Nanoparticle carriers are widely used for nucleic acid delivery, yet the dominant surface modifier poly(ethylene glycol) (PEG) increasingly raises concerns due to immunogenicity. Zwitterionic polymers are emerging as promising PEG alternatives, but their performance is rarely evaluated in primary immune cells or across biological sexes. Here, we synthesized pH-responsive PEG- and zwitterion-grafted cationic nanoparticles, including PEG methyl ether methacrylate (PEGMMA), carboxybetaine methacrylate (CBMA), sulfobetaine DMAPS, and polymeric PDMAPS, and assessed their cytotoxicity and siRNA delivery in primary male and female murine macrophages. All formulations enabled intracellular siRNA uptake and suppressed tumor necrosis factor–α (TNF-α) expression. However, sex-dependent effects varied by surface chemistry and biological endpoint: zwitterionic nanoparticles exhibited sex-specific effects in cytotoxicity and gene silencing despite similar uptake, whereas PEGMMA showed higher uptake in females but comparable knockdown across sexes. These findings demonstrate that uptake alone does not predict functional efficacy and highlight the importance of sex-informed evaluation when developing nanomedicines.

Materials:

Monomers 2-(dimethylamino)ethyl methacrylate (DMAEMA) (Cat. No. 234907), bis(2-methacryloyl)oxyethyl disulfide (DSDMA), Poly(ethylene glycol) methyl ether methacrylate solution (PEGMMA2k, Mn = 2000) (Cat. No. 457876), ethyl α-bromoisobutyrate (EBiB, 98%) (Cat. No. 252271), and L-ascorbic acid (AA) (Cat. No. A7506) were purchased from Sigma-Aldrich. Hexyl methacrylate (Cat. No. M0503) and Tris(2-pyridylmethyl)amine (Cat. No. T2671) were purchased from TCI America. Brij 30 (Cat. Code 216725000), Brij 35 (Cat. No. 228345000), and copper (II) bromide (99 + %) (Cat. No. 206331000) were purchased from Acros Organics. Tetrahydrofuran, acetone, ethanol, and hydrochloric acid were purchased from Fisher Scientific. Carboxybetaine methacrylate (CBMA) was purchased from MedChem Express. 2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA),  4-((((2-Carboxyethyl)thio) carbono thioyl) thio)-4-cyanopentanoic acid (CTA), 4,4-azobis(4-cyanopentanoic acid) (ACVA), and sodium chloride (NaCl) were used as received from Sigma Aldrich and Fisher Scientific. RPMI 1640 media, penicillin-streptomycin, and fetal bovine serum (FBS), lipopolysaccharide (LPS), and macrophage colony-stimulating factor (MCSF) were all purchased from Fisher Scientific. MTS reagents (CellTiter 96 AQueous One Solution Cell Proliferation Assay) were obtained from Promega Corporation. Mission siRNA Fluorescent Universal Negative Control #2, 6-FAM (catalog #SIC008-5X1NMOL) was purchased from Sigma Aldrich. Mouse TNF-ɑ siRNA was purchased from Thermo Scientific (catalog #4390771), and the Mouse TNF-ɑ/TNFA/TNFSF2 ELISA Pair Set was sourced from Sino Biological (catalog #SEK50349-5).

Table 1: Reagents Required for Nanocarrier Synthesis

Synthesis via RAFT Polymerization:

The synthesis of PSBMA was carried out by RAFT polymerization using a method previously described with slight modifications. To obtain a similar molecular weight to what was used for the PEG NPs, the ratio between SBMA and the CTA was carefully tailored. SBMA (1396.75 mg, 5 mmol, 7 equiv.), CTA (219.58 mg, 0.71 mmol, 1 equiv.), and ACVA (40 mg, 0.14 mmol, 0.2 equiv.) were dissolved in a 0.5 M NaCl solution (50% wt. with SBMA). The pH was adjusted to 7, and the solution was purged with nitrogen for 40 min. The solution was then left to polymerize at 70°C for 19h. Finally, after the reaction was finished, the polymer was lyophilized and stored until further use.

Synthesis of Cationic Nanoparticles via ARGET-ATRP:

PEGMMA, CBMA, SBMA, and PSBMA NPs were synthesized using a one-pot oil-in-water emulsion polymerization approach via ARGET-ATRP process. Emulsions were created by mixing all reagents in a 1-dram glass vial in 2-mL batches and homogenizing them for 20 minutes with a 24-tip microtip sonicator probe (10s on/10s off cycle) (Sonicator Ultrasonic Processor XL from QSonica). Subsequently, the samples were transferred into an MBraun Glovebox LABmaster 130 under inert nitrogen conditions (O₂<0.1 ppm; H₂O <0.1 ppm) to initiate the reaction by adding ascorbic acid. After 24 hours, the reaction was stopped by exposing the samples to air and diluting them to a 0.5 N concentration by adding 4N HCl. Unreacted monomers and impurities were removed from the NPs using ionomer collapse, which involves acidifying the samples and precipitating them with a low dielectric constant solvent like tetrahydrofuran. The nanogels were precipitated through ionomer collapse three times and collected after each cycle by centrifugation at 4000g for 5 minutes. Following the final centrifugation, the NPs were resuspended in 5 mL of deionized water and dialyzed against deionized water for at least 7 days to eliminate any residual tetrahydrofuran ³⁸. CBMA and SBMA particles were synthesized at two different monomer input volumes to investigate the effect of zwitterionic concentration. CBMA and SBMA were synthesized using 11 μL and 13 μL of 2 mg/mL zwitterionic stock solutions, respectively.

Nuclear Magnetic Resonance:

The ¹H^^ Nuclear Magnetic Resonance (NMR) spectrum of PSBMA was obtained using a Bruker 500 MHz NMR spectrometer. PSBMA was dissolved in D₂O at a 5 mg/mL concentration and analyzed using the MestReNova 14.3.0 software.

Fourier-Transformed Infrared Spectroscopy:

The Fourier-Transformed Infrared (FTIR) Spectroscopy spectrum of PSBMA was acquired using an Attenuated Total Reflectance FTIR through a germanium crystal on a Nicolet iS10 spectrometer with a SMART iTX accessory, both from Thermo Scientific. The lyophilized sample was analyzed by placing it on the crystal after the background spectrum was collected. The spectrum was analyzed using Omnic software.

Characterizing Nanoparticle Size & Electrophoretic Potential:

The hydrodynamic diameters and zeta potentials of the NPs were determined using dynamic light scattering (DLS) and zeta potential measurements on a Malvern ZetaSizer (NanoZS). For DLS characterization, the stock NPs solution was diluted 66.67 times in 1X PBS (75 μL NP: 5 mL PBS) to a final volume of 5 mL. Measurements were taken at pH 3-5 (acidic), pH 6-7 (neutral), and pH 9 (basic) to assess NPs size and monodispersity (expressed as the polydispersity index, PDI). The volume swelling ratio was calculated by:

where V is the respective volume at each pH, which simplifies to each diameter (d) at each pH cubed. Zeta potential measurements were performed on diluted NPs (150 μL NP: 6 mL 5 mM sodium phosphate buffer) at pH 3-5 (acidic) and pH 6-7 (neutral). Folded capillary zeta cells were used for these measurements. All DLS and zeta potential readings were conducted in triplicate using standardized protocols optimized for cationic NPs.

Monocyte Isolation & Culture:

Bone marrow-derived monocytes were isolated from the hindlimbs of male and female FVBn mice (all littermates, 6-8 weeks old; N=3 per sex) and cultured as described by Rybalko et al. Bones from the hindlimbs of the mice were extracted, and marrow was subsequently isolated in the cell culture hood by flushing bones with sterile PBS. Cells were centrifuged at 500g for 5 min and resuspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin, along with MCSF (10 ng/mL) at a density of 1 × 10⁶ cells/mL. For 96-well plates, 100 μl of cells (1 × 10⁶ cells/ml) were added to each well. Media with fresh MCSF was added on days 3, 5, and 7 to differentiate monocytes into macrophages. Experiments were performed beginning on day 7.

All experiments (cytotoxicity, uptake, and TNF-α knockdown) included N = 3 biological replicates per sex, with n = 6–9 technical replicates per condition. Samples were not pooled; each biological replicate corresponds to cells derived from a single mouse. The same batch of cells from each animal was used across all assays to ensure consistency between endpoints.

Cytotoxicity:

The cytotoxicity of the NPs was assessed using the colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay kit. This kit uses a soluble tetrazolium salt, [3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethyl phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS), which is bio-reduced by viable cells into a purple formazan product. The level of MTS bio-reduction correlates with the metabolism of the cells, which is often used as a measure of cell viability. NPs were tested on primary murine monocytes grown to 40,000 cells per well. On day 7, NPs were added to the cell culture medium at concentrations of 12.5, 50, 125, and 500 μg/ml. After 24 hours of NP exposure, the cells were washed twice with DPBS and resuspended in assay media (16.67% MTS reagent, 83.33% growth media without phenol red). The plates were incubated for 2 hours at 37°C and 5% CO₂, after which absorbance readings were taken at 490 using a Biotek Cytation 3 Plate Reader.

Fluorescent siRNA Loading and Uptake in Primary Monocytes:

NPs (5000 ng/ml) were loaded at a 10:1 NP: siRNA ratio with 500 ng/ml fluorescent siRNA (fluorescent siRNA or anti-TNF-ɑ siRNA) in sterile 1% DEPC-treated PBS at pH 5.5, vortexed, and allowed to incubate for one hour in the dark. Loaded NPs were centrifuged at 15,000 x g for 10 min, and the loading supernatant was removed and replaced with 500 μL of cell culture medium. Loaded NPs solutions were then added to the cells at 12.5 μg/ml for 24 hours. Wells were then washed twice with DPBS and placed in phenol-free RPMI 1640 culture medium. Wells were imaged using an Incucyte S3 Live Cell Analysis Instrument (Sartorius). The phase contrast and green fluorescent channels were used to measure the cells and siRNA, respectively, with three wells per condition. The Incucyte software was used to measure cell confluency and fluorescence intensity within the cells. Relative uptake was then calculated via:

 (Fluorescence Intensity per Area)/(Cell Confluency)=Relative Uptake

 A control condition using NPs without fluorescent siRNA (blank NPs) was included to account for background fluorescence. The average signal from this control was subtracted from all uptake values.

TNF-ɑ Knockdown:

NPs loaded with anti-TNF-ɑ siRNA were centrifuged at 15,000g for 10 min, and the loading supernatant was removed and replaced with 500 μL of cell culture medium. Simultaneously, mouse macrophages were cultured as previously described and were treated with LPS at 50 ng/ml overnight to induce TNF-ɑ production. Macrophages were then treated with NPs at 12.5 μg/ml. After 24 hours, the treated cell media was collected. TNF-ɑ content was assessed with an enzyme-linked immunosorbent assay kit manufactured by Sino Biological. The standard curve was plotted using a 4-parameter logistic curve. Percent TNF-ɑ knockdown was calculated by normalizing to the LPS-stimulated control of the specified sex.

Statistical Analyses:

All experiments were run in triplicate. Primary cell experiments were done with biological triplicates (n=3) for each sex (male, female), with technical replicates for each assay ranging from 3-6. For technical replicates, the mean values were found. Values are reported as the biological mean ± standard deviation unless otherwise noted in the figure caption. Data were plotted in Microsoft Excel and GraphPad Prism. All statistics were run in GraphPad Prism as t-tests, one-way ANOVA, or two-way ANOVA (sex + nanoparticle treatment considered multiple variables) with a post-hoc analysis using Tukey HSD or Šidák’s. Reported significant results are p < 0.05 unless otherwise stated.