Ex vivo effect of gold nanoparticles on porcine synovial membrane

Abstract

Gold nanoparticles (AuNPs) have great potential as carriers for local drug delivery and as a primary therapeutic for treatment of inflammation. Here we report on the AuNP-synovium interaction in an ex vivo model of intra-articular application for treatment of joint inflammation. Sheets of porcine femoropatellar synovium were obtained post mortem and each side of the tissue samples was maintained in a separate fluid environment. Permeability to AuNPs of different sizes (5-52 nm) and biomarker levels of inflammation were determined to characterize the ex vivo particle interaction with the synovium. Lipopolysaccharide or recombinant human interleukin-1β were added to fluid environments to assess the ex vivo effect of pro-inflammatory factors on permeability and biomarker levels. The synovium showed size selective permeability with only 5 nm AuNPs effectively permeating the entire tissues' width. This process was further governed by particle stability in the fluid environment. AuNPs reduced matrix metalloproteinase and lactate dehydrogenase activity and hyaluronic acid concentrations but had no effect on prostaglandin E2 levels. Exposure to pro-inflammatory factors did not significantly affect AuNP permeation or biomarker levels in this model. Results with ex vivo tissue modeling of porcine synovium support an anti-inflammatory effect of AuNPs warranting further investigation.

Keywords: arthritis; ex vivo; gold; nanogold; nanoparticles; permeability; synovitis; synovium; ussing.

Figures

Figure 1. Experimental Protocol. (A) Schematic of the experimental groups. (B) Schematic of the experimental procedures (AuNPs = gold nanoparticles, Au = elemental gold, LPS = lipopolysaccharide, LPSV = LPS vehicle, IL-1β = interleukin-1β, IL1V = interleukin-1β vehicle, MMP = matrix metalloproteinase, LDH = lactate dehydrogenase, HA = hyaluronic acid, PGE2 = Prostaglandin E2)

Figure 2. Size dependent permeation of the synovial membrane assessed by histomorphometry. Bars represent the average surface area of the gold specific tissue stain (pixel2) for each size of gold nanoparticle (AuNP) used (5, 10, 20 and 52 nm). Significance was reached for all comparisons with 5 nm AuNPs (p < 0.001). Error bars represent the standard error for the mean.

Figure 3. Time dependent permeation of 5 nm gold nanoparticles through the synovial membrane. A significant difference in synovial AuNP permeation was observed between chambers receiving IL-1β vehicle and LPS or LPS vehicle. Significantly different permeation curves (p < 0.05) are indicated by different letters. Stars above time points indicate when and for what group sample concentrations were significantly different from baseline values. Error bars represent the standard error for the mean. (LPS = Lipopolysaccharide, IL-1β = Interleukin-1β, V = Vehicle)

Figure 4. Representative images of the size dependent permeation of the synovial membrane. Tissues had been exposed to (A) 5 nm AuNPs, (B) 10 nm AuNPs, (C) 20 nm AuNPs and (D) 52 nm AuNPs prior to gold enhancement. AuNPs of 5 nm size can be seen penetrating all tissue layers. Elemental gold appears black. (AuNPs = gold nanoparticles; scale bar = 50 µm)

Figure 5. Representative transmission electron microscopic images of AuNP localization. By enlarge AuNPs were scattered throughout the cytoplasm and organelles of synoviocytes including the smooth endoplasmatic reticulum and mitochondria (C and D). Extracellular location of large particle agglomerates were also appreciated (A and B). Evidence for larger AuNPs agglomerates in endosomes (white arrow) is illustrated in (D).

Figure 6. Molecular biomarkers by experimental group Group AuNP/P-Infl: Tissues exposed to AuNPs and pro-inflammatory factor; Group AuNP/V: Tissues exposed to AuNPs and vehicle; Group C/IL1V: Synovium exposed to Interleukin-1β vehicle only; Group C/IL-1β: Synovium exposed to Interleukin-1β. Error bars represent the standard error for the mean. Bars above group comparisons indicate significance (p < 0.05).

Figure 7. Representation of synovial ulceration. The degree of synovial ulceration was graded based on the estimated percentage of synovium missing over the length of the sample evaluated. (A) shows a representation of the synovial lining at 60 × magnification that was considered normal; a continuous lining of synovial cell is present. (B) represents tissue at the same magnification with ulceration of the synovium; a discontinuous lining of synovial cell is observed over the subsynovial connective tissue. (Bar = 30µm).