GHE (MW = 298 g/mol; Sigma-Aldrich, St Louis, MO) and low molecular weight branched PEI (MW = 800 g/mol; Sigma-Aldrich, St Louis, MO) were mixed in a glass vial at a molar ratio of 8:1. The vial was sealed and the samples were stirred at 300 rpm for 24 hours at 40°C. Afterward, the sample was cooled at room temperature and the obtained white solid was broken down into a fine powder and stored at 4°C.

NPs were prepared by a modified solvent extraction/evaporation method. Briefly, 25 mg of PEI lipid, 15 mg of Poly(D,L-lactide-co-glycolide) (50/50) (PLGA; LACTEL Absorbable Polymers, Birmingham, AL) and 2 mg of 1,2-Dimyristoyl-sn-glycerol, Methoxypolyethylene Glycol (DMG-PEG; NOF America, White Plains, NY) were dissolved in 0.5 mL of dichloromethane (Sigma-Aldrich, St Louis, MO). 0.3 mg Rhodamine 6G (Sigma-Aldrich, St Louis, MO) was added for intracellular trafficking experiments. The solution was mixed with 2.5 mL DD water then sonicated to obtain a fine emulsion. The emulsion was further stirred overnight at room temperature then filtered with an Amicon Ultra-4 centrifugal filter (molecular weight cutoff of 100 kDa; Sigma-Aldrich, St Louis, MO). The suspension was adjusted to 2.5 mL with DD water and stored at 4°C.

The “Helper-free” AAV system pAAV-IRES-hrGFP was purchased from Agilent (Santa Clara, CA). Human IL-4 was cloned into pAAV-IRES-hrGFP to produce IL-4 plasmid DNA. The correct orientation of the inserted sequences and the resulting plasmid DNA were respectively checked by DNA sequencing analysis and agarose gel electrophoresis.

Changes in the morphologies of the obtained NPs were observed by using a SEM (JEOL JSM-6010LA, Tokyo, Japan). In order to avoid break down of the liposomes and NPs, 50 µl of liposomes and NPs were placed onto the cover glass, and one drop of 2% of uranyl acetate (Cat. No. 22400, Electron Microscopy Sciences Hattfield, PA) was added for 15 minutes. The slide was then washed out with DI water, and the sample was kept in a vacuum desiccator to dry out for 24 hours. The dried samples were coated with gold/palladium using Gold/Palladium Target 57 mm diameter×0.1 mm thick (Electron Microscopy Sciences, Hatfield, PA) with research grade argon gas to observe SEM, which was operated at 15 kV with top view.

AFM was used to observe NPs morphology. NPs of 1.2 μg/ml in phosphate-buffered saline (PBS) were deposited onto glass slides and then mounted on the scanner of a stand-alone AFM microscope (Park Systems, Model: XE-100, Santa Clara, CA). Typical scan sizes were 1 - 10 μm at a resolution of 512 × 512 data points. Images were taken and then analyzed with Femtoscan software.

A zetasizer (Malvern Instruments, Model: Zetasizer Nano ZS, Malvern, United Kingdom) was used to measure the size distribution, zeta potential, and heterogeneity of the obtained NPs. For each sample, the NPs were tested three times at 25°C in double distilled water at a concentration 1 mg/mL.

NPs were loaded with human IL-4, human IL-4/GFP, mouse IL-4 or mouse IL-4/GFP pDNA (Sino Biological, Wayne, PA) based on the molar ratios of PEI nitrogen (N) to DNA phosphate (P) (N/P ratio). The following equation was used to determine the N/P ratio, where the molecular mass (MM) of the repeating unit for branched PEI is 43 Da, the number of primary nitrogen’s in the repeating unit of branched PEI is 4, and the MM of DNA used was 330 Da (30, 31).

DNA encapsulation by NPs was investigated by agarose gel electrophoresis. NPs were loaded with pDNA at different N/P ratios and ran on a 1% agarose gel for 30 minutes. pDNA was visualized with an Amersham Imager 680 blot and gel imager (GE Healthcare, Chicago, IL).

The IL-4/GFPpDNA-NPs were made at different N/P ratios in PBS. NPs suspension and IL-4pDNA solutions were prepared before each experiment at various N/P ratios. First, 1 μl of NP suspension (including of 0.1 μg of PEI) was mixed with 20 μl of PBS to disperse the NP suspension. Second, the prepared IL-4pDNA solution was added into the NP suspension with various amounts to meet the different N/P ratio. Third, IL-4pDNA solution was well mixed with the NP suspension, then the mixture was incubated for 30 minutes at room temperature before transfection.

For human monocyte-derived macrophage (MDM) cultures, peripheral blood CD14+ monocytes were cultured in DMEM supplemented with 10% heat-inactivated pooled human serum (Thermo Fisher Scientific, Waltham, MA), 1% penicillin-streptomycin (P/S), 2 mM L- Glutamine, and 1000 U/ml recombinant human MCSF at 37°C with 5% CO₂. Monocytes were seeded into a standard tissue culture-treated plate at a density of 1 million cells per ml. Human peripheral blood monocytes were cultured up to 7 days, allowing differentiation into macrophages, then subsequently used for experiments.

For mouse bone marrow derived macrophage (BMM), the femur was collected from a Balb/C mouse (male 4-6 weeks) and the bone marrow was dissociated into single-cell suspensions. Bone marrow cells were cultured in DMEM with fetal bovine serum (10%), 1% penicillin-streptomycin (P/S), 2 mM L- Glutamine (Thermo Fisher Scientific, Waltham, MA), and 1000 U/ml highly purified recombinant macrophage colony stimulating factor (MCSF; R&D Systems, Minneapolis, MN) at 37°C with 5% CO₂. Cells were allowed to differentiate into macrophages for 12-14 days, then samples were used for experimentation. All experiments were carried out in compliance with the ARRIVE guidelines. The animals used in this study were approved by Institutional Animal Care and Use Committee, Texas Tech University Health Sciences Center, El Paso.

For transfection of MDM, human IL-4pDNA/GFP-NPs were added to cells at a 1:1, 1:4, 1:10, 1:20 and 1:50 NPs/media ratio. The media was changed after four hours, and the cells were further cultured for 9 days. For transfection of BMM, 1.5 ng/mL of mouse IL-4pDNA/GFP-NPs were added to cells at a 1:20 NPs/media ratio. The media was changed after 4 hours, and the cells were further cultured for 5 days. Cells were imaged with a Nikon Ti fluorescence microscope and analyzed for GFP expression with Image Pro Plus software.

MDM cells were seeded in a 96 well plate and transfected with IL-4pDNA/GFP-NPs for 24 hours. Non-treated cultures served as the control. After 24 hours, cell viability was assayed using the Quick Cell Proliferation Colorimetric Assay Kit (BioVision, Milpitas, CA) according to the manufacturer’s protocol. Samples were analyzed with the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at 450 nm, and the data collected was normalized to the controls. The experiment was repeated in triplicate.

To visualize intracellular transportation of IL-4pDNA-NPs, dual labeling of IL-4pDNA with YOYO-1 iodide (green, Thermo Fisher Scientific, Waltham, MA) at 1 dye molecule to 50 pDNA base pairs for 24 hours, and MDM with LysoTracker Red (Thermo Fisher Scientific, Waltham, MA) to label lysosome or CellBrite™ Fix555 Red (Biotium, Fremont, CA) to label cell membranes.

For better imaging of YOYO-IL-4pDNA-NPs uptake by macrophages to cross the membranes and enter the nucleus, the cultures were incubated with CellBrite™ Fix555 to label the cell membranes (red) and DAPI (blue) to illustrate the nucleus. The labeled cultures were treated to YOYO-IL-4pDNA-NPs (green) for live cell imaging by fluorescent microscopy automatically every 30 minutes. The live images at 10, 40, 100, 160, and 250 minutes were used to determine the interaction of YOYO-IL-4pDNA-NPs and cellular membranes.

To track intracellular transportation, MDM were cultured in an 8 well chamber slide and treated with YOYO-IL-4pDNA-NPs at a NPs/media ratio of 1:20. Then, YOYO-IL-4pDNA-NPs treated MDM were stained with LysoTracker Red and DAPI (blue) and analyzed at 0, 2, 2.5, 3, 3.5, and 4 hours post transfection. Cells were then fixed with 4% paraformaldehyde for co-imaging of SEM and confocal microscopy assay. First , the triple labeled 8 well chamber slides were scanned by a confocal microscope (Nikon Ti Eclipse microscope, Nikon Instruments Inc., Melville, NY). Then , to co-image with SEM, the fractured cells were obtained from confocal microscopy imaged slides by adhesion of cover glass. In brief, another cover glass was attached very tightly to the sample cover slips, then pulled out mechanically and dried in the vacuum desiccator for 24 hours at RT. The dried samples were coated with gold/Palladium using Gold/Palladium Target 57 mm diameter x 0.1 mm thick (Gold/Palladium Target: Electron Microscopy Sciences, Hatfield, PA) with research grade argon gas. SEM were operated at 15 kV with top view. Finally , Co-imaging of confocal microscopy with SEM were used to analyze the intracellular trafficking of YOYO-IL-4pDNA-NPs in the MDM.

ELISA was performed on culture media to determine the secretion of cytokines using ELISA Kits purchased from eBioscience (San Diego, CA). The supernatant was collected at 1, 3, and 5 days post transfection and were analyzed according to the manufacturer’s protocol.

The mouse model of inflammation was established with intraperitoneal injection of lipopolysaccharide (LPS; 100 μg) in Balb/C mice. The pre-treated group was administered 100 μL of mouse IL-4pDNA-NPs through the tail vein 24 hours before LPS injection. The post-treated group was given IL-4pDNA-NPs 24 hours after LPS injection. LPS only and normal mouse served as the control. All experiments in Balb/C mice were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council. The protocols were approved by the Texas Tech University Health Sciences Center El Paso (TTUHSC EP) Institutional Animal Care and Use Committee. All mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 4 weeks and were raised and maintained by the Laboratory Animal Resource Center at TTUHSC.

Mice were humanely euthanized at day 7 and the spleen was collected and the sections were prepared (5 µm thick) for staining. The immunostaining was performed with antibodies of IL-4 (1:200; Novus Biologicals, Littleton, CO), iNOS (1:200; Abcam, Cambridge, United Kingdom), Arginase-1 (1:200, Thermo Fisher Scientific, Waltham, MA), CD11b (1:200; abcam, Cambridge, Ma, USA). Secondary antibodies used were goat anti rat Alexa Fluor 488 and goat anti rabbit Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA). The nuclei were stained with DAPI. Immunofluorescence images were observed under a microscope (Nikon Instruments Inc., Melville, NY).

MDM cells treated with human IL-4pDNA/GFP-NPs were scraped and fixed in 2% paraformaldehyde (Sigma-Aldrich, St Louis, MO) and analyzed on a BD FACS Canto II flow cytometer (BD, Franklin Lakes, NJ). Data on 5 x 10⁵ cells were collected and the percentage of GFP positive cells and fluorescence intensity between conditions were analyzed with FlowJo software (FlowJo, LLC, Ashland, OR).

For in vivo studies, the spleen was collected from mice and was broken down through a 40 μM strainer. Cells were centrifuged for 5 minutes at 1200 rpm, and then the supernatant was discarded. Cells were resuspended in 5 mL ACK buffer and were incubated at room temperature for 5 minutes. 5 mL’s of DPBS was added to stop the reaction, and the cell suspension was strained through a 40 μM strainer. The solution was centrifuged for 5 minutes at 1200 rpm, then the cells were resuspended in blocking buffer (3% BSA in PBS) and counted. 1 x 10⁶ cells were blocked for 1 hour per condition, then samples were incubated for 30 minutes with CD45 PE-Cy7, CD11b Alexa Fluor 700, iNOS APC, and Arginase 1 PE (Thermo Fisher Scientific, Waltham, MA) at 4°C. Samples were then washed 3 times in wash buffer (1X BSA in PBS) and fixed (1X BSA, 2% PFA, in PBS). Samples were immediately analyzed on a Gallios Flow Cytometer (Beckman Coulter, Brea, CA). Data on 5 x 10⁵ cells were collected and analyzed with FlowJo software. 2 – 3 mice were analyzed per condition, and the experiment was repeated in triplicate.

SPSS software was used for all statistical analyses and all data are presented as the mean ± standard error of the mean (SEM). In all groups, P values <0.05 were considered statistically significant. For comparison of groups, we used one-way analysis of variance (ANOVA) Student’s t-test. All experiments were repeated in triplicate.

The cationic branched PEI lipid was first synthesized by reacting low molecular weight, branched PEI (MW = 800 g/mol) with GHE (MW = 298 g/mol) at a molar ratio of 1:8 respectively. This ratio was experimentally determined in our previous work to enhance transfection efficiency while decreasing cytotoxicity (29). A nanoformulation was made with this cationic branched PEI lipid shell and PLGA core (NPs) through a modified solvent evaporation procedure. The polar hydrophilic headgroup of highly positive cationic lipids enable the condensation of negative charged IL-4 pDNA. A plasmid containing full length IL-4 pDNA tagged with GFP was loaded into NPs (IL-4pDNA/GFP-NPs). SEM and AFM assays ( Figures 1A, B ) indicated that these NPs had a smooth surface, are spherically shaped, and are monodispersed, suggesting a successful nanoformulation. The particle size, polydispersity index (PDI) and surface charge were also characterized by a zetasizer. IL-4 pDNA/GFP-NPs at an N/P ratio of 1:10 showed a mean diameter of 282.3 ± 6.4 nm ( Figure 1C ). Note that the particle size increased with loading the IL-4 pDNA. This is consistent with the data presented in the SEM images in Figure 1A . The PDI, a measure of the broadness of the molecular weight distribution, is 0.481 ± 0.009. PDI values under 0.5 indicates that the NPs are monodispersed in the solution (32). Finally, the zeta potential, a measure of stability, showed IL-4pDNA-NPs were +36 ± 7.13 mV. Values larger than ±35 mV suggest the NPs are stable (33, 34), while the positive charge represents the cationic nature of the PEI lipid (35, 36). Together, these data indicate our newly synthesized NPs are cationic and stable in a colloidal suspension, and are homogeneous in size and shape. A facile method to synthesize cationic lipids using low molecular weight PEI and NPs successfully delivered IL-4 pDNA (29) to enhance the transfection. Furthermore, the stability of NPs were up to 3 months (data not shown).

To determine the encapsulation capacity, NPs were loaded with IL-4pDNA at various molar ratios of PEI nitrogen’s (N) to DNA phosphates (P) up to N/P=10. Encapsulation capacity of IL-4pDNA/GFP-NPs was determined by agarose gel electrophoresis at N/P ratios of 4, 2.6, 2, 1.8, and 1.6 ( Figure 1D , Lane 2-6). DNA ladder was used as the size marker ( Figure 1D , Lane 1). The results indicated that IL-4pDNA-NPs at N/P ratios of 4 and 2.6 were fully encapsulated after 30 minutes of incubation. By increasing IL-4pDNA concentration to N/P ratios of 2, 1.8, and 1.6, a positive band was detected on the gel that indicates the over-loading of unencapsulated IL-4pDNA. Further, the loading capacity was confirmed using unloaded IL-4pDNA as control to run the gel ( Supplementary Figure 1 )

Transfection of MDMs with IL-4pDNA/GFP-NPs was evaluated by examining GFP expression ( Figure 2A , green). MDMs were cultured up to 7 days with 1000 units of human MCSF, and were allowed to differentiate into macrophages. MDMs were then treated with IL-4pDNA/GFP-NPs at N/P ratios of 10, 6, 3, and 1.5 with NPs to media ratios of 1:1, 1:4, 1:10, 1:20, and 1:50. GFP+ transfected MDM were seen in all conditions ( Figure 2A ). An increase of GFP+ MDM was seen with a NPs to media ratio from 1:1 up to 1:20. Interestingly, the transfection efficiency decreased with a NPs to media ratio of 1:50. The absolute numbers of GFP+ MDM were counted ( Figure 2B ), indicating successful transfection in all conditions. The highest GFP+ MDM counts occurred with transfection of IL-4pDNA/GFP-NPs at N/P ratios of 6 and 3 with a NPs to media ratio of 1:20. The percentage of GFP+ MDM was significantly increased in IL-4pDNA/GFP-NPs at N/P ratio 3 with NPs to media ratio of 1:20 ( Figure 2C ). Flow cytometry confirmed the increases of GFP+ MDM following treatment with IL-4pDNA-NPs at ratios of 1:1, 1:4, 1:10, 1:20 and 1:50 at day 5 ( Supplementary Figure 2 ). The merged flow cytometry histograms illustrate there is GFP+ expression in MDM in all conditions.

Intracellular trafficking of IL-4pDNA was performed by a real-time cell imaging assay. IL-4pDNA was labeled with the green dye YOYO-1 iodide (YOYO-IL-4pDNA), then loaded into NPs (YOYO-IL-4pDNA-NPs). The membranes of the cells were labeled with red dye CellBrite Fix555 and the nucleus was tracked by DAPI. Triple labeling of YOYO-IL-4pDNA-NPs, the cell membranes and the nucleus was initially imaged at 10 minutes followed by automatic scanning every 30 minutes. Live cell images showed that YOYO-IL-4pDNA-NPs ( Figure 3 , green) crossed the cell membranes ( Figure 3 , red, narrow head) and entered the cytoplasm at 40 minutes. The levels of YOYO-IL-4pDNA-NPs consistently increased inside of cells up to 250 minutes. The nuclear envelop ( Figure 3 , red, arrow) was labeled at 160 minutes after exposure to CellBrite Fix555, while “green” YOYO-IL-4pDNA traversed across the “red” nuclear membranes to enter into the “blue” nucleus. Importantly, consistent nuclear accumulation of YOYO-IL-4pDNA was obtained from 160 to 250 minutes ( Figure 3 ). We observed YOYO-IL-4pDNA-NPs were quickly taken up by macrophages that are actively transported throughout the cytoplasm, allowing nuclear accumulation of YOYO-IL-4pDNA for effective delivery.

IL-4pDNA escape from lysosomes to enter the nucleus is key for gene delivery and plasmid transfection. Lysosomes are cellular organelles responsible for digestion and removal of foreign objects in cells. As professional phagocytes, lysosomes in MDM are largely activated during uptake of “foreign” IL-4pDNA-NPs ( Supplementary Figure 3A ). We detected the lysosomal responses to intracellular IL-4pDNA-NPs in mouse bone marrow-derived macrophages (BMM) at different time-points ( Supplementary Figure 3B ). Maximal increase of lysosomes in BMM was detected at 1 day of IL-4pDNA-NPs treatment ( Supplementary Figure 3C ). This presents a major challenge to develop a carrier that can escape from lysosomes and traverse to the nucleus.

We developed a co-imaging assay using fluorescence microscopy and SEM for microstructural trafficking of YOYO-IL-4pDNA-NPs. MDM were treated with YOYO-IL-4pDNA (green) and lysosomes were labeled with LysoTracker (red). To locate the nucleus, DAPI (blue) staining was applied. The microstructures of MDM were co-imaged to reveal the escape of YOYO-IL-4pDNA from lysosomes for nuclear ingress. The image was taken for 2 hours after YOYO-IL-4pDNA-NPs treatment. Three phases of cytoplasmic transportation ( Figure 4 ) show the uptake of YOYO-IL-4pDNA-NPs ( Figure 4A , green) by MDM, the separation of “green” YOYO-IL-4pDNA-NPs from “red” lysosomes ( Figure 4-b ) and the transportation of YOYO-IL-4pDNA integration into the nucleus ( Figure 4-c ). Escape of YOYO-IL-4pDNA-NPs from lysosomes showed the effective delivery of pDNA by NPs. Nuclear ingress of YOYO-IL-4pDNA within 2 hours indicated that YOYO-IL-4pDNA-NPs can effectively cross the cytoplasmic membranes and the nuclear envelop to deliver pDNA for MDM transfection.

Time-dependent intracellular trafficking of IL-4 pDNA/GFP-NPs in MDM was assessed by the co-imaging assay ( Figure 5 ). MDM were treated with YOYO-IL-4pDNA-NPs for 0.5, 1, 2, 4 hours. Lysosomes and nuclei were labeled with LysoTracker and DAPI, respectively ( Figure 2C ). Co-images from the SEM and confocal microscopes revealed YOYO-IL-4pDNA ( Figure 5 , green) were taken in by MDM at 30 minutes, and were immediately captured within lysosomes ( Figure 5 , red) indicated by “yellow”. Separation of “green” YOYO-IL-4pDNA from “red” lysosomes occurred at 1 and 2 hours, showing successful escape of YOYO-IL-4pDNA-NPs from lysosomes. YOYO-IL-4pDNA traversed into the nucleus within 2 hours where it remained for the next 2 hours. At 4 hours, MDM continued to uptake YOYO-IL-4pDNA-NPs within the cytoplasm. These results strongly indicate that PLGA/PEI NPs facilitated MDM uptake to continually maintain the intracellular levels of IL-4pDNA for effective transfection.

NPs were labeled with red dye Rhodamine 6G (rNPs) and loaded with GFP-tagged IL-4pDNA (IL-4pDNA/GFP-rNPs). The intracellular red fluorescence signals were imaged to determine the uptake and the degradation of IL-4pDNA/GFP-rNPs in MDM. GFP expression (green) in MDM revealed successful transfection. IL-4pDNA/GFP-rNPs were internalized into MDM within 1 day and gradually decreased from days 2 to 9 ( Figure 6A , red). IL-4/GFP expression continually increased from day 2 with the highest GFP expression seen on day 5 ( Figure 6A , green). IL-4/GFP expression subsequently decreased at days 6 to 9. To identify the stability of IL-4/GFP expression, GFP fluorescence intensity was examined in a time-period of 30 days. Peak GFP intensity occurred on day 5 that decreased by day 10, with sustained levels up to 30 days ( Figure 6B and Supplementary Figure 4A ).

Cytotoxicity is a major concern when developing a nanoparticle-based gene delivery system. Specific toxicity of different ratios of NPs to culture media for various concentrations of IL-4pDNA was evaluated to assess the safety of IL-4pDNA/GFP-NPs. MDM were treated with 1, 1.5, 3, and 6 ng/ml of IL-4pDNA-NPs at NPs to culture media ratios of 1:1, 1:4, 1:10, 1:20, and 1:50. The viability assay was performed on day 5 ( Figure 6C ) post transfection. The cultures treated with IL-4pDNA/GFP-NPs at 1:20 and 1:50 demonstrated greater viability. In contrast, higher concentrations of IL-4pDNA/GFP-NPs treated to cultures displayed increased toxicity at NPs to media ratios of 1:10, 1:4 and 1:1. The data suggest higher concentrations of IL-4pDNA/GFP-NPs were toxic to the cells.

Next, we studied how IL-4pDNA-NP transfection manipulates macrophage immunological functions. Because macrophages can produce IL-4, we verified the production of induced human IL-4 in IL-4pDNA/GFP-NP transfected mouse bone marrow derived macrophages (BMM). The secretion of human IL-4 from mouse BMM was evaluated by ELISA using an antibody specific to human IL-4. BMM and NPs without pDNA treatment served as controls. The culture medium from day 1, 3 and 5 indicated significant production of human IL-4 from transfected mouse BMM ( Figure 7A ). A large increase of human IL-4 was obtained at day 3 and 5 post transfection. A series of human IL-4pDNA-NPs were treated to human MDM. The levels of IL-4 in the culture media ( Figure 7B ) were analyzed by ELISA at day 5. The highest level of IL-4 was obtained at an N/P ratio of 6 with NPs to media ratio of 1:20 (p<0.01 to all conditions except N/P ratio of 10 with NPs to media ratio of 1:20). IL-4pDNA-NPs can achieve gene delivery in macrophages.

The regulatory role of IL-4pDNA-NPs transfection was investigated by examining LPS-induced inflammatory MDM (M1) polarization into immunosuppressive M2. The pro-inflammatory cytokines TNF-α and IL-6, and the anti-inflammatory cytokine TGF-β were used to characterized M1/M2 polarization. Non-treated MDM served as the control. Examination of the M2 marker TGF-β was significantly increased in IL-4pDNA-NP treated inflammatory M1 MDM cells when compared to the control and LPS-induced M1 cells ( Figure 7C ). This suggests IL-4pDNA-NPs were capable of repolarizing inflammatory macrophages by overexpression of anti-inflammatory cytokine TGF-β. LPS treated inflammatory M1 MDM showed an increase in the M1 markers IL-6 ( Figure 7D ) and TNF-α ( Figure 7E ). With IL-4-pDNA-NPs treatment, there was a 23% reduction of IL-6 production compared and a significant reduction in the level of TNF-α to LPS alone (p < 0.01). Our data suggests IL-4pDNA-NPs upregulated the M2 cytokine TGF-β and downregulated LPS-induced inflammatory cytokines TNF-α and IL-6, leading to functional regulation of M1/M2 cytokine signaling pathways for MDM repolarization.

We further characterized IL-4-pDNA-NPs inhibition of the inflammatory marker iNOS in MDM by flow cytometry. MDM were either pre-treated with IL-4pDNA-NPs followed by exposure to LPS (pre-NPs+LPS), or post-treated (NPs+LPS) with IL-4pDNA-NPs 1 day after LPS exposure. MDM were exposed to LPS to induce the overexpression of iNOS. Flow cytometry results revealed a decrease of iNOS+ MDM in pre- and post-IL-4pDNA-NP treated cultures ( Supplementary Figure 4B ) . There was significant inhibition of iNOS in IL-4pDNA-NPs pre- and post-treated inflammatory MDM ( Supplementary Figure 4C ), suggesting IL-4pDNA-NPs inhibited LPS-induced overexpression of iNOS in MDM.

The in vivo delivery effectiveness of IL-4pDNA-NPs was tested in Balb/C mouse. All experiments in Balb/C mice were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council. The protocols were approved by the Texas Tech University Health Sciences Center El Paso (TTUHSC EP) Institutional Animal Care and Use Committee. Mice were administered IL-4pDNA/GFP-rNPs via the tail vein at a NP to blood volume ratio of 1:20. The spleens were collected at day 5 and fresh spleen sections were scanned by fluorescence microscopy. The images suggest that IL-4/GFP expression ( Supplementary Figure 5 , green) co-localized with the cells that internalized IL-4pDNA/GFP-rNPs ( Supplementary Figure 5 , red). Specifically, the “green” GFP+ and “red” rNPs were present in the border of the marginal zone and outer periarteriolar lymphatic sheaths of the spleen ( Supplementary Figure 5 , merge). This indicates that splenic macrophages can uptake IL-4pDNA/GFP-rNPs in vivo.

We further investigated the anti-inflammatory role of IL-4pDNA/GFP-rNPs in an LPS-induced inflammation mouse model. For comparison, pre-treatment mice were given IL-4pDNA-NPs 24 hours before LPS injection (pre-NP+LPS). The post-treatment group received IL-4pDNA-NPs 24 hours after LPS-induction (post-NP+LPS). Normal mice and LPS only mice served as the control (Ctl and LPS, respectively). The spleen sections were collected at day 7 post LPS-induction. The spleen sections were stained with antibodies against arginase-1 (Arg1, M2 marker) and iNOS (M1 marker). IL-4 staining was used to evaluate IL-4pDNA-NPs transfection in mouse spleen. Immunofluorescence images revealed an overexpression of IL-4 ( Figure 8A , green) in mice treated with IL-4pDNA-NPs, suggesting successful transfection of IL-4 in vivo. The M2 marker Arg-1 ( Figure 8A , red) was largely increased in both pre-NP+LPS and post-NP+LPS groups compared to the control and LPS only groups. The greatest Arg-1 expression was seen in post-NP+LPS mice that also showed the highest IL-4 positive staining. Quantitative imaging confirmed the statistical increase of Arg-1 in LPS, pre-NP+LPS and post-NP+LPS groups compared to the control ( Figure 8B ). Immunostaining for the M1 marker iNOS in spleen sections showed increases in all conditions compared to the control ( Figure 8C ). Importantly, IL-4pDNA-NPs treatment decreased the LPS-induced iNOS over-expression. These results suggest that administration of IL-4pDNA-NPs can polarize M1 macrophages towards an M2 phenotype against inflammation in vivo.

To confirm these results, fresh spleen cells were isolated and analyzed by flow cytometry. The splenic macrophages were gated on CD45+ and CD11b+. The CD11b+/Arg-1+ and CD11b+/iNOS+ macrophages ( Figure 8D ) were analyzed to reveal M2 and M1 subpopulations. Notably, a significant increase of CD11b+/Arg-1+ M2 macrophages was obtained in post-NPs+LPS mice when compared to control ( Figure 8E ). This result indicated IL-4pDNA-NPs polarized macrophages towards the M2 phenotype after inflammation. The increase of iNOS expression was seen in all conditions when compared to the control ( Figure 8F ) similar to the spleen sections with immunostaining. Together, these data further support the evidence of M2 polarization by IL-4pDNA-NPs after inflammation in vivo.