MicroRNA-223 Induced Repolarization of Peritoneal Macrophages Using CD44 Targeting Hyaluronic Acid Nanoparticles for Anti-Inflammatory Effects - Fig 4

<p>(a) Comparative transfection study of hyaluronic acid-poly(ethyleneimine) (HA-PEI) / miR-223 duplexes nanoparticles (NPs) and HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223) NPs in J774A.1 at 24 h, 48 h, and 72 h post-transfection. (b) miR-223 expression in primary peritoneal macrophages at 24 h and 48 h post-transfection of HA-PEI/miR-223 and Lipofectamine^®/miR-223. miR-223 expression was quantified by Taqman miRNA assay specific for miR-223; U6snRNA was used as a house keeping gene. *p<0.05 compared to untreated macrophages, n = 3.</p

<i>In vitro</i> anti-inflammatory study in peritoneal macrophages: expression of pro-inflammatory cytokines (a) TNF-α, (b) IL-1β, and (c) IL-6 mRNA level at 48 h post-transfection of macrophages with hyaluronic acid-poly(ethyleneimine) (HA-PEI)/miR-223 duplexes nanoparticles (NPs) or Lipofectamine^®/miR-223, followed by stimulation with LPS (100 ng/mL) for 6 h.

<p>qPCR was used to quantify mRNA levels. *p<0.05 compared to LPS-treated group, n = 3.</p

Repolarization of Tumor-Associated Macrophages in a Genetically Engineered Nonsmall Cell Lung Cancer Model by Intraperitoneal Administration of Hyaluronic Acid-Based Nanoparticles Encapsulating MicroRNA-125b

Tumor-associated macrophages (TAMs) acquire a pro-tumor (M2) phenotype, which promotes tumor growth, angiogenesis, and metastasis. Certain microRNAs (miRs), such as miR-125b, can reprogram TAMs into an antitumor/pro-inflammatory (M1) phenotype. Using CD44 targeting hyaluronic acid-poly­(ethylenimine) (HA-PEI)-based nanoparticles encapsulating miR-125b, we have herein shown macrophage-specific delivery and transfection upon intraperitoneal (i.p.) administration. We have exploited the inherent ability of peritoneal macrophages to migrate toward the inflammation/injury and demonstrated that following intraperitoneal administration of HA-PEI nanoparticles, there is an accumulation of HA-PEI nanoparticles in the macrophage-ablated lung tissues of both naïve and KRAS/p53 double mutant genetically engineered (KP-GEM) nonsmall cell lung cancer (NSCLC) mouse model. Additionally, upon transfection with miR-125b, we observed a >6-fold increase in the M1 to M2 macrophage ratio and 300-fold increase in the iNOS (M1 marker)/Arg-1 (M2 marker) ratio in TAMs as compared to the untreated control group. The results of these studies show that i.p. administered macrophage-specific HA-PEI nanoparticles can successfully transfect TAMs in lung tissues of both naïve mice and a KP-GEM NSCLC mouse model. Successful TAM repolarization toward the M1 phenotype has significant implication in anticancer immunotherapy

Assessment of cellular toxicity as measured by viability of J774A.1 macrophages incubated with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles at different concentrations from 1 μg/mL to 1,000 μg/mL for 24 hours.

<p>Assessment of cellular toxicity as measured by viability of J774A.1 macrophages incubated with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles at different concentrations from 1 μg/mL to 1,000 μg/mL for 24 hours.</p

<i>In vitro</i> polarization study.

<p>(a) iNOS-2 (M1 marker) and Arg-1 (M2 marker) in M1 J774A.1 macrophages treated with hyaluronic acid-poly(ethyleneimine) (HA-PEI)/miR-223 duplexes nanoparticles (NPs) or HA-PEI/plasmid DNA expressing miR-223 ((pDNA-miR-223) NPs for 48 h. (b) iNOS-2 (M1 marker) and Arg-1 (M2 marker) in M1 peritoneal macrophages treated with HA-PEI/miR-223 or Lipofectamine^®/miR-223 for 48 h. *p<0.05 compared to M1 macrophages which were obtained by stimulating macrophages with lipopolysaccharide (LPS, 100 ng/mL) combined with interferon-gamma (100 ng/mL) for 16 h, n = 3.</p

Peritoneal Macrophage-Specific TNF‑α Gene Silencing in LPS-Induced Acute Inflammation Model Using CD44 Targeting Hyaluronic Acid Nanoparticles

The main goal of this study was to evaluate tumor necrosis factor-alpha (TNF-α) gene silencing in peritoneal macrophages upon activation with lipopolysaccharide (LPS), using CD44-targeting hyaluronic acid (HA)-based nanoparticles encapsulating TNF-α-specific small interfering RNA (siTNF-α). HA nanoparticles were formulated by blending hyaluronic acid-poly­(ethylene imine) (HA-PEI), hyaluronic acid-hexyl fatty acid (HA-C6), and hyaluronic acid-poly­(ethylene glycol) (HA-PEG) in 3:2:1 weight ratio, and encapsulating siTNF-α to form spherical particles of 78–90 nm diameter. Following intraperitoneal (IP) administration in LPS-treated C57BL/6 mice, the nanoparticles were actively taken up by macrophages and led to a significant downregulation of peritoneal TNF-α level. Downregulation of peritoneal macrophage-specific TNF-α also had a significant impact on other pro-inflammatory cytokine and chemokine levels in the serum. The C57BL/6 group of mice challenged with 5 mg/kg LPS had a significantly higher survival rate when they were treated with 3 mg/kg siTNF-α, either prior or simultaneously with the LPS administration, as compared to the LPS-challenged mice, which were treated with controls including the scrambled siRNA formulation. Overall, the results of this study demonstrate that CD44 targeting HA nanoparticles can selectively deliver siTNF-α to peritoneal macrophages leading to downregulation of pro-inflammatory cytokines in the peritoneal fluid and in the serum. This RNAi strategy could potentially provide an important therapeutic modality for acute inflammatory diseases, such as septic shock

Fluorescence microscopy analysis of uptake of miR-223-encapsulated in hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles in J774A.1 alveolar and primary peritoneal macrophages and macrophages at 2 h post-incubation.

<p>Prior to cellular uptake study, HA-PEI was conjugated with a red fluorescence dye, Cy5.</p

Cosilencing Intestinal Transglutaminase‑2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an <i>in Vitro</i> Model of Celiac Disease

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol–water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an <i>in vitro</i> model of celiac disease by utilizing immunogenic α-gliadin peptide p31–43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and −6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an <i>in vitro</i> model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31–43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease

<i>In vivo</i> histological evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system on plaque elastin and smooth muscle cell content (A) Elastin staining of the aortic valves and plaque elastin analysis quantified using Image J (n = 4 independent animals per group).

<p>There was no significant difference in the elastin content across treatment groups. (B) SMA staining of the aortic valves and plaque SMC content analysis quantified using Image J (n = 4 independent animals per group). There was no significant difference in the elastin content across treatment groups.</p

<i>In vivo</i> safety profile evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system.

<p>(A) Body weights of control mice fed a high-fat diet and mice fed a high-fat diet and treated with either the blank nanoemulsion, 17-βE in solution, or the 17-βE-loaded CREKA-peptide-modified nanoemulsion. (n = 8 independent animals per group). (B) Histology of liver and kidney tissues isolated from the control (untreated) and specified treatment groups. (n = 6 independent animals per group).</p