used in biomaterials as they enhance the shelf life and improve the release kinetics of N-diazeniumdiolates.13,14
Gelatin, derived from the hydrolysis of collagen, and is a widely used biomacromolecule within both the food and pharmaceutical industries, due to its biocompatibility and cost-effectiveness.15 In clinical settings, gelatin is commonly used as a vaccine stabiliser, such as in the inhaled influenza vaccine to improve the stability and shelf-life of the product.16 As gelatin is derived from collagen its structure comprises of multiple amino acid groups, this allows for many chemical modifications and covalent attachment, making it an ideal candidate for use as a drug delivery vehicle.17 The use of gelatin-derived biomaterials has been well documented for a range of applications, including tissue engineering, cancer treatments and wound healing.18–20 Furthermore, Li et al.21 has shown that electrospun polycaprolactone (PCL)/gelatin blended wound dressings (PCL:
G. 25
:
75 wt%) can be functionalised with N-diazeniumdiolates, as the polyamine composition of gelatin provides multiple tethering sites for the NO donor. The electrospun wound dressing significantly reduced the presence of both Pseudomonas aeruginosa and Staphylococcus aureus, reducing the risk of infections.
Gelatin nanoparticles (GNPs) have been extensively studied as a drug delivery system for a range of applications, including cancer treatment,22 tissue engineering,23 and vaccine delivery.24 However the use of GNP for the delivery of NO has not been investigated, therefore this study assessed the antimicrobial efficacy of N-diazeniumdiolate-releasing gelatin nanoparticles (GNP/NO).
We investigated the antimicrobial and antibiofilm capabilities of GNP/NO against three common hospital-acquired pathogens. This proof-of-concept study outlines the synthesis of homogenous GNP/NO, which demonstrated antimicrobial and antibiofilm capabilities against, Staphylococcus aureus, Escherichia coli and Candida albicans within a cytocompatible range, in both nutrient-rich and nutrient-poor conditions. Notably, the nanoparticles maintained their antimicrobial efficacy after 6 months of storage. These results emphasise the potential of GNP/NO as an effective infection treatment, which may help to mitigate the current global AMR threat caused by antibiotic overuse.
The particles were centrifuged at 10500 rpm (Thermo-Scientific Heraeus Megafuge 16R) for 10 min and washed in 30% acetone, this step was then repeated a further 3 times. After the final wash, GNPs were resuspended in dH2O and frozen before lyophilisation (ScanVac Cool Safe, La60 Gene). The particles were then stored in a refrigerator at 5 °C until required. A schematic representation of this method is depicted in Fig. 1.
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Fig. 1 Schematic of the two-step desolvation method used to synthesise gelatin nanoparticles. Created with http://BioRender.com. |
To measure the zeta potential of the particles, 2 mg of lyophilised GNPs were suspended in 1 mL dH2O, placed into a capillary cuvette (Malvern Panalytical), and measured using a Zetasizer Nano ZS.
All images were processed using ImageJ software, and an average particle size was obtained from each image (n = 30).
Measurements were conducted at 0 (T = 0), 3 (T = 3) and 6 months (T = 6) time points to assess the stability of NO release from GNP/NO, between time points GNP/NO were stored in a freezer at −20 °C. Measurements at T = 0 and T = 3 were conducted in triplicate (N = 3), whilst those measurements at t = 6 were performed only once (N = 1).
Microbial cultures were grown in 15 mL LB and incubated at 37 °C with agitation (150 rpm) for 18 h, except for C. albicans which was grown in 15 mL TSB. The cultures were centrifuged at 3500 rpm for 10 min, and the supernatant was discarded. The pellet was resuspended in sterile PBS, to achieve an OD600 = 1 (Hitachi, U-2900, Tokyo, Japan). The bacterial suspension was diluted 1:
100 in PBS for nutrient-poor conditions, and in LB or TSB for nutrient-rich condition to obtain a final cell concentration of ∼106 CFU mL−1. Following this, 1 mL cell suspension was placed into microcentrifuge tubes containing either 2.5, 5 or 7 mg GNP/NO, referred to as GNP/NO2.5, GNP/NO5 and GNP/NO7. Non-functionalised GNP of the same weight were used as controls (GNP2.5, GNP5 and GNP7), and an additional positive control containing bacterial suspension only was used and incubated at 37 °C with agitation (150 rpm).
Antimicrobial efficacy was evaluated at 4 h to assess the immediate antimicrobial effects of GNP/NO and at 24 h to examine its sustained effects on bacterial and fungal populations. These time points were chosen as they align with common antimicrobial susceptibility testing methods. Following incubation, 100 μL was taken from each microcentrifuge tube and serially diluted 1:
10 six times in PBS. A 20 μL aliquot from each dilution were plated onto Luria broth agar (LBA; Sigma-Aldrich) in triplicate and incubated for 24 h at 37 °C, except for C. albicans which was plated onto tryptic soya agar (TSA; BD Difco). The number of visible colonies was counted to determine the total number of viable microorganisms per mL (CFU mL−1). All experiments were conducted in triplicate (N = 3).
This experiment was repeated at T = 0, T = 3 and T = 6 to assess the long-term stability all the GNP/NO concentrations and to fully optimise the antimicrobial efficacy of GNP/NO.
The plate lid was subsequently rinsed with PBS and immersed in a new 96-well plate containing 200 μL media. The plate was sonicated (VWR, USC100 TH Ultrasonic Bath) for 10 min to remove biofilms from the pegs. A 20 μL aliquot was removed from each test well and serially diluted (1:
10) to a factor of eight, and 20 μL of each dilution was plated onto LB agar and incubated for 24 h at 37 °C. The number of visible colonies were counted, and CFU mL−1 was calculated.
Antimicrobial efficacy was evaluated at T = 0, T = 3 and T = 6 to assess the longevity antimicrobial stability of the particles. However, these experiments were not conducted against C. albicans due to the inability of this strain to form biofilms.
Scanning electron microscopy was used to characterise the morphology and size of the GNP after lyophilisation (Fig. 3a–d). The particles were homogenous and spherical in structure. The particle diameter of the lyophilised GNP, measured via SEM was calculated to be 190 ± 8 nm.
As stability is an important factor to consider when assessing the potential of new drug candidates29 the long-term stability and shelf life of the particles was also investigated. The NO release from GNP/NO was measured at t = 0 (initial measurement) and after t = 3 months and t = 6 months of storage. The release profiles of GNP/NO for each medium at t = 0 is shown in Fig. 5.
Sample | Month | [NO]tota (μmol mg−1) | [NO]maxb (nmol mg−1) | [NO]tmaxc (min) | [NO]tdd (h) |
---|---|---|---|---|---|
a The total NO release from the sample over time. b The highest release burst from the sample. c The time taken for a sample to achieve [NO]max. d The total duration of release for a sample. | |||||
GNP/NO2.5 | 0 | 9.21 ± 3.80 | 1.79 ± 0.76 | 0.35 ± 0.23 | 12.83 ± 6.66 |
3 | 8.83 ± 0.75 | 10.33 ± 4.17 | 0.47 ± 0.19 | 10.21 ± 0.83 | |
6 | 0.46 | 4.26 | 0.20 | 0.34 | |
GNP/NO5 | 0 | 6.09 ± 3.16 | 11.02 ± 4.83 | 0.12 ± 0.02 | 8.14 ± 0.68 |
3 | 22.18 ± 4.51 | 12.93 ± 0.00 | 0.185 ± 0.01 | 11.35 ± 0.01 | |
6 | 2.25 | 8.16 | 0.27 | 3.23 | |
GNP/NO7 | 0 | 7.65 ± 4.68 | 23.29 ± 29.77 | 0.44 ± 0.23 | 7.95 ± 4.54 |
3 | 2.19 ± 0.34 | 10.46 ± 4.75 | 0.16 ± 0.10 | 4.96 ± 2.58 | |
6 | 0.83 | 4.77 | 0.38 | 1.72 |
T = 0: at t = 0, it was revealed that the [NO]tot was not proportional to the GNP/NO concentration as GNP/NO2.5, GNP/NO5 and GNP/NO7 released 9.21 ± 3.80, 6.09 ± 3.16 and 7.65 ± 4.68 μmol mg−1, respectively. As well as demonstrating the highest [NO]tot release, GNP/NO2.5 also exhibited the longest [NO]td (12.83 ± 6.66 h) in PBS.
T = 3: at t = 3 a reduction in [NO]tot concentration for GNP/NO2.5 (8.83 ± 0.75) and GNP/NO7 (2.19 ± 0.34 μmol mg−1) was observed, however interestingly the opposite was seen for GNP/NO5 (22.18 ± 4.51 μmol mg−1).
T = 6: the decrease in NO release from GNP/NO over time was also reflected in the results at t = 6, which found that the [NO]tot for all GNP/NO concentrations decreased considerably compared with those at t = 0.
Sample | Month | [NO]tota (μmol mg−1) | [NO]maxb (nmol mg−1) | [NO]tmaxc (min) | [NO]tdd (h) |
---|---|---|---|---|---|
a The total NO release from the sample over time. b The highest release burst from the sample. c The time taken for a sample to achieve [NO]max. d The total duration of release for a sample. | |||||
GNP/NO2.5 | 0 | 14.55 ± 11.21 | 44.45 ± 19.91 | 0.16 ± 0.02 | 7.63 ± 4.82 |
3 | 6.06 ± 5.63 | 12.80 ± 0.18 | 0.24 ± 0.06 | 7.50 ± 7.23 | |
6 | 1.64 | 7.38 | 0.27 | 2.73 | |
GNP/NO5 | 0 | 19.09 ± 0.67 | 19.00 ± 5.66 | 0.21 ± 0.12 | 9.30 ± 1.67 |
3 | 5.97 ± 5.25 | 6.66 ± 2.08 | 0.21 ± 0.06 | 7.57 ± 5.03 | |
6 | 1.18 | 5.95 | 0.14 | 1.94 | |
GNP/NO7 | 0 | 19.96 ± 1.76 | 22.4 ± 9.75 | 0.09 ± 0.06 | 19.55 ± 2.31 |
3 | 8.27 ± 6.42 | 13.11 ± 3.61 | 0.16 ± 0.01 | 9.48 ± 3.36 | |
6 | 2.17 | 7.43 | 0.1 | 5.34 |
T = 0: the [NO]tot release in LB was found to be proportional to GNP/NO concentration, with increasing concentrations leading to an increase in [NO]tot release (GNP/NO2.5 (14.55 ± 11.21 μmol mg−1), GNP/NO5 (19.09 ± 0.67 μmol mg−1) and GNP/NO7 (19.96 ± 1.76 μmol mg−1)), unlike the results revealed under nutrient-poor conditions.
T = 3: at t = 3, a drastic decrease in the [NO]tot and [NO]max release for all GNP/NO concentrations was observed. A decrease was also seen in the [NO]td release for GNP/NO7 (9.48 ± 3.36 h), however GNP/NO2.5 (7.50 ± 7.23 h) and GNP/NO5 (7.57 ± 5.03 h) remained largely unchanged.
T = 6: a further reduction in [NO]tot, [NO]max [NO]td was observed for all GNP/NO concentrations at t = 6 compared to t = 0, though [NO]tmax remained largely unchanged throughout the test period.
Sample | Month | [NO]tota (μmol mg−1) | [NO]maxb (nmol mg−1) | [NO]tmaxc (min) | [NO]tdd (h) |
---|---|---|---|---|---|
a The total NO release from the sample over time. b The highest release burst from the sample. c The time taken for a sample to achieve [NO]max. d The total duration of release for a sample. | |||||
GNP/NO2.5 | 0 | 12.41 ± 0.48 | 10.48 ± 4.48 | 0.19 ± 0.06 | 9.29 ± 0.84 |
3 | 8.15 ± 2.96 | 17.40 ± 9.16 | 0.28 ± 0.12 | 9.09 ± 0.44 | |
6 | 2.80 | 10.78 | 0.19 | 2.01 | |
GNP/NO5 | 0 | 11.35 ± 1.70 | 17.48 ± 1.18 | 0.41 ± 0.12 | 3.74 ± 1.92 |
3 | 4.92 ± 1.18 | 14.51 ± 8.20 | 0.17 ± 0.05 | 6.05 ± 3.85 | |
6 | 8.53 | 6.94 | 0.21 | 11.82 | |
GNP/NO7 | 0 | 37.92 ± 21.60 | 45.63 ± 24.94 | 0.20 ± 0.06 | 12.57 ± 4.84 |
3 | 2.47 ± 1.67 | 3.81 ± 0.70 | 0.89 ± 0.54 | 4.53 ± 4.27 | |
6 | 5.71 | 5.18 | 0.23 | 11.80 |
T = 0: similar to the results in LB, with increasing GNP/NO concentration, the [NO]tot and [NO]max release also increased in TSB, however interestingly this was not observed for [NO]td.
T = 3: following 3 months of storage the [NO]tot release substantially decreased for GNP/NO2.5, GNP/NO5 and GNP/NO7, 8.15 ± 2.96 μmol mg−1, 4.92 ± 1.18 μmol mg−1 and 2.47 ± 1.67 μmol mg−1, respectively.
T = 6: at t = 6, the [NO]tot release for GNP/NO2.5 decreased further compared to the t = 0 and t = 3 timepoints (2.80 μmol mg−1), however interestingly this was not seen for the GNP/NO5 (8.53 μmol mg−1) or GNP/NO7 (5.71 μmol mg−1) samples, which were found to increase slightly compared with the t = 3 timepoint.
Sample | [NO]tota (μmol mg−1) | [NO]maxb (nmol mg−1) | [NO]tmaxc (min) | [NO]tdd (h) |
---|---|---|---|---|
a The total NO release from the sample over time. b The highest release burst from the sample. c The time taken for a sample to achieve [NO]max. d The total duration of release for a sample. | ||||
GNP/NO2.5 | 10.22 ± 3.56 | 6.12 ± 3.28 | 0.25 ± 0.08 | 7.98 ± 1.03 |
GNP/NO5 | 6.86 ± 3.13 | 15.31 ± 10.13 | 0.97 ± 1.45 | 7.93 ± 0.89 |
GNP/NO7 | 3.08 ± 0.73 | 5.74 ± 3.67 | 0.17 ± 0.06 | 6.44 ± 1.13 |
As GNP/NO demonstrated no cytotoxicity at t = 0, the release of NO from GNP/NO in DMEM was measured at t = 0, alone. Similarly to results in PBS, the [NO]tot release in DMEM was not proportional to GNP/NO concentrations, it was found that GNP/NO2.5 had a [NO]tot of 10.22 ± 3.56 μmol mg−1 and GNP/NO5 and GNP/NO7 released 6.86 ± 3.13 μmol mg−1 and 3.08 ± 0.73 μmol mg−1, respectively.
As shown in Fig. 6, all GNP/NO concentrations completely eradicated S. aureus within 4 h under nutrient-poor conditions. At t = 3, the antimicrobial efficacy of GNP/NO decreased, with only GNP/NO7 causing complete eradication of S. aureus at 4 h. After 24 h however, incubation with GNP/NO5 demonstrated a significant reduction in S. aureus (4.3log). Interestingly, the antimicrobial efficacy of GNP/NO significantly decreased at t = 6, and GNP/NO was unable to completely eradicate S. aureus, although GNP/NO7 did cause a 2.36
log reduction after 24 h.
The antimicrobial efficacy of GNP/NO against E. coli at t = 0, t = 3 and t = 6 is shown in Fig. 6. Surprisingly unlike S. aureus, GNP/NO2.5 was revealed to be ineffective against E. coli, and was unable to significantly reduce the bacterium at any timepoint. However, incubation with both GNP/NO5 and GNP/NO7 caused complete eradication of E. coli after 24 h at both t = 0 and t = 3. A significant reduction in the antimicrobial efficacy was observed at t = 6, with GNP/NO5 and GNP/NO7 only reducing E. coli by 1.28 and 1.24log reductions, respectively after 4 h.
In addition to examining the antibacterial properties of GNP/NO, we extended our research to examine its antifungal properties, through exposure of GNP/NO to C. albicans, a prevalent yeast species.
Across all time points, it was evident that a higher dosage of GNP/NO (i.e. ≥GNP/NO5) was required to significantly reduce C. albicans, as exposure to GNP/NO2.5 proved to be ineffective against the fungi (Fig. 6). At t = 0, both GNP/NO5 and GNP/NO7 led to the complete eradication of the fungi after 24 h. Similar to results observed against S. aureus and E. coli at t = 3, it was found that the antimicrobial efficacy of GNP/NO decreased against C. albicans over time, and only GNP/NO7 caused complete eradication of the fungi at either 4 h or 24 h. This reduction in efficacy was shown to decrease further at t = 6, as only the GNP/NO7 sample elicited a significant reduction in C. albicans (3log) after 24 h.
At t = 0 (Fig. 7), 4 h incubation with GNP/NO2.5 (0.8log), GNP/NO5 (1.6
log) and GNP/NO7 (1.8
log) all demonstrated significant antimicrobial efficacy against S. aureus under nutrient-rich conditions (LB). Interestingly however, a regrowth in S. aureus was observed for GNP/NO2.5 after 24 h. Similar to results observed under nutrient-poor conditions the antimicrobial efficacy of GNP/NO was found to decrease over time, with only GNP/NO7 demonstrating a 0.9
log reduction at 4 h and a 2.17
log reduction after 24 h under nutrient-rich conditions at t = 3. Interestingly, at t = 6 both GNP/NO5 (1.5
log) and GNP/NO7 (2.4
log) led to a reduction in S. aureus after 24 h of incubation.
It was revealed that under nutrient-rich conditions (LB), both GNP/NO5 (1.7log) and GNP/NO7 (3.1
log) significantly reduced E. coli at 4 h (Fig. 7), with a further reduction of 7.8
log reduction by GNP/NO7 after 24 h. A significant reduction to the antimicrobial efficacy of GNP/NO was observed at both t = 3, where GNP/NO7 resulted in a 0.9
log reduction after 4 h, and at t = 6 GNP/NO was no longer effective against E. coli.
Investigation of the antimicrobial efficacy of GNP/NO against C. albicans under nutrient-rich conditions (TSB) revealed that at t = 0, both GNP/NO5 (0.9log) and GNP/NO7 (1.2
log) caused a significant reduction in the CFU mL−1 counts of C. albicans after 24 h. However, no significant antimicrobial activity was exhibited by GNP/NO after this time point.
Biofilms were established over a 24 h period and subsequently washed to remove planktonic organisms. The established biofilms were then incubated in nutrient-poor conditions (PBS) with GNP/NO, and the remaining viable microorganisms were counted after 4 and 24 h of incubation. The antibiofilm efficacy of the particles was investigated after 0, 3 and 6 months of storage to evaluate their shelf life.
The results showed that only 24 h incubation with the highest concentration, GNP/NO7 led to significant dispersal of S. aureus biofilms at t = 0 and t = 3 under nutrient-poor conditions (Fig. 8), suggesting a higher NO dosage may be required against S. aureus biofilms compared to planktonic S. aureus. At t = 6, GNP/NO particles no longer exhibited antibiofilm activity against established S. aureus biofilms.
Fig. 8 illustrates the antibiofilm efficacy of GNP/NO against the established E. coli biofilms under nutrient-poor conditions. At t = 0, GNP/NO7 caused complete dispersion of E. coli biofilms after 4 h of incubation, and incubation with GNP/NO5 led to a 0.9log reduction in E. coli biofilms after 24 h. At t = 3, only GNP/NO7 resulted in the complete dispersion of E. coli biofilms after 24 h, however at t = 6 the nanoparticles were no longer effective against established E. coli biofilms, similar to results seen against S. aureus biofilms.
Fig. 9 shows the effects of GNP/NO on established S. aureus biofilms under nutrient-rich conditions. GNP/NO7 caused significant biofilm disruption after 24 h, reducing the biofilm by 4.7log. At t = 3, both GNP/NO5 and GNP/NO7 reduced S. aureus biofilms within 4 h, resulting in 0.8
log and 0.7
log reductions, respectively. However, biofilms incubated with GNP/NO5 reformed after 24 h. At t = 6, GNP/NO did not demonstrate antibiofilm activity against established S. aureus biofilms.
The antibiofilm capabilities of GNP/NO against established E. coli biofilms under nutrient-rich conditions, over a 6-month period were also investigated (Fig. 9). It was revealed that both GNP/NO5 (1.9log) and GNP/NO7 (complete dispersal) significantly reduced the presence of E.coli biofilms at 4 h, however, a biofilm reformation was observed in both samples at 24 h. The results at t = 3, showed that incubation with GNP/NO7 caused a 4.4
log biofilm reduction after 4 h. Interestingly, despite reformation of the biofilm at 24 h, GNP/NO7 still remained significantly lower than the control (1.1
log). Surprisingly, after 6-months of storage, GNP/NO7 maintained its antimicrobial efficacy leading to a 3.4
log reduction to the E.coli biofilms after 4 h, although the biofilm fully reformed after 24 h.
This study assessed the effects of GNP/NO leachates on L929 cells formed by the incubation of GNP/NO in DMEM for 4 and 24 h (Fig. 10). As no sample reduced the cell viability below 70%, it suggests that GNP/NO is biocompatible based on the ISO10993-22 protocol. Surprisingly, many samples demonstrated an increase in cell viability, as much as 35% for the GNP2.5, 4 h leachate.
The synthesis of the nanoparticles was carried out using a two-step desolvation method, which resulted in smooth, homogeneous particles with a diameter of ∼200 nm and a PDI of 0.09. The PDI indicates the size distribution within a population, with a PDI ≥1.00 representing a polydisperse particle size distribution, and a PDI of 0.00 indicating a monodispersed size population.33 Within polymer nanomaterial research a PDI ≤0.2 is generally considered acceptable for use in medical therapeutics,34 suggesting that the GNP synthesised in this study form relatively uniform particle size distributions (monodispersed) within the therapeutic range. Additionally, comparison of different synthesis batches found minimal variation in the size distribution (Fig. S1†), indicating the 2-step desolvation method to be a robust and repeatable method for GNP synthesis.
Interestingly, a discrepancy was identified between particle diameter measurements obtained through DLS (226.9 ± 18.8 nm) compared to SEM (190 ± 8 nm), approximately 36.3 nm difference in diameter. This coincides with previous reports by Hassani Besheli35 which showed the hydrodynamic diameter of GNP, measured by DLS to be larger than SEM measurements. This is likely due to the hydrophilic nature of gelatin,36 which cause the particles to swell in aqueous solutions. To further validate this, lyophilised GNP were rehydrated and the hydrodynamic diameter was remeasured every 5 min over a 24 h period via DLS (Fig. S2†), the results revealed the GNP diameter to increase by ∼40 nm within the first 5 min of measurements, closely aligning with the variations observed between DLS and SEM measurements.
The pH of all media in this study was tested to evaluate their effects on the NO release from GNP/NO. This is essential as NO release is dependent on pH, and the different buffering capacities of each medium can significantly affect the decomposition of N-diazeniumdiolates. All media examined in this study exhibited a pH between 6.9–7.5, however, the addition of GNP/NO (GNP/NO7), led to a decrease in pH. This is due to the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are rapidly produced by the reaction of NO in the presence of O2 and H2O. One product of this reaction is N2O3, this can react with H2O to form H+ and NO2−, increasing the concentration of H+ present, thus lowering the pH of the medium.37 Subsequent pH measurements at 2, 4, 18, and 24 h revealed that the pH of each medium gradually returned to its original pH, which was likely due to the presence of buffers in the media.
Chemiluminescence is widely regarded as the gold standard for NO detection due to its sensitivity with a detection range of 0.5 ppb to 500 ppm,38 and a detection rate of 10−7 L mol−1 s−1, allowing for near real-time analysis NO analysis. Given these advantages, this method was chosen to measure the NO payload and release kinetics of GNP/NO (GNP/NO2.5, GNP/NO5 and GNP/NO7) under nutrient-poor and nutrient-rich conditions. The choice of medium significantly influenced the NO release with considerable differences in both the total release ([NO]tot) and initial burst release ([NO]max) concentrations. Interestingly, at t = 0 the [NO]tot release from GNP/NO2.5, was as follows: PBS < DMEM < TSB < LB, with PBS releasing the least. This strongly correlates with the initial pH measurements of each medium, where PBS showed the highest pH (pH 7.83 ± 0.07), followed by DMEM (pH 7.42 ± 0.02), TSB (pH 7.12 ± 0.16), and LB (pH 6.93 ± 0.19). These findings align with previous studies Salmon39 which indicated that NO release from N-diazeniumdiolates is mediated by pH. Furthermore, Tai et al.40 demonstrated that even small decreases in pH (from 7 to 6) can increase NO release by 2-3-fold. In this case, a higher pH would indicate a lower proton concentration, leading to lower protonation of N-diazeniumdiolates, resulting in less NO being released and a reduced [NO]tot.
The shelf life of GNP/NO was determined by assessing the NO payload from the particles in PBS, LB, and TSB at t = 0, t = 3 and t = 6 to evaluate its practicality as a therapeutic agent. The NO release kinetics in DMEM were not evaluated over time, as GNP/NO was found to be cytocompatible at t = 0, and therefore it was deemed unnecessary to assess the NO release past this point. A general decrease in the [NO]tot concentrations was observed over time for all media tested, with an average reduction of 75% between t = 0 and t = 6. Though, the decomposition of N-diazeniumdiolate is predominantly pH-mediated, it can also occur through thermal dissociation,39 therefore throughout this study GNP/NO was stored at −20 °C to improve the storage capabilities of the nanoparticles. Evidence suggests that the stability of N-diazeniumdiolates can be prolonged by storage in a nitrogen environment. Batchelor41 demonstrated that lipophilic dialkyldiamine-based diazeniumdiolates maintained 99% of their NO release capability when stored in a dry nitrogen environment, compared to 62% when stored under ambient conditions after 4 weeks. The authors attributed the slow decomposition to the presence of water vapour within the polymer, resulting in NO release from the biomaterial, which may explain the decline in [NO]tot concentrations over time. Despite this decrease, the results of this study have shown GNP/NO continues to release NO for up to six-months when stored at −20 °C. Further research is required to fully understand the effects of storage conditions on GNP/NO, and to improve the nanoparticle's shelf life.
To evaluate the broad-spectrum antimicrobial potential of GNP/NO, the antimicrobial efficacy of GNP/NO2.5, GNP/NO5 and GNP/NO7 was assessed against three common healthcare-associated pathogens, S. aureus, E. coli and C. albicans. These concentrations were evaluated over a six-month to determine the longevity of their effectiveness. Additionally, as our results indicated that the medium significantly impacted the NO release kinetics from GNP/NO, we also examined the antimicrobial efficacy of the nanoparticles under both nutrient-poor and nutrient rich conditions to determine the effect on the nanoparticles' performance.
Although, the NO release kinetics showed the [NO]tmax of GNP/NO to be <1 min, an incubation period of up to 24 h was often necessary for the antimicrobial GNP/NO to take effect. This discrepancy is expected, as NO is a free radical with a short half-life,42 therefore the molecule itself does not typically act on the microorganism. Instead, its antimicrobial activity primarily arises from the formation of RNS and ROS through interactions with oxygen and superoxide (˙O2−). This effect is particularly evident when comparing the antimicrobial efficacy of GNP/NO at 4 and 24 h. The initial reduction observed at 4 h results from the oxidative and nitrosative stress within the cell, driven by ROS and RNS such as peroxynitrite (ONOO−), nitrogen dioxide (NO2) and dinitrogen tetroxide (N2O3). The further decrease at 24 h is largely attributed to the secondary effects of these reactive species, including DNA damage, inactivation of proteins and lipid peroxidation. These processes ultimately lead to cell death, even after NO concentrations have diminished.43
Nitric oxide exhibits antimicrobial and antibiofilm properties in a concentration dependent manner, concentrations of >1 μM are generally considered sufficient to exert bactericidal effects. Conversely, sublethal NO concentrations of ∼0.5 nM NO induce the dispersal of biofilms, causing bacteria to revert to their planktonic state.26 In our study, the NO release kinetics observed suggest GNP/NO can achieve concentrations within these effective ranges, indicating both antibiofilm and antimicrobial capabilities. However, the antimicrobial results observed are more complex, suggesting additional factors may influence the antimicrobial and antibiofilm efficacies of GNP/NO.
Initial results at t = 0 revealed there to be differences in the antimicrobial efficacy of GNP/NO between microorganisms, it was found that the nanoparticles were more effective against S. aureus compared to E.coli or C. albicans, as the lowest concentration (GNP/NO2.5) was able to significantly reduce the bacteria at t = 0, under both nutrient-poor and nutrient-rich conditions. This coincides with reports in the literature, showing NO to be more effective against Gram positive bacteria.44,45 This can be attributed to the ability of Gram negative bacteria such as E. coli to synthesise flavohemoglobins, which convert NO radicals to NO3− ions via nitrosylation and can counteract the nitrosative stress caused by RNS produced by NO.46 Although, some studies have reported the contrary, suggesting Gram negative bacteria are more susceptible to NO,47–49 it is likely that the susceptibility of bacteria to ROS and RNS is independent of a bacterium's Gram classification and is affected more by the species itself, the NO dose concentration, and the method of NO delivery.
The antimicrobial efficacy of GNP/NO was evaluated against C. albicans, a common healthcare-associated pathogen known to cause candidiasis and bloodstream infections.50 The study found that C. albicans exhibited a greater resistance to GNP/NO under nutrient-poor conditions compared to S. aureus and E. coli, as higher concentrations of GNP/NO (>GNP/NO5) were required to significantly reduce the fungi, consistent with previous studies.51 The antifungal mechanisms of NO have been found to be similar to its antibacterial actions, with the ROS and RNS generated by NO interacting with DNA, lipid membranes and proteins9,52 ultimately leading to cell death. As the lipid cell membrane in yeast are encased within a cell wall,53 this may provide some protection from cell damage through lipid peroxidation by increasing the distance that ROS and RNS, generated by NO must travel to reach the membrane.
Given the variations in NO payload observed by GNP/NO in nutrient-rich conditions, we extended our investigation to assess the antimicrobial efficacy of GNP/NO using two biologically relevant media, LB and TSB. The shelf life of the particles was again investigated over a six-month period (t = 0, t = 3 and t = 6).
Although GNP/NO released higher [NO]tot concentrations under nutrient-rich conditions (LB and TSB) compared to nutrient-poor conditions (PBS), suggesting a potential increase to its antimicrobial capabilities, the results did not reflect this. In contrast, GNP/NO exhibited reduced antimicrobial activity under nutrient-rich conditions. While incubation with GNP/NO under nutrient-rich led to significant microbial reductions, the particles did not achieve complete eradication.
This reduced antimicrobial efficacy in nutrient-rich conditions may be due to the presence of proteins, trace metal ions and amino acids present, which have been shown to inhibit or sequester NO. For instance, LB contains tryptone, which comprises many amino acids, including tryptophan, cysteine and methionine, all of which have been identified as free-radical scavengers or antioxidants.54 Furthermore, TSB contains high concentrations of glucose, a known NO scavenger across both the animal and plant kingdom, which plays a role in modulating signalling pathways.55 Therefore, it can be hypothesised that these scavengers in LB and TSB quench the NO radicals, along with any generated RNS or ROS before they induce nitrosative and oxidative stress in the organisms.
Furthermore, studies have indicated that nutrient-poor conditions can increase a microorganism's susceptibility to antimicrobial agents.56 This increased susceptibility to GNP/NO under nutrient-poor conditions may occur due to a lack of resources. For example, superoxide dismutase (SOD), an enzyme that mitigates oxidative stress in microorganisms, requires Mn, Fe, Cu or Zn ions for its catalytic activity.57 However, these ions are also essential for protein synthesis and lipid metabolism in microorganisms.58 Due to resource limitations in nutrient-poor conditions, ions that would typically be used to help counteract nitrosative and oxidative stress are instead preferentially used to maintain minimum or essential metabolic functions,59 making microorganisms more susceptible to GNP/NO under nutrient-poor conditions. These results highlight the importance of assessing the antimicrobial efficacy of NO-releasing biomaterials in various environments to fully understand their potential.
This study also assessed the antimicrobial efficacy of GNP/NO after 0, 3 and 6 months of storage. The antimicrobial efficacy of GNP/NO decreased over time in both nutrient-poor and nutrient-rich conditions against all microorganisms tested, consistent the NO release kinetics results. Despite this reduction, GNP/NO7 was still found to significantly reduce planktonic S. aureus under nutrient-poor conditions at t = 6, indicating that GNP/NO still maintained partial antimicrobial efficacy after 6 months of storage.
Given that many hospital-associated chronic infections are often the result of biofilm formation, we investigated the antibiofilm efficacy of GNP/NO. This study assessed the ability of GNP/NO particles to disperse established S. aureus or E. coli biofilms, under both nutrient-poor and nutrient-rich conditions, at t = 0, t = 3 and t = 6.
Incubation of GNP/NO with established S. aureus and E. coli biofilms resulted in complete biofilm dispersion after 24 h under nutrient-poor conditions, up to t = 3. This finding is consistent with many studies demonstrating that NO is an effective biofilm dispersant.60,61 Interestingly, GNP/NO2.5 was unable to cause significant biofilm disruption at any time point for either S. aureus or E. coli biofilms, suggesting that higher concentrations of GNP/NO may be required to combat biofilms than planktonic bacteria.
Under nutrient-rich conditions, it was revealed that despite GNP/NO causing significant reductions in E. coli biofilms at 4 h, reformation was observed after 24 h. NO-mediated biofilm dispersal in Gram-negative bacteria, such as E. coli has been attributed to the activation of phosphodiesterase enzymes by NO, which hydrolyses cyclic-di-GMP. This reduction in cyclic-di GMP triggers a shift from a non-motile to a motile state, leading to biofilm dispersal.62 However this shift does not eradicate the bacteria, allowing biofilms to reform over time,63 aligning with the findings of this study. In contrast, NO-mediated dispersal of Gram-positive, such as S. aureus appears to occur independently of cyclic-di-GMP, though its exact mechanisms remains largely unknown.60 Notably, in this study the reformation of S. aureus biofilms was only observed once at t = 3, under nutrient-rich conditions, potentially supporting the idea that NO disperses S. aureus biofilms through an alternative pathway.
Finally, we study assessed the cytotoxic effects of GNP/NO on L929 cells according to ISO-10993. The results demonstrated that GNP/NO was cytocompatible, with no reduction in cell viability below 70%. Interestingly, several tested samples displayed increased cell viability. As gelatin is known to promote cell proliferation,64 and this observation is likely due to increased cell proliferation.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4na01042f |
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