Silver nanoparticles can be sampled by ultrafiltration probe but elution into & recovery from plasma and DPBS differs in vitro

We compared 1) the influence of elution fluid on rate, pattern, and completeness of silver nanoparticle (AgNP) elution, and 2) ultrafiltration (UF) probe and direct sampling in vitro. Six specimens (2.5ml of 0.02mg/ml 10nm AgNP and 5.0ml of 30% poloxamer 407) contained in a dialysis tube (12-14kDa pores) were placed in 100ml Dulbecco’s Phosphate Buffered Saline (DPBS) (n=3) or canine plasma (n=3) for 96h on a stirred hot plate (37°C and 600rpm) and sampled 20 times. Six pipette and UF probe samples were taken of a 0.001mg AgNP/ml DPBS or plasma solution. Inductively coupled plasma mass spectrometry was used to analyze Ag. Stock plasma contained Ag. At 96h, 5/6 dialysis tubes had not fully released AgNP. One peak in hourly Ag increase was present in DPBS (10-13h), and two peaks in plasma (6-8h and 10-13h). The hourly Ag increase in plasma decreased earlier than in DPBS. UF probe sampling was possible in both DPBS and plasma and resulted in higher Ag concentrations but with more variation than pipette samples. While in vitro use of DPBS might be more cost effective, plasma should be considered due to difference in elution and recovery.


Introduction
With the increase in antimicrobial resistance in veterinary medicine [1,2], novel strategies and antimicrobials to combat infections are (re)gaining favor.One strategy might be to deliver a high local dose of antibiotics [3][4][5][6][7].Another might be to explore the use of non-antibiotic antimicrobials, such as silver (Ag), especially silver nanoparticles (AgNP).They have received considerable interest for their use against micro-organisms [8,9] and in wound care [10].
Poloxamer 407 is a versatile reverse gelatinating polymer that is safe for implantable use [11,12], and has found use for delivery of antifungals [12], chemotherapeutics [11,13], and antibiotics [14].It also might have inherent antimicrobial properties of its own [15].Prior silver elution studies have been performed into phosphate-buffered saline (PBS) from poloxamer 407 [16], into deionized water baths (from silver oxide films) [17], or aqueous samples with varying NaCl concentrations (from solid coated specimens) [18].Plasma might be a closer in vitro fluid approximation to an in vivo environment than PBS and could mimic protein interactions that might be encountered.The elution into human plasma from an antibiotic containing polymethyl methacrylate (PMMA) construct as well as the effect plasma on PMMA properties was reported [19].However, plasma has not been evaluated for suitability of Ag release studies, and whether the benefit could outweigh the increased cost of commercial canine plasma (~$2,400/500ml; Innovative research, Novi, MI) over PBS (~$30/500ml; Gibco, Thermofisher Scientific, Billings, MT).
The ultimate goal of sustained release compounds assessed by elution studies is their in vivo application.Ultrafiltration (UF) probes have been used successfully in dogs [20,21] and are used to sample compounds present in the tissue, without the need for more invasive sampling methods, such as tissue biopsies [22].Establishing the feasibility of sampling Ag with UF probes would be preferable prior to their use to investigate the in vivo pharmacokinetic profile of Ag containing local delivery products.However, the possibility of sampling AgNP via UF probe has not been assessed, nor has a comparison of canine UF probes and direct sampling been reported for AgNP.
We aimed to 1) determine and compare the influence of elution fluid on rate, pattern, and completeness of AgNP elution in vitro, and 2) compare UF probe sampling with direct samples.
We hypothesized that 1) elution of AgNP into Dulbecco's PBS (DPBS) will be similar to elution in canine plasma, and that 2) UF probe sampling will be constant over time and yield similar results as direct sampling.

Materials and Methods
An observational elution study and repeat sampling study were performed separately.Canine Ultrafiltration Probes (BASi Instruments, West-Lafayette, IN) were assembled as per manufacturer instructions [23].

Elution
A commercial 0.02mg/ml 10nm silver nanoparticles (AgNP) in aqueous buffer with sodium citrate as stabilizer (Sigma Aldrich, Saint Louis, MO) was used for the study.Six elution specimens were prepared.Each contained 2.5ml of the 0.02 mg/ml 10nm AgNP stock solution mixed with 5.0ml 30% poloxamer 407 (Pluronic® F-127, Sigma Aldrich) (1:2 ratio with a total Ag content of 0.05mg/specimen).The specimens were prepared in individual 12ml syringes <2 hours before use and stored refrigerated and shielded from light.Individual 150ml crystallization dishes (Synthware glass, Pleasant Prairie, WI) with 100ml fluid were prewarmed for 2 hours prior to starting the elution study.Three dishes had Dulbecco's phosphate-buffered saline without Calcium chloride or magnesium chloride added (DPBS, Gibco, Thermofisher Scientific, Billings, MT) and three had canine plasma with dipotassium EDTA (K2-EDTA) (IGCNPLAK2E500ml Canine Plasma lot 41273, Innovative research, Novi, MI).A 10 multiposition hotplate with magnetic stir bars was used (RT 10, IKA Magnetic Stirrers, Wilmington, NC) with settings at 37°C and 600 rpm throughout the experiment.Room temperature was set at 68°F.Specimens were created using a 10cm long strip of 1 inch dialysis tubing with 12-14kDa pores (Carolina Biological Supply Co, Burlington, NC) [24]).The distal free end was folded up length wise and secured in folded position using 2 large surgical stainless steel hemoclips (Hemoclips® Teleflex, Morrisville, NC) in opposite direction [16].The 1:2 AgNP-poloxamer mix was then placed in the tube, and the proximal free end of the tube folded and closed in similar fashion.All specimens were created in one batch and then placed in the prewarmed DPBS or canine plasma within 5 minutes after assembly.The t=0 sample was taken immediately after all specimens were submerged in the same order of assembly and placement.Twenty samples (0.15ml each) were taken over 96 hours with a decrease in frequency (0, 1, 2, 3, 4, 5, 6, 8, 10, 13, 17, 22, 27, 34, 42, 48, 58, 66, 72, 96 hours) with the sampling order consistent throughout.At 96 hours, an additional sample (by needle aspiration through the tube) of the fluid contained within the dialysis tube was taken.

Ultrafiltration probe sampling
Two specimens were prepared immediately prior to sampling in a 50ml conical centrifuge tube (Thermofisher Scientific) using graduated pipettes (Thermofisher Scientific) and a 3ml syringe (Covidien, Mansfield, MA).Each specimen contained 0.03mg of AgNP (1.5ml of the commercial 0.02mg AgNP stock solution) total in 28.5ml of either DPBS (n=1) or canine plasma (n=1) for a targeted fluid concentration of 0.001mg/ml of AgNP.Ultrafiltration probes were tested for patency and sampling using DPBS prior to use for the study.
Full submerging of the probe near the lowest point of the tube was ensured, and positioning was checked after each manipulation.Vacutainers without additives (Vacuette Blood collection tube, 3.0ml, no additive, Greiner Bio-One, Sigma Aldrich, St Louis, MO) were used to collect the probe samples and an additional 10ml of air was removed to increase negative pressure in each vacutainer to increase sampling speed.The initial sample of the study specimen was discarded to avoid risk of dilution by plain DPBS.Repeat samples using both a UF probe and direct sampling using a pipette from the same area as the membrane of the UF probe were taken at 0, 5, 10, 15, 30 and 60 minutes from the plasma and DPBS specimen.
Samples of commercial stock AgNP solution, DPBS and canine plasma were taken prior to the start of the study.All samples were stored at -78°C until batch analysis.

Sample and data analysis
The quantity of Ag in each sample was determined via inductively coupled plasma mass spectrometry (ICP-MS) as previously described [16].DPBS samples were diluted in 2% HNO3 and plasma samples were digested over night at 70°C in the 1:1 mixture of 70% HNO3 and H2O2 and analyzed using ICP-MS (Perkin Elmer NexION 300D) to determine the concentration of silver within each sample.The short-term precision was less than 3% relative standard deviation (RSD), and the long-term stability was <4% relative standard deviation over 4 hours.
Isotope-ratio precision was less than 0.08% relative standard deviation.The Ag detection limit was 0.001ng/ml, and quantification limit at 0.002ng/ml, and all samples below this limit were recorded as 0ng/ml.Silver concentrations were expressed in parts per billion (ppb), with 1ppb=1ng/ml.Hourly increase in Ag was calculated as the difference between the measured values at two subsequent time points divided by the hours between time points (expressed as ppb/hr).The data of the 3 elution specimens will be expressed graphically as mean±SD.The values of the repeat sampling experiment will be reported as individual results and a mean±SD (RSD) for both specimens in an observational manner without further statistical analysis.The RSD was calculated by dividing the SD by the mean and will be expressed as a %.

Results
Silver concentrations for stock solutions used in this study were: DPBS stock 0.19ppb Ag; plasma stock 4.83ppb Ag and the commercial 0.02mg/ml AgNP solution contained 24,940ppb Ag.Assembled UF probes initially did not reliably yield appropriate negative suction to obtain a sample, and probes were not re-usable for repeat experiments.Additional negative pressure applied to the vacutainers together with sealing connecting points with glue (3g Tube, The Gorilla Glue Company, Cincinnati, OH) allowed single use sampling.

Elution
No leakage of any dialysis tubes was observed under gentle pressure immediately after assembly.Five out of 6 dialysis tubes were fully filled at 96 hours, the sixth was not fully filled when retrieved.The five fully filled specimens still contained more Ag than the surrounding fluid at 96 hours (Table 1), and release of Ag was not complete at 96 hours.A burst release of Ag was seen both into DPBS and plasma in the first 13 hours (DPBS, light grey) and 8 hours (plasma, dark grey), with the baseline amount in plasma higher than in DPBS (Fig 1AB)   Measured Ag content in both the fluid and Ag:poloxamer specimen remnant at 96 hours were less in plasma than DPBS, with the specimens still higher in Ag content than the surrounding DPBS or plasma.

Ultrafiltration probe sampling
Anticipated Ag content based on the stock (24,940ppb Ag (AgNP stock) diluted in 30ml) was 1,247ppb Ag of the diluted fluid.Ultrafiltration probes were able to collect Ag in both DPBS and plasma, with Ag content in fluid obtained via UF probe sampling higher than the corresponding pipette samples (Fig 3).However, the UF probe-obtained samples had more variation and values differed between sampling times and decreased over time (Table 2).Samples obtained by both methods had a measured Ag lower than anticipated, with the underestimation greater in DPBS than plasma.119.9±53.9(45%) Probes were allowed to sample for 4 minutes to obtain enough sample volume.Direct samples were taken using a pipettor at the time point.UF probe samples were taken by attaching a vacutainer to the probe for 4 minutes at the start time.DPBS= Dulbecco's Phosphate Buffered Saline, UF= ultrafiltration.The mean is provided with both standard deviation (SD) and relative standard deviation (RSD) for each sampling method over time from the same fluid.
. The increase of Ag measured in plasma decreased earlier, between 8-60 hours (dark grey, Fig 1AB), and the measured Ag decreased thereafter.The hourly increase of Ag in DPBS (light grey) tapered between 27-84 hours but continued throughout the study (Fig 1C).The highest hourly increase in Ag was between 10-13 hours in both DPBS (light grey) and plasma (dark grey) (Fig 1D).A clear single peak in DPBS (20.66ppb/hr) whereas two peaks were present for plasma: between 6-8 hours (12.31ppb/hr) and between 10-13 hours (13.85ppb/hr) (Fig 1D).The amount of Ag measured at 96 hours was higher in the remaining Ag:poloxamer mix contained within the tube than the surrounding fluid (both DPBS and plasma) (Fig 2).The Ag concentration measured at 96 hours in DPBS was higher (both specimen and fluid) than in plasma (Fig 2).The mean±SD total amount of Ag removed via sampling for the three DPBS fluid set ups was 330±51ng Ag and for the three plasma fluid set ups 198.1±71ngAg.

Fig 1 :
Fig 1: A. Elution of Ag into either DPBS (light grey) or plasma (dark grey), expressed as ppb

Fig 2 :
Fig 2: Ag amount (ppb) in elution fluid (DPBS or plasma) and remainder of the

Table 1 :
Silver (Ag) at 96 hours expressed as parts per billion (ppb) for the remaining elution fluid outside of the dialysis tube and the remaining specimen contained within the dialysis tube.Ag at the start of the experiment.*denotes the specimen that was not fully filled and the surrounding elution fluid.Silver at 96 hours was analyzed once for six specimens (3 each in DPBS and plasma) and elution fluid.

Table 2 :
Repeat pipette and ultrafiltration (UF) probe sampling of a planned solution of 1,247ppbAg in DPBS and plasma solution (one specimen each).