All materials were purchased from Sigma-Aldrich unless otherwise noted. Monomethyl ether hydroquinone inhibitors were removed from dimethylaminoethyl methacrylate (DMAEMA) and butyl methacrylate (BMA) using an activated basic aluminum oxide column. All DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). For in vivo biodistribution studies, double stranded DNA (dsDNA) designed to be length-matched to therapeutic IκBα siRNA and conjugated with a cyanine-5 (Cy5) fluorophore was used. IκBα siRNA sequence was based on previous studies and the scrambled siRNA sequence was randomized from the IκBα sequence and analyzed via the Basic Local Alignment Search Tool (BLAST, NCBI) to ensure no off-target effects with our sequence [27, 29]. Oligonucleotide sequences are listed in Supplemental Table 1.
The mannose-poly (ethylene glycol)-(DMAEMA-co-BMA) (MnPEGDB) polymer was fabricated as previously described [27, 36, 40, 41]. Nucleophilic substitution of propargyl bromide with D-mannose to form mannose-alkyne was performed and characterized previously . The core of the micelle comprises a diblock copolymer fabricated using 4-cyano-4-(ethylsulfanylthiocarbonyl)-sulfanylpentanoic acid (ECT) as a chain transfer agent (CTA) conjugated to an azide-functionalized PEG (Az-PEG). The Az-PEG-ECT was then RAFT polymerized to DMAEMA and BMA as previously described to create a “smart” polymer capable of encapsulating anionic siRNA and inducing endosomal escape upon uptake [27, 41, 44]. The AzPEGDB was then reacted with Mn-alkyne (1:3 azide:alkyne molar ratio) via copper-catalyzed azide-alkyne cycloaddition (CuAAC) using a previously optimized copper catalyst concentration of 0.75 mM with 5 mM of sodium ascorbate . Chemical structures and 1H-NMR for all reaction steps are shown in Supplemental Figs. S1-4, and mannose conjugation was verified with Fourier transform infrared (FTIR) spectroscopy as previously described (Supplemental Fig. S5) . All chemical structures were made using ChemDraw (PerkinElmer). FTIR spectroscopy was performed at the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE) and all 1H-NMR experiments were performed at the Vanderbilt Small Molecule NMR Facility on a 400 MHz spectrometer (Bruker).
Nanoscale polymeric complexes were fabricated as previously described [27, 30, 41]. To form mannosylated-nanoparticles (MnNPs) with oligonucleotides, MnPEGDB was dissolved in 90% (v/v) 10 mM citrate buffer (pH = 4) with 200-proof ethanol (EtOH). MnNPs were complexed with either Cy5-dsDNA, IκBα siRNA, or scrambled siRNA for 30 minutes to form micelles before adding 5× volume 10 mM phosphate buffer (pH = 8) for a final solution pH of 7.4. The micelle N+/P− ratio, determined by mole ratio of protonated amines on the DMAEMA polymer to the number of phosphates on the oligonucleotides, was chosen as 10:1 based on previous studies [27, 41]. Nanoparticle size and zeta potential were evaluated using a Malvern Zetasizer located at VINSE, and the results are shown in Supplemental Fig. S6. All in vitro treatments were conducted with a final concentration of 50 nM oligonucleotides and all in vivo treatments were performed using a dose of 1 mg/kg (1 mg of oligonucleotide/kg of mouse weight). For in vivo NP preparation, the pH = 7.4 solution was diluted in sterile phosphate buffered saline (PBS) without magnesium and calcium and centrifuged in 5000 MWCO Amicon® Ultra-15 Centrifugal Filters (Millipore Sigma; UFC905024) at 2000×g for 30 minutes. The concentrated NPs were diluted in PBS (−/−) and centrifuged again before adding PBS to get to the appropriate volume for a 1 mg/kg concentration. The final preparation was filtered through a 0.45 μm syringe filter before being used for intraperitoneal injection.
Cell culture and tumor induction
Unless otherwise noted, all cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose, pyruvate, Gibco; 11,885,084) supplemented with 10% fetal bovine serum (FBS, certified, Gibco; 16,000,044) and 1% penicillin/streptomycin (P/S) at 37 °C in a 5% CO2 humidified atmosphere. Luciferized ID8 ovarian tumor cells and TBR5 genetically modified ovarian tumor cells were used as previously described [45,46,47,48]. TBR5 cells were from Dr. Sandra Orsulic  and luciferized ID8 cells were from Dr. Balkwill . All animal work was reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC). The ID8 cells were used in syngeneic C57Bl/6 background mice while TBR5 cells were used in syngeneic FVB background mice. For tumor induction, cells were resuspended in sterile PBS at a concentration of 5 × 106 cells/200 μL. A sterile 3 mL Luer-Lok™ syringe with an 18G needle was used to inject of 5 × 106 cells tumor cells in 200 μL PBS intraperitoneally (IP) into each mouse. At endpoint, all mice were euthanized via carbon dioxide inhalation with secondary cervical dislocation as approved by IACUC protocols.
Primary bone marrow-derived macrophages (BMDMs) were isolated from healthy female wild-type FVB mice and immortalized NGL-BMDMs were previously derived from NF-κB green fluorescent protein (GFP)-luciferase (NGL) reporter transgenic mice on an FVB background [51,52,53]. BMDMs were used for background in vitro assays and M2-polarized BMDMs are commonly used as surrogates for TAMs as a practical substitute [27, 29, 54].
Culturing and treating bone marrow-derived macrophages
Immortalized NGL-BMDMs were used from frozen stocks for in vitro experiments . NGL-BMDMs were plated in 6-well plates at 1 × 106 cells/well in 2 mL of DMEM (ThermoFisher; 11,995,073) supplemented with 10% FBS and 1% P/S. The macrophages were polarized to M1 with 10 ng/mL each of IFN-γ and LPS for 24 hours and to M2 with 10 ng/mL of IL-4 for 48 hours. M2-polarized NGL-BMDMs were treated with Scr-MnNPs or IκBα-MnNPs for 24 hours before collecting cells for luminescent measurements. Samples were prepared for luminescence readings using a Luciferase Assay System (Promega; E4030) following the manufacturer’s instructions.
In vivo biodistribution studies in ovarian tumor-bearing mice
For the preliminary 24-hour delivery study, 6 female C57Bl/6 mice were injected IP with 5 × 106 ID8 ovarian tumor cells in 200 μL PBS. Tumors developed for 8 weeks before treatment. Control mice were injected with 200 μL PBS and treatment mice received 200 μL PBS containing MnNPs loaded with Cy5-dsDNA at the previously listed dosage. After 24 hours, the mice were sacrificed and the ascites, tumors, and spleens were collected. The ascites was centrifuged at 1500 rpm for 5 minutes, supernatant collected, and red blood cells (RBCs) lysed with 5 mL Geyz lysing buffer (4.15 g NH4Cl, 0.5 g KHCO3 in 500 mL MilliQ water) for 5 minutes at 37 °C. This step was repeated as needed until a clear pellet was obtained. The resulting cells were resuspended in PBS with 1% BSA and snap-frozen in liquid nitrogen for RNA isolation or protein analysis. The solid tumors were collected into 3 mL DMEM (MT-10-13-CV) containing 10% FBS and 1% P/S for 1 hour on ice. The tissue was cut into small pieces and resuspended in 3 mL DMEM with 200 μL Collagenase A (Roche; 10,103,578,001), 300 μL hyaluronidase (Sigma; H4272), 500 μL DNase I (Sigma; D5025), and 30 μL amphotericin B (ThermoFisher; 15,290,026) and placed at 37 °C for 2 hours with frequent vortexing. The solution was filtered through a 70 μm strainer to form a single cell suspension used for flow cytometry. Tumors were then treated with the same RBC lysis buffer as the ascites. Similarly, spleens were collected in DMEM (10% FBS, 1% P/S) for 1 hour on ice, chopped into small pieces, and immediately filtered through a 70 μm strainer twice. The final single cell suspension was treated with RBC lysis buffer before flow analysis.
The long-term biodistribution study was performed in the TBR5 ovarian tumor model. 10 female FVB mice received IP injections of 5 × 106 TBR5 cells in 200 μL PBS (day 0). Tumors developed for 7 days before starting treatment on day 7. Mice either received IP injections of 100 μL PBS or 100 μL Cy5-MnNPs. Treatments were performed on days 7, 10, 14, and 17 before takedown on day 18. Single cell suspensions were made from tumors, ascites, and spleen as previously described and used for flow cytometry analysis.
Flow Cytometry of in vivo biodistribution
Single cell suspensions were obtained from the tumors, ascites fluid, and spleens of either ID8 or TBR5 tumor-bearing mice. For ID8 24-hour biodistribution, the cells were resuspended in flow buffer (PBS with 2 mM EDTA and 0.5% (v/v) BSA) at 1 × 106 cells/50 μL buffer. The following anti-mouse antibodies were used: CD45 PE-Cy7 (eBioscience; 25-0451-82), F4/80 PE (eBioscience; 12-4801-82), and Gr-1 Alexa Fluor 700 (eBioscience; 53-5931-82). After staining for 30 minutes, the cells were rinsed in PBS and resuspended in flow buffer before running flow analysis at the Vanderbilt Flow Cytometry Shared Resource. All flow analysis was performed using FlowJo v10.7.1. Flow gating strategy is shown in Supplemental Fig. S7.
For the TBR5 biodistribution study, flow cytometry was performed as previously described . The cells were incubated in an Fc block (BD Biosciences; 553,142) for 10 minutes at RT, stained for surface markers for 15 minutes at RT, washed with a FACS buffer containing PBS with 2% (v/v) FBS, and resuspended in the FACS buffer for flow analysis on a Miltenyi MACSQuant Analyzer 10 or 16. The eBioscience™ Foxp3/transcription factor staining buffer kit (Fisher Scientific; 00-5523-00) was used for intracellular staining. After surface staining, the cells were fixed and permeabilized for 20 minutes at 4 °C before staining for intracellular markers for 30 minutes at 4 °C. To quantify cell viability, a Ghost Dye Red 780 viability marker (1:4000, Cell Signaling Technology; 18452S) was used. The following anti-mouse antibodies were used: CD45 BV510 (1:1600, BioLegend; 103,138), CD3 FITC (1:200, BioLegend; 100,204), CD4 PerCP-Cy5.5 (1:600, BioLegend; 100,540), CD8a PE (1:800, eBioscience; 12-0081-82), B220 e450 (1:400, ThermoFisher; 48-0452-82), NKp46 PE-Cy7 (1:200, BioLegend; 137,618), CD11c PE (1:1000, BioLegend; 117,308), CD11b e450 (1:1600, ThermoFisher; 48-0112-82), F4/80 PE-Cy7 (1:800, BioLegend; 123,114), Ly6C FITC (1:4000, BioLegend; 128,006), and Ly6G PerCP-Cy5.5 (1:800, BioLegend 127,616). Flow cytometry data were analyzed with FlowJo v10.7.1. Representative gating strategies of ascites, tumors, and spleens are shown in Supplemental Figs. S8-10.
In vivo tumor studies
Treatment of the ID8 ovarian tumor model was formulated as a late-stage disease treatment. Similar to uptake studies, 5 × 106 ID8 ovarian tumor cells in 200 μL PBS were IP injected into 15 female C57Bl/6 mice (day 0) and allowed to develop tumors for 7 weeks. Starting on day 49, mice received IP injections of 100 μL PBS, Scr-MnNPs, or IκBα-MnNPs for 3 consecutive days. MnNPs were given at a dose of 1 mg/kg. The mice were euthanized 1 day after the final treatment. Blood samples were collected for liver (aspartate aminotransferase, AST, and alanine aminotransferase, ALT) and kidney (blood urea nitrogen, BUN) enzyme measurements and the ascites volume was measured and collected. Normal ranges for serum AST, ALT, and BUN levels were referenced from the Vanderbilt University Translational Pathology Shared Resource (TPSR). Tumors and spleens were then harvested. The ascites fluid was collected, centrifuged, and the supernatant stored for protein serum concentration analysis as described above. The remaining ascites cell pellet was further processed with RBC lysis as described above until the cell pellet was clear. The cell pellet was frozen at − 80 °C for RNA isolation. Tumors were weighed, cut in half, and one half was snap frozen in liquid nitrogen for RNA isolation. The other tumor half and the entire spleens were fixed in 10% formalin for 48-72 hours for histology before being switched to 70% EtOH. Processing, embedding and sectioning, and hematoxylin and eosin (H&E) staining of tumor tissue, as well as blood chemistry analyses, were performed by the Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resource (TPSR) core. H&E-stained tissues were imaged using the EVOS XL Core microscope (ThermoFisher) on 4x and 10x brightfield magnification.
The faster developing TBR5 model was used to model a more aggressive, early-stage treatment strategy. 5 × 106 TBR5 ovarian tumor cells in 200 μL PBS were IP injected into 15 female FVB mice (day 0) and allowed to develop tumors for 7 days. A biweekly treatment was adopted to combat the aggressive growth. Mice received IP injections of 100 μL PBS, Scr-MnNPs, or IκBα-MnNPs on days 7, 10, 14, 17, and 21 and the mice were sacrificed on day 22. For the extended study, additional MnNP treatments were administered on days 24 and 28 before euthanizing mice on day 29. Similar to the ID8 model, blood samples and ascites were collected immediately after takedown before surgically removing the spleens and tumors. The ascites was centrifuged and supernatant collected. The tumors were weighed and then split into two samples: one for fixation and one for snap freezing for RNA isolation. Spleens were fixed as previously described. The same analyses performed on the ID8 ovarian tumors were repeated here. For flow cytometry analysis, CD3 APC (1:200, BioLegend; 100,236) and CD8a AF488 (1:1600, BioLegend; 100,723) were used in place of CD3 FITC and CD8a PE used for biodistribution. The rest of the panel was the same with the addition of the following anti-mouse antibodies: CD206 APC (1:500, BioLegend; 141,708), FOXP3 PE (1:125, ThermoFisher; 12-5773-82), and PD-1 PE (1:100, BioLegend; 135,206). Flow gating strategy was repeated as previously shown. To visualize tumor cells populations, cells were gated on forward scatter (FSC) vs side scatter (SSC), single cells (FSC-area vs FSC-height), live/dead, and CD45-. The CD45- cells were visualized again as FSC vs SSC where a clear population of “big” SSC-high (SSChi) cells were present only in cells from the solid tumors and ascites, but not the spleen. Representative tumor cells gating is shown in Supplemental Fig. S11.
Immunofluorescent staining for confocal imaging of tumor sections
The protocol of immunofluorescent staining and analysis has been previously described . To evaluate CD8 T cell infiltration, the primary antibody rat anti-mouse CD8 (Novus Biologicals; BP1-49045SS, 1:100) was used with a secondary goat anti-rat IgG Alexa Fluor 488 (Abcam; ab150157). Previously sectioned tumor samples were deparaffinized in Xylenes 2x for 10 minutes each. The samples were then rehydrated in 100% EtOH 2x for 2 minutes each, and then once each in 90, 80, and 70% EtOH (v/v in DI water) for 2 minutes. The slides were then incubated on a shaker in 0.1% Sudan Black Dye solution (diluted in 70% EtOH) for 20 minutes before one more 2-minute wash in 50% EtOH. The sections were then permeabilized in 1X Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) for 20 minutes, rinsed in TBS for 5 minutes, and finally rinsed briefly with Milli-Q DI water. Antigen retrieval was performed by placing slides in a rice cooker with a 10 mM sodium citrate solution (pH = 6.0) with 0.1% Tween 20 and heated for 15-20 minutes before cooling at RT for 30 minutes. The slides were then washed 3x with TBS for 5 minutes each. Slides were treated with a blocking buffer comprised of 4 mL 0.5% TBST, 0.04 g bovine serum albumin, and 250 μL goat serum (Abcam; ab7481) for 1 hour in a humidified chamber at RT. The blocking buffer was aspirated and 100 μL of primary antibody was added and incubated at 4 °C overnight. The primary antibody was aspirated and each slide washed 3x for 10 minutes each in TBST and then rinsed a final time with TBS for 5 minutes while shaking. The TBS was aspirated and 100 μL of secondary antibody was diluted in blocking buffer (1:1000) and incubated at RT in the dark for 2 hours. The slides were washed 3x for 10 minutes each in TBST followed by a single 5-minute wash in TBS on a shaker. DAPI (0.1 μ/mL) was added to each slide and incubated at RT for 5 minutes to stain cell nuclei. The slides were washed 2x for 3 minutes each in TBS and then 20 μL ProLong™ Gold Antifade Mountant (ThermoFisher; P36930) was added to preserve fluorescent signal. Slides were stored at 4 °C until imaging was performed via fluorescent microscopy (Nikon C1si + confocal microscope system on Nikon Eclipse Ti-0E inverted microscope base, Plan APO VC 20× objective, 405/488 dichroic mirror). Images were analyzed using Fiji in ImageJ .
RNA extraction for quantitative reverse transcription polymerase chain reaction (qRT-PCR)
For in vitro BMDM experiments, RNA was isolated using the RNeasy Mini Kit (Qiagen; 74,106) and residual DNA was removed using the RNase-Free DNase set (Qiagen; 79,256). cDNA was synthesized with SuperScript IV reverse transcriptase kit (Invitrogen; 18,090,050). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad; 1,725,270) on a CFX96 real-time PCR instrument and software (Bio-Rad) through the VUMC Molecular Cell Biology Resource (MCBR) core.
For in vivo experiments, RNA was extracted from both the ascites cells and tumor cells using TRIzol™ (Invitrogen; 15,596,026) and a Direct-zol RNA Miniprep kit (Zymo Research; R2050). Snap frozen tumor samples were ground into small pieces with a mortar and pestle before suspending in 300 μL TRIzol solution. The ascites pellet was resuspended in 300 μL TRIzol solution. Both solutions were used with the Direct-zol RNA Miniprep kit following the manufacturer’s instructions. Final RNA concentration was measured with a NanoDrop 200 spectrophotometer (Biotek). cDNA fabrication and qRT-PCR were performed as described above. All primer sequences are listed in Supplemental Table 2. For all experiments, target genes were normalized to a housekeeping gene (B2M or GAPDH) to obtain the ΔCT value. All qRT-PCR data is shown as relative expression using the 2-ΔΔCT method.
Protein isolated from ascites cells in the TBR5 experimental model was used for western blot analysis of IκBα expression. Whole protein isolation, western blotting, and signal detection were performed as previously described . Primary antibodies used were rabbit polyclonal anti-IκBα (1:100 dilution, Cell Signaling Technology; 9242). Equal loading was confirmed using mouse monoclonal anti-histone H3 (1:1000 dilution, Cell Signaling Technology; 14269S) as a loading control. To image loading control and experimental antibody on the same blot, the gel was cut prior to hybridization. The pieces of the uncropped blot are shown in Supplemental Fig. S12.
All statistical analyses were performed using a one-way ANOVA with Tukey’s multiple comparison test in the case of two or more groups, a two-way ANOVA with Tukey’s multiple comparison test in the case of two or more groups in two or more sets, or a two-tailed student’s t-test in the case of only two groups, all with α = 0.05. Statistical analyses were performed using GraphPad Prism v8.4.3. All figures were made using Adobe Photoshop 2020 v21.2.2.