Fig 1: Evaluating the safety profile of microcapsule delivery for localized immunomodulation in healthy Yucatan Pigs.A) Demonstration of capsule delivery in pig cadaver lungs. Capsules were dyed blue for visualization B-C) Quantification of bronchoalveolar lavage (BAL) fluid and plasma concentrations of IL-1Ra and IL-10 before instillation, two days after, and at euthanasia on day 28 D) Triglycerides, Total Cholesterol, and Glucose blood levels on days 0, 2, and 28, showing no significant change. E) Albumin blood levels as a marker of liver function showing no significant change. F) Saturation of peripheral oxygen and end-tidal carbon dioxide at the start and end of each procedure G) H&E-stained sections of the lung. Scale bar, 2 mm and inset, 200 mm. Data represented as mean ± SEM (n=3).
Fig 2: Evaluation of Micro-Capsules in an LPS model of ARDS.A) Total number of cells collected from the BAL fluid 24 hours after LPS and capsule treatment determined by Trypan Blue stain. LPS versus RPE-IL10 ****P<0.0001; LPS versus RPE-IL1Ra ****P<0.0001 B)The concentration of TNF-α and C) MCP-1 in BAL fluid was measured by ELISA from rats receiving treatment as described and harvested 24 hours after LPS administration. TNF-α: LPS versus RPE-IL10 ****P<0.0001; LPS versus RPE-IL1Ra *P=0.0481; MCP1: LPS versus RPE-IL10 *P=0.0191; LPS versus RPE-IL1Ra *P=0.00751; D) Quantified histology of the inflammation area at 1 day following LPS administration; LPS versus RPE-IL10 ****P<0.0001; LPS versus RPE-IL1Ra ****P<0.0001 E) Scanned images of H&E-stained lungs 24 hours following LPS administration and treatment. Scale bar, 2 mm. Inset is a magnification of a representative section from each lung and depicts the pathology described in the scoring metrics. Scale bar, 200 mm. All data is represented as mean ± SEM (n = 7–12 rats/group), calculated using one-way ANOVA followed by Tukey’s multiple comparison test
Fig 3: Characterization of a microcapsule delivery platform for localized immunomodulation.A)Schematic of piggyBac transposon-mediated engineering technique used to develop a library of therapeutic proteins with schematic of intratracheal instillation of alginate hydrogels loaded with retinal pigment epithelial (RPE) cells. Inset shows interaction between alginate capsules engineered to produce immunomodulatory proteins and local immune cells within bronchioles. B)Representative brightfield image of alginate capsules and cell-laden capsules. Scale bar, 400 mm C) Ex vivo fluorescent imaging of dextran-loaded capsules distributed throughout the lungs immediately after instillation. Scale bar, 1 mm. D) MicroCT scans of rat lungs after instillation of empty or barium sulfate-loaded capsules. E) In vivo bioluminescent imaging of firefly luciferase expression cell capsule retention versus an equivalent dose in unencapsulated, free cells. 60s exposure time (n=9–10 rats/group) F) In vivo bioluminescent total flux analysis 24-hours post-instillation. Analyzed using an unpaired two-tailed Welch’s t-test. *P=0.0342 (n = 9–10 rats/group) G-K) Quantification of bronchoalveolar lavage (BAL) fluid and plasma concentrations of IL-10, IL-1Ra, FGF-21, IL-13, and IL-4 24 hours post-instillation of 100 ml of 1E7 cells/mL capsules. The dashed line represents basal levels. L-M) IL-10 dose escalation measured in BAL and plasma 24 hours post-instillation with L) increasing capsule volume (saline, 50, 100, and 150 μL of 1E7 cells/mL RPE-IL-10 capsules) and M) increasing cell density (100 μL of empty capsules, 1E4, 1E5, 1E6, 1E7 cells/mL of alginate) N) BAL and plasma concentrations of IL-10 as a function of time with a dose of 100 μL of 1E7 cells/mL RPE-IL-10 capsules. Dashed line represents basal levels. O) Representative histology images corresponding to time points in panel M. Scale bar, 200 mm and 25 mm P)BAL and plasma concentrations of IL-10 24 hours post-instillation with RPE-IL-10 capsules and upon repeat dose at 30 days. (n=8 rats/group). Unless otherwise specified, all other quantification data are represented as mean ± SEM (n=5 rats/group).
Supplier Page from Abcam for Rat IL-1Ra ELISA Kit