Multidrug resistance (MDR) is a worldwide public health threat that is increasing costs and morbidity for both human and veterinary patients.1–3 Antimicrobial-resistant infections are diagnosed in > 2 million people annually in the United States,4 and retrospective evaluation of companion animal antibiograms has revealed frequencies of up to 40% for various methicillin-resistant staphylococcal infections.5 As a result of its numerous bactericidal effects, silver was a mainstay of infection management prior to the introduction of antimicrobials in the early 20th century,6 and microbial resistance against silver is rare.7 The in vitro antimicrobial effects of silver nanoparticles (AgNPs) against multiple bacteria, viruses, and eukaryotic organisms,8 including Escherichia coli,9,10 Staphylococcus aureus,10 and Pseudomonas aeruginosa,11 have been well documented. In aqueous solutions, AgNPs release silver ions that interfere with cell wall and cell membrane formation and various intracellular functions.12 Metallic silver is available in a variety of commercial formulations for use in veterinary medicine, such as silver sulfadiazine 1% cream and silver-impregnated hydrofiber dressings. A commercial product containing alginate and AgNPs (Acticoat, Smith+Nephew) has been shown to reduce microbial growth of a variety of gram-positive and gram-negative bacteria over a 24-hour period.13 To our knowledge, however, no studies have been published on the use of sustained-release AgNP constructs for ongoing local treatment following wound closure.
Various carriers have been evaluated for use as sustained-release constructs in veterinary medicine. Antimicrobial-impregnated calcium sulfate hemihydrate (CSH) beads are commonly used in orthopedic procedures to help prevent osteomyelitis and the development of implant-associated biofilms.14,15 These beads allow for high local tissue concentrations of antimicrobials and become inapparent radiographically within 5 weeks.15 Poloxamer 407 is a nontoxic, biodegradable synthetic hydrogel that has been used for controlled drug delivery and mesenchymal stem cell encapsulation applications.16 This thermosensitive gel can be mixed with other liquids when cooled and develops into a gel at room temperature.16 Compressed absorbable gelatin sponges are frequently used intraoperatively as focal hemostatic agents. These absorptive, biologically inert, malleable sponges have been shown to dissolve nearly completely dissolve within 40 days.17
Development of a widely available sustained-release AgNP construct may allow for earlier wound closure, reduced treatment costs, and greater success with treatment of MDR infections. Identification of the rate, pattern, and duration of elution of AgNPs from such a construct could be used to guide clinical recommendations for the integration of nanotechnology into clinical practice and treatment of MDR infections. The objectives of the study reported here were to determine and compare the rate, pattern, and completeness of AgNP elution in vitro over 7 days from 3 carrier media (CSH beads [AgNP–CSH beads], poloxamer 407 [AgNP–gel], and a gelatin sponge [AgNP–sponge]) when incubated in PBSS. We hypothesized that (1) complete elution of AgNPs from the AgNP–gel and AgNP–sponge constructs would occur within 24 hours and that (2) maximum elution from the AgNP–CSH bead constructs would occur within 7 days.
Materials and Methods
Carrier media preparation
Three commercially available carrier media were used: CSH beads (Kerrier LLC), poloxamer 407 gel (Pluronic F-127, Sigma Aldrich), and a compressed absorbable gelatin sponge (Vetspon, Elanco). Ten-nanometer-diameter AgNPs (0.02 mg/mL; Sigma Aldrich) were added to each carrier medium to create constructs with 0.01 mg AgNP content/construct. Each type of carrier medium was evaluated in quadruplicate, resulting in a total of 12 constructs: 4 AgNP–CSH bead constructs, 4 AgNP–sponge constructs, and 4 AgNP–gel constructs. Constructs were placed in 1-inch-diameter dialysis tubing with 12- to 14-kDa pores (Carolina Biological Supply Co), and surgical stainless steel clips (Hemoclips, Teleflex) were used to close each end of the tubing. The tubing was then submerged in 25 mL PBSS (ThermoFisher Scientific; Figure 1) in a jar, and the jars were placed in an incubator (38°C; 5% carbon dioxide).
Photograph of silver nanoparticle (AgNP) constructs created with 3 carrier media (left to right: calcium sulfate hemihydrate beads, a compressed absorbable gelatin sponge, and poloxamer 407) and submerged in PBSS. Each construct was placed in 1-inch-diameter dialysis tubing with 12- to 14-kDa pores, and surgical stainless steel clips were used to close each end of the tubing. No attempt was made to remove air from the tubing at the time of closure.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.21.12.0211
A single piece of dialysis tubing that had been sealed in the same fashion as the carrier medium constructs was used as a negative control. Prior to carrier medium construct preparation, a similar construct was prepared, submerged in PBSS at room temperature, and observed visually for 2 weeks to evaluate for gross changes to the solution, clips, and dialysis tubing.
CSH beads were made in accordance with directions provided by the manufacturer. The total powder in the kit was divided by weight into 3 equal portions. The manufacturer recommended adding a total volume of 4.0 mL liquid to the total amount of power provided in each kit. Therefore, 1.33 mL liquid was added to each of the 3 equal portions of powder. This liquid consisted of 0.83 mL of the saline solution provided in the kit and 0.5 mL of the AgNP solution (0.2 mg AgNPs/mL). The saline solution was added first and gently mixed for 60 seconds with the provided mixing tool; the AgNP solution was then added and mixed for an additional 30 seconds. Each of these 3 mixtures, each containing 0.01 mg AgNPs, was then packed into separate 5-mm-diameter bead mats provided by the manufacturer. The bead mixtures were allowed to set at room temperature for 120 minutes. A second kit was used to create another 3 sets of CSH beads in the same manner, resulting in 6 sets of AgNP–CSH beads, each containing 0.5 mL AgNP solution. Four of these bead sets were used in our study, with the remaining 2 sets of AgNP–CSH beads reserved as backup in case of specimen loss. Each of the 4 sets of AgNP–CSH beads was packaged individually in a piece of dialysis tubing. The tubing was cut to length to accommodate the beads, with approximately 1 cm of tubing available on either end to allow closure of the construct with 2 alternating clips on either end of the tubing.
The poloxamer solution was stored as a liquid at 4°C, as specified by the manufacturer, until the time of mixing with the AgNP solution. Each poloxamer 407 gel sample was prepared by mixing 2.5 mL 30% gel and 0.625 mL AgNP solution (0.02 mg of AgNPs/mL) in a 6-mL syringe. The contents of the syringe contained 0.005 mg AgNPs/mL of gel. Each syringe was inverted gently for 5 minutes to allow mixing of the liquid gel and AgNP solution. The mixture was then left to sit at room temperature for 15 minutes to allow solidification of the gel. A slightly greater quantity of sample was prepared than was ultimately used to create each construct to account for sample that would be left behind in the mixing syringe. A total of 2.5 mL AgNP–gel containing 0.0125 mg AgNPs was then injected into a piece of dialysis tubing that had been sealed on 1 end with 2 alternating clips, and the other end of the tubing was then sealed with 2 alternating clips.
Each of 4 compressed absorbable gelatin sponges (2 X 0.6 X 7 cm) were placed in a piece of dialysis tubing of appropriate length sealed on 1 end with 2 alternating clips, and 0.5 mL AgNP solution (0.02 mg AgNPs/mL) was injected into the center of the sponge, so that each sponge contained 0.01 mg AgNPs. The other end of the tubing was then sealed with 2 alternating clips.
Construct sampling
Immediately prior to assembly of the AgNP–carrier medium constructs (time 0), samples of the stock PBSS (1.0 mL) and the AgNP solution (0.1 mL) were collected to verify initial AgNP concentrations. A sample (1.0 mL) was also collected from the control construct after 2 weeks of submersion in PBSS.
For the test constructs, the same sampling order was maintained throughout the study. Each jar containing a construct was removed briefly from the incubator at the time of sampling, and the jar was agitated gently for 20 seconds prior to sample collection. A 1.0-mL volume of eluate was removed from each jar and stored in a 1.5-mL Eppendorf tube at –80°C until analysis. All samples were collected in duplicate. After sample collection, the entire remaining eluate was removed from each jar and replaced with 25 mL fresh, prewarmed PBSS. Jars containing the constructs were then returned to the incubator.
Samples were collected from each of the 12 test constructs and the control construct at 2, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 hours. In addition, jars were agitated gently for 30 seconds at 60, 84, 108, 132, and 156 hours to disperse the eluate evenly at consistent time points.
Data analysis
One full set of the duplicate samples was analyzed. The quantity of AgNPs in each sample was determined by means of acid digestion followed by inductively coupled plasma mass spectrometry (ICP-MS; NexION 300D, Perkin Elmer), as previously described.18,19 The short-term precision was < 3% of a relative SD, and the long-term stability was < 4% of a relative SD over 4 hours. Isotope ratio precision was < 0.08% of a relative SD. The AgNP detection limit was 0.047 ng/mL, and all samples with concentrations less than this limit were recorded as 0 ng/mL. ICP-MS results were used to calculate the total quantity and percentage of silver eluted into the PBSS for each construct at each time point and cumulatively over the course of the 168-hour study period. A mixed-effects model was used to evaluate differences in release of AgNPs over time and among carrier media.
Calculations
The 0.1-mL stock solution sample evaluated with ICP-MS had a concentration of 0.017 mg/mL, rather than the marketed concentration of 0.02 mg/mL, and all results were adjusted for this lower AgNP stock solution concentration. Mean quantity of AgNPs eluted from each of the 4 constructs for each of the 3 carrier media was calculated for each time point. Values for each time point were then added to determine the total cumulative quantity of AgNPs released over the evaluation period. On the basis of an initial quantity of 8,500 ng of AgNPs/construct, the cumulative quantity of AgNPs released was expressed as a percentage. The pattern of elution was illustrated by calculating the percentage of the total quantity of AgNPs released from each construct over the 168-hour evaluation period at each time point. The rate of AgNP release from each of the 3 carrier media was calculated for each time point.
Results
Samples were obtained at all time points for all constructs, and all samples were analyzed. No AgNPs were detected in the stock PBSS, the control construct, or the dialysis tubing suspended in PBSS for 2 weeks. No gross changes to the PBSS, clips, or dialysis tubing were seen after 2 weeks of submersion in PBSS. All test constructs remained intact without visual changes noted.
The total amount of AgNPs released over the 7-day study period varied significantly (P < .001) among the 3 carrier media, with incomplete release of AgNPs from all 3 carrier media after 168 hours (Figure 2). The total amount of AgNPs released was 87.82 ng (1.03%) for the AgNP–CSH bead constructs, 8,401.02 ng (98.84%) for the AgNP–gel constructs, and 1,503.45 ng (17.69%) for the AgNP–sponge constructs. All 3 carrier media released AgNPs with an initial burst followed by a gradual decrease in release rate; however, the amount of AgNPs released at each time point differed significantly (P < .001). Most of the total AgNPs that eluted from the CSH beads (64.99%) and gel (88.20%) constructs were detected within the first 24 hours; however, most of the total AgNPs that eluted from the sponge constructs (61.90%) were detected within the first 72 hours (Figure 3). Although the gel and sponge constructs continued to release AgNPs until the end of the 168-hour evaluation period, no additional elution was detected from the CSH beads after the first 72 hours. Release rates (nanograms of AgNPs per hour) were greatest within the first 2 hours for all 3 carrier media: CSH beads, 3.29 ng AgNPs/h; gel, 1,466.66 ng AgNPs/h; and sponge, 66.74 ng AgNPs/h.
Mean ± SEM quantity of silver nanoparticles (AgNPs) eluted from AgNP–carrier medium constructs (n = 4 constructs/carrier medium) at each sampling time (A), total cumulative quantity of AgNPs released over time (B), and cumulative percentage of initial quantity of AgNPs (8,500 ng) eluted over time (C). Sustained release of AgNPs was seen from all sustained-release AgNP constructs for a minimum of 72 hours.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.21.12.0211
Mean percentage of the total quantity of silver nanoparticles (AgNPs) released from AgNP–carrier medium construct during the 168-hour study period (A; AgNP–gel constructs released 98.84% of the total initial AgNPs, AgNP–sponge constructs released 17.69%, and AgNP–CSH bead constructs released 1.03%); rate of AgNP release from AgNP–CSH bead constructs over time (B); rate of AgNP release from AgNP–gel constructs over time (C); and rate of AgNP release from AgNP–sponge constructs over time (D).
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.21.12.0211
The greatest quantity of AgNPs released at a given time point and greatest release rates after 2 hours differed among carrier media. The greatest quantity of AgNPs released from CSH beads (20.34 ng, 1.70 ng AgNPs/h) at a single time point occurred at 36 hours (Table 1). Of the total amount of AgNPs released from the AgNP–CSH bead constructs over the study period, 64.99% were detected within the first 24 hours, 96.40% were detected within the first 48 hours, and 100.0% were detected within the first 72 hours (Figure 3). The greatest quantity of AgNPs released from the gel (2,933.32 ng, 1,466.66 ng AgNPs/h) at a single time point occurred at 2 hours. Of the total amount of AgNPs released from the AgNP–gel constructs over the study period, 88.20% were detected within the first 24 hours, 94.19% were detected within the first 48 hours, and 96.69% were detected within the first 72 hours. The greatest quantity of AgNPs released from the sponge (228.59 ng, 9.53 ng AgNPs/h) at a single time point occurred at 72 hours. Of the total amount of AgNPs released from the AgNP–sponge constructs over the study period, 29.50% were detected within the first 24 hours, 46.69% were detected within the first 48 hours, and 72.73% were detected within the first 72 hours.
Mean ± SEM quantity of silver nanoparticles (AgNPs)released and rate of AgNP release for AgNP–carrier medium constructs (calcium sulfate hemihydrate [CSH] beads, poloxamer 407, and a compressed absorbable gelatin sponge; 4 constructs/carrier medium) submerged in PBSS.
| CSH beads | Poloxamer 407 | Gelatin sponge | ||||
|---|---|---|---|---|---|---|
| Time (h) | Quantity released (ng) | Release rate (ng/h) | Quantity released (ng) | Release rate (ng/h) | Quantity released (ng) | Release rate (ng/h) |
| 2 | 6.57 ± 0.21 | 3.29 | 2,933.32 ± 33.96 | 1,466.66 | 133.48 ± 3.66 | 66.74 |
| 6 | 7.61 ± 0.18 | 1.90 | 2,140.29 ± 8.35 | 535.07 | 45.182 ± 1.58 | 11.30 |
| 12 | 14.60 ± 0.28 | 2.43 | 1,519.03 ± 11.57 | 253.17 | 114.85 ± 3.93 | 19.14 |
| 18 | 15.15 ± 0.27 | 2.53 | 557.00 ± 6.09 | 92.88 | 76.49 ± 2.81 | 12.75 |
| 24 | 13.14 ± 0.19 | 2.19 | 259.66 ± 3.56 | 43.28 | 73.56 ± 1.57 | 12.26 |
| 36 | 20.34 ± 0.34 | 1.70 | 320.88 ± 4.71 | 26.74 | 139.18 ± 2.18 | 11.60 |
| 48 | 7.25 ± 0.17 | 0.60 | 182.39 ± 2.92 | 15.20 | 119.27 ± 1.39 | 9.94 |
| 72 | 3.17 ± 0.07 | 0.13 | 210.64 ± 3.89 | 8.78 | 228.59 ± 1.32 | 9.53 |
| 96 | 0 | 0 | 114.16 ± 2.13 | 4.76 | 162.94 ± 0.55 | 6.79 |
| 120 | 0 | 0 | 75.82 ± 1.40 | 3.16 | 138.56 ± 0.37 | 5.77 |
| 144 | 0 | 0 | 49.99 ± 0.94 | 2.08 | 134.90 ± 1.03 | 5.62 |
| 168 | 0 | 0 | 37.86 ± 0.82 | 1.58 | 136.47 ± 1.01 | 5.69 |
Discussion
An initial burst of release of AgNPs followed by a continued release of AgNPs for a minimum of 72 hours was seen with all 3 carrier media used in our study. The AgNP–CSH bead constructs reached maximum elution within 72 hours, and any additional release was less than detectable limits. An initial burst release of AgNPs was seen with the AgNP–gel and AgNP–sponge constructs, followed by continued release throughout the study period beyond the first 24 hours.
The AgNP–CSH bead constructs released the lowest quantity of AgNPs, with only 1.03% of the AgNPs released within 72 hours, and no additional release seen after this time point. Although the exact reason for this limited release is unknown, a variety of factors may have contributed. One of the antimicrobial mechanisms of silver ions includes interaction with sulfhydryl groups leading to inactivation of enzymes.16 Although no amino acids were present in the CSH beads, AgNP elution may have been limited as a result of degradation or interaction between the silver ions and calcium sulfate. Furthermore, the mixing technique for the AgNP–CSH beads was based on nonstandard liquid mixing instructions provided by the manufacturer; however, an approximate set time was not available from the manufacturer because of the novel nature of this technique. To increase the chances of appropriate solidification and handling, the AgNP–CSH beads were allotted 120 minutes to set, which was approximately 25% longer than the longest set time provided by the manufacturer. It is possible that a different set time or mixing technique may have resulted in a different elution profile. In addition, a small portion of the AgNP–CSH bead mixture was lost between wells on the bead mat at the time of bead preparation as a result of the described manufacturer technique of spreading the mixture into the wells of the bead mat. Although the amount of mixture lost between wells was minimal, direct injection of the mixture into each of the bead wells may have minimized sample loss and resulted in a slightly greater total percentage of AgNP release. Furthermore, previous studies have documented the influence of bead size on elution pattern,20 and it is possible that a larger CSH bead size may have resulted in an elution period beyond 72 hours. Although the maximum elution from AgNP–CSH beads was reached within 7 days as expected, the low total quantity of AgNPs released from this construct likely limits its value as a sustained-release carrier medium when prepared as described.
The AgNP–gel constructs had the greatest initial burst release of all constructs. Although these constructs showed near-complete release of the total initial AgNPs by the end of the evaluation period, 88.195% of the initial AgNPs were released within the first 24 hours. The AgNP–gel constructs continued to release AgNPs throughout the study period; however, the rate of release declined steadily from an initial release rate of 1,466.66 ng/h to a final rate of 1.58 ng/h. Although the clinical antimicrobial effects of AgNPs on specific strains of bacteria should be evaluated on a case-by-case basis, a previous study21 showed > 90% sustained inhibition of ampicillin-resistant E coli and MDR P aeruginosa after initial exposure to high concentrations (50 mM) of AgNPs. The clinical efficacy of the release pattern of the AgNP–gel constructs was beyond the scope of our study; however, this construct may be considered for clinical scenarios requiring a large initial release of AgNPs. The poloxamer solution was stored as a liquid at 4°C until the time of mixing with the AgNP solution. The poloxamer and AgNP solutions were mixed together easily as liquids, but formed a thick gel within approximately 5 minutes of mixing. This gel was more technically challenging to handle than the CSH beads and sponge. Therefore, an excess of the AgNP-infused poloxamer gel was made, and a proportionate amount was placed in the dialysis tubing to ensure that an equal quantity of the AgNP solution was placed into the dialysis tubing for each of the AgNP–gel constructs. In a clinical setting, the poloxamer solution could be mixed aseptically with the solution of interest at the desired concentration prior to its anticipated time of use and then left to solidify at room temperature. After the gel has solidified into a gel at room temperature, it could be injected into tissue or spread to cover a wound bed.
The AgNP–sponge constructs released an intermediate quantity of AgNPs (17.69% of the initial quantity), but had the greatest release rate by the end of the study. AgNPs have been shown to bind irreversibly to proteins in vitro.22 The gelatin sponges were manufactured from purified porcine skin, and the incomplete release of AgNPs from these constructs can likely be attributed to irreversible binding of the AgNPs to these proteins.23 Although these constructs provided the longest duration of sustained release, a previous study24 evaluating the antimicrobial effects of AgNPs showed that AgNP antimicrobial effects are largely concentration dependent. Therefore, the gel constructs may have a greater clinical antimicrobial benefit.
Some variation can be seen between the methods used in our study and the wide array of methodologies used in other elution studies. Incomplete removal of PBSS at the time of sampling has been used to avoid an acute change in concentration gradient25,26; however, this has been speculated to result in degradation of the eluate. Therefore, complete removal of PBSS and replacement with fresh PBSS at each sampling time was elected. Previous studies23,25,26 have reported wide ranges in the volume of PBSS used per sample (6 to 300 mL); however, full submersion of the construct without excessive PBSS appeared to be the consistent factor for determining the ideal PBSS volume. Therefore, 25 mL PBSS was chosen to submerge all AgNP constructs fully without adding excessive fluid volume and causing unnecessary AgNP dilution.
Limitations of our study include those associated with the use of an in vitro elution study to mimic in vivo conditions, such as the lack of local and systemic patient factors (eg, stage of wound healing, fluid flow dynamics, pH, and temperature) that might influence elution.27 Constant, continuous agitation of the samples was desired during the study design phase to allow even contact of the entire volume of PBSS with the AgNP constructs, but was unavailable in an incubator environment. Temperature control of the samples was prioritized; however, each sample was agitated gently immediately prior to sampling and at a minimum of every 12 hours to allow for mixing of the eluate and to encourage even distribution of AgNPs in the PBSS prior to sample collection. Wound beds have the potential to have a more acidic pH than healthy, uninjured tissue, and it is possible that the elution profile may be influenced by variations in pH. Another limitation of our study was the lack of construct evaluation beyond 168 hours. Quantification of the remaining AgNPs in each construct would have allowed for further evaluation of potential loss or contamination and may have allowed for confirmation of suspected AgNP protein binding. In addition, there is no reference regarding the use of dialysis tubing with AgNPs, and therefore there is a possibility that the dialysis tubing interacted with the constructs and limited the elution of AgNPs into the PBSS.
The AgNP–CSH bead constructs in our study showed no additional release of AgNPs after the first 72 hours and had a low total percentage of AgNPs released. The AgNP–sponge and AgNP–gel constructs continued to release AgNPs for the complete duration of the study. Although the AgNP–sponge constructs allowed for the most consistent release of AgNPs, the AgNP–gel constructs released the greatest total amount of AgNPs and provided the highest rate of release and highest concentration of AgNPs. The poloxamer gel appears to be a suitable medium for the production of sustained-release AgNP constructs as result of an initial high release, followed by continued release over 168 hours, resulting in a high percentage of total AgNP release. Additional studies are needed to evaluate the clinical efficacy of sustained-release AgNP constructs for the treatment of MDR infections in veterinary medicine.
Acknowledgments
The authors thank Abhijit Mukopadhyay of the Purdue University Veterinary Clinical Sciences Research Laboratory for assistance with material collection and Dr. George E Moore of the Purdue University Department of Veterinary Administration for assistance with study design and statistical analysis. The Nanomedicines Characterization Core Facility at the Center for Nanotechnology in Drug Delivery assisted with data analyses.
The study was internally funded by a VCS Graduate Student Research fund, but no external funding was used. The authors declare that there were no conflicts of interest.
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