1. IntroductionMacrophages are highly plastic in nature and thus they can be polarized into several subsets under different stimuli. Bacterial lipopolysaccharide (LPS) can stimulate macrophages to their inflammatory state while IL-4 can stimulate them into anti-inflammatory phenotype [1,2]. A highly complex set of regulatory network governs this macrophage polarization [3]. Proinflammatory macrophages generally kill pathogens and present their antigens to the adaptive immune system [4]. Subsequently, the anti-inflammatory cells resolve inflammation and repair the damage [5]. The balance of the two phenotypes is crucial during inflammation or injury. In acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and during foreign body response (FBR) this balance is somewhat disrupted [6,7]. Continuous M1 polarization can release excessive proinflammatory cytokines like interleukin-1 (IL-1), nitric oxide (NO), tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) to induce a severe inflammatory response [6,8]. Prostaglandin E2 (PGE2) significantly influences the progression of the inflammatory response by macrophages [9]. Their production is markedly increased in inflamed tissues, and they help to produce the key symptoms of severe inflammation [10]. Recently, PGE2 was reported to augment M1 polarization. Suppression of PGE2 also promoted M2 macrophage polarization and decreased the allergic airway inflammatory cell infiltration in Abx-treated mice [11]. Therefore, PGE2 can be a potential therapeutic target to attenuate M1 polarization. Some new strategies are in progress to down-regulate the expression of PGE2 and prevent M1-induced severe inflammation [12]. Small-interfering RNAs (siRNAs), also known as short interfering RNA or silencing RNA, are a type of double-stranded non-coding RNAs that functions through the RNA interference (RNAi) pathway. They are usually 20–24 base pairs (commonly 21 bp) long, making them similar to miRNA [13]. They have recently been demonstrated to induce transcriptional gene silencing in humans [14]. An RNA-induced silencing complex (RISC) system (an endogenous enzyme system), initiates gene silencing with siRNA. In theory, specific siRNA can target and silence any gene. Thus, it can be used to down-regulate the expression of PGE2. However, several significant obstacles prevent siRNA from being delivered for therapeutic applications. First, in biological fluids, "naked" siRNAs are unstable and easily are destroyed by nucleases, resulting in their poor accumulation at target sites [15]. Second, because of its large size, (13 to 15 kDa), and negative charges, siRNA cannot cross the cell membrane [16]. Moreover, systemically injected siRNAs can accumulate in the liver and kidney. The kidneys remove siRNA from circulation and excrete them [17]. To get over these aforementioned obstacles, suitable delivery vehicles are consequently required. Lipid-based nanoparticles (LNPs) such as liposomes are frequently used for nucleic acid delivery within target cells. The LNPs are biocompatible, biodegradable, less toxic, structurally flexible, and can be easily produced on a large scale [18,19,20]. Therefore, LNPs can be an ideal vehicle for the delivery of siRNAs.

This work was designed to combat acute inflammation and control the proinflammatory M1 polarization by silencing PGE2 through LNP-mediated Si-RNA delivery.

2. Materials and Methods 2.1. Chemicals and Reagents

RAW264.7 (The murine macrophage cell line) was procured from NCCS, Pune, India. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA), FBS (heat inactivated) (Gibco, Grand Island, NY, USA) and 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA) were purchased. Trizol reagent (Ambion, Elk Grove, CA, USA, cDNA synthesis kit (verso, cDNA synthesis kit, Thermo Scientific, Waltham, MA, USA), SYBR green (PowerUp™ SYBR™ Green Master Mix, Applied Biosystems, Thermo, Waltham, MA, USA) were purchased. All routines chemicals were purchased from local supplier.

2.2. Synthesis of NanoparticlesSynthesis of lipid nanoparticles (LNPs) is done by a thin layer evaporation method [21]. In brief, DSPC, cholesterol, and polyethylenimine (PEI) (4:1:2) were dissolved in Chloroform and Methanol (1:1) and kept on magnetic stirrer for 4 hr at 25 °C and 200 rpm. Next, the solvent was evaporated using a rotary evaporator (DLAB RE-100 Pro) at 50 °C, 40 RPM, to obtain a thin layer. The obtained thin layer of lipids was dispersed in doubled distilled water by sonication (Sonics, Vibra cell VCX 500) at 50 amplitude (10 s on-off cycle) for 10 min and extruded using a 200 nm then 100 nm polycarbonate membrane at 50 °C to control the LNPs size. 2.3. Characterization of LNPs

Nano Zetasizer system (Malvern Instruments) was used for dynamic light scattering (DLS) analysis at 25 °C, 0.8872 mPas medium viscosity, and 1.59 medium refractive index for the evaluation of hydrodynamic size distribution of the particles, zeta potential and disparities in the colloidal sample. The analysis was done after loading the material into a quartz microcuvette and measurements was done accordingly.

Transmission electron microscopy (TEM-Philips, EM-410LS, JEOL, Osaka, Japan) and Scanning electron microscopy were used to examine the morphology of LNPs (SEM). For TEM analysis, a little drop of the highly diluted sample was evenly distributed throughout the copper grid and dried at room temperature. Similarly, samples were prepared for SEM analysis followed by gold coating in a sputter coater to examine the samples. Micrographs were captured on SEM (Nova NanoSEM 450, at accelerating voltage of 5 keV.

2.4. Cell Culture and Maintenance

RAW264.7 (murine macrophage cell line) was procured from NCCS, Pune, India, and maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (heat inactivated) (Gibco, Grand Island, NY, USA) and 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Cells were incubated at 37 °C in a humidified incubator saturated with 5% CO2. These macrophages were stimulated by LPS treatment (100 ng/mL).

2.5. Biocompatibility of the LNPsBiocompatibility was assessed using MTT assay [22,23]. Briefly, 7 ⤫ 103 cells (RAW264.7) in their exponential growth phase were seeded in a 96-well plate and kept in an CO2 incubator at 37 °C for 24 h. Different concentrations of LNPs were used to treat the cells and after 24 h, 20 μL MTT reagent was added in each well and incubated for 4 h. Formazan crystals were dissolved by adding 150 μL of DMSO in each well. Absorbance was measured using a microplate reader at 590 nm (imark BIO-RAD microplate reader). The percentage viability of cells was calculated using following equation:

% Viability = 100 − [(Absorbance of control − Absorbance of treated)/Absorbance of control] * 100

2.6. Binding Efficiency of siRNA with LNPs through Gel Retardation Assay

Different quantities of LNPs (1–7 µg) were made to react with a fixed amount of siRNA (10 µg) for 5 min at room temperature. The mixture was run on the denaturing agarose gel (1.5 %) at 100 V for 20 min to find the best combination ratio of siRNA:LNP.

2.7. RNA Extraction and Real-Time Quantitative PCRRealtime PCR was performed to check the mRNA expression of target genes. Following the manufacturer’s instructions, RNA was extracted from the siRNA treated RAW264.7 cell line using Trizol reagent (Ambion, Elk Grove, CA, USA). Then, using a cDNA synthesis kit (verso, cDNA synthesis kit, Thermo Scientific, Waltham, MA, USA) cDNA was synthesized. To perform qPCR, cDNA was diluted 1:20 times. With the use of SYBR green (PowerUp™ SYBR™ Green Master Mix, Applied Biosystems, Thermo, Waltham, MA, USA), the mRNA levels were assessed. The Real-time PCR System QuantStudio 6 Flex (Applied Biosystems, Waltham, MA, USA) was used for the experiment, and the thermocycling settings were as follows: initial denaturation 95 °C for 5 min, denaturation 95 °C for 30 s, annealing (54 °C) for 35 s, and extension 72 °C for 40 s. Data was analyzed by the Livak (ΔΔCt) method [23]. Following primers are used to detect the expression of target genes by qRT-PCR analysis: PGE2: Forward-5′ GAA GGA CTG AGA TCA AAT TCT C 3′, Reverse-5′ ATG ACA GAG GAG TCA TTG AG 3′, Β-ACTIN: Forward- 5′ TGA CCC AGA TCA TGT TTG AG 3′, Reverse- 5′ ATC CCA TCA CAA TGC CTG 3′, iNOS: Forward- 5′ TCC TGG AGG AAG TGG GCC GAA G 3′, Reverse- 5′ CCT CCA CGG GCC CGG TAC TC 3′,IL-1β: Forward- 5′ TCA GGC AGG CAG TAT CAC TC 3′, Reverse- 5′ CAT GAG TCA CAG AGG ATG GG 3′. 2.8. Protein Extraction and Western Blot

RIPA lysis buffer (Thermo scientific, USA) supplemented with protease and phosphatase inhibitor (Thermo scientific, USA) was used to extract the protein. A Bio-Rad kit was used to quantify the extracted protein and was quantified by Bradford assay kit (Bio-Rad, Hercules, CA, USA). SDS-PAGE was used to resolve the protein, which was subsequently transferred onto PVDF membrane. After the membrane had been blocked with 5% skimmed milk, the primary mouse monoclonal antibody, Anti-iNOS (BioLegend, USA) and Anti-IL-1β antibodies (sc-12742, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added onto the membrane for an overnight incubation at 4 °C. It was followed by an incubation with HRP-conjugated secondary antibody at room temperature for 1 h. Thereafter, protein bands were visualized using Chemi-Doc XRS (Bio-Rad) by ECL substrate (Bio-Rad).

2.9. Statistical Analysis

All the experiments were run in triplicate. Data were expressed as the mean ± SEM. GraphPad Prism 3.0 software (San Diego, CA, USA) was used for the statistical analyses. One-way ANOVA and Dunnett tests were further used. Values of p < 0.05 were considered statistically significant

4. DiscussionPGE2 is derived from arachidonic acid and is a major physiologically active lipid. PGE2 is abundantly produced during acute inflammation and edema and is blocked by NSAIDs, can resolve the inflammation [33,34]. But these drugs can cause serious effects on gastrointestinal and cardiovascular system [35]. To downregulate PGE2 here we designed or synthesized lipid nanocarriers using DSPC:Cholestrol:PEI system. Synthesis was con-firmed by DLS, Zeta analyzer SEM and TEM analysis. 2:5 ratio of NPs and siRNA was a suitable ratio identified by gel retardation assay. The procedure successfully deliver siR-NA to the macrophage cells RAW264.7 to silence PGE2 gene and further inflammatory pathways and macrophage polarization markers were assessed. PGE2 is involved in macrophage metabolism during inflammation and these cells are crucial for immunolog-ic response, pathogen response, tissue repair, regeneration [36], and metabolism thus maintaining tissue homeostasis [33,37,38,39]. Similarly, M1 macrophages also release the potent pro-inflammatory cytokine IL-1β. Due to the activation of the NF-kB and MAPK cascades, very high quantities of IL-1β are present in M1 polarized macrophages, while M2 polarized macrophages do not contain any IL-1β protein [40,41]. Downregulation of IL-1β expression is an indication towards anti-inflammatory M2 macrophage polarization and reduction in M1 polarization [42]. IL-1β can be produced and induce inflammation in response to the Toll-like receptor 4-ligand lipopolysaccharide (LPS). Recently, in murine bone marrow-derived macrophages, it has been reported that PGE2 can induce an inflammatory response by stimulating the production of 1L-1β through cAMP/protein kinase signaling and inhibiting TNF-alpha [43]. PGE2 was also reported to boost the ability of LPS to induce pro-1L-1β expression. Similarly, suppression of PGE2 can affect the LPS-induced 1L-1β expression adversely through a positive feedback loop. Similar observations are also made in our study. We observed that nanoparticle-mediated silencing of PGE2 significantly decreased LPS-induced 1L-1β expression and inflammatory response.

The study indicates that expression of IL-1β also decreased in siLPS + LNPs as compared to scLPS + LNPs. These results affirmed that siRNA mediated silencing of PGE-2 can decrease proinflammatory polarization of macrophages via affecting iNOS and IL-1β expressions.

Limitation of this study, PEI-based liposomes were synthesized along with DSPC which is known for its lower toxicity. However, in the current scenario toxicity of nanoparticles was only checked against the cell line and further work needs to be done on the animal model in order to acquire the complete information related to their siRNA leakage, accumulation in the reticuloendothelial system, release from the body and stability in the circulatory system.