Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo

Component: Polycationic Polymer; Description: Branched low molecular weight PEI (4-10 kDa, PEI-F25 LMW) — 2.5 μg/well for in vitro; 50 μg/2×10⁷ cells for in vivo

Component: Liposomal Reagents; Description: • Ibafect — 3 μg/well (PromoKine)<br>• Lipofectamine™ 2000 — 1.5 μg/well (Life Technologies)

Component: Magnetic Nanoparticles; Description: • Matra-A (PromoKine) — 1 μL/well for magnetofection<br>• FluidMag (PEI enhancement) — 1:1000 ratio<br>• MA Lipofection Enhancer (Ibafect enhancement) — 0.5 μg/well<br>• CombiMag (Lipofectamine enhancement) — 0.5 μg/well

Component: Nucleic Acid Payloads; Description: • Nonsense AON (2'-O-Me-PTO-RNA, 5'-caagggcauuacacuaccu-3') — 0.5 μg/2.5×10⁵ cells; FAM-labeled<br>• Nonsense siRNA (5'-cguacgcggaauacuucga-3') — 0.5 μg/2.5×10⁵ cells; Alexa488-labeled

Component: Cell Types; Description: • Primary human PBMCs (total and naPBMCs; n=4 donors)<br>• Jurkat T cell line (human)<br>• RLD1 T cell line (murine)<br>• Murine splenocytes (C57Bl/6)

Component: In Vivo Model; Description: GvHD: C57Bl/6 → BALB/c (H-2ᵇ → H-2ᵈ); irradiation (8 Gy); BM + splenocyte transplantation

Component: Stimulus; Description: Phytohaemagglutinin (PHA, 5 mg/mL) — T cell mitogen

Parameter: In Vitro Transfection; Details: PBMCs or naPBMCs (2.5×10⁵ cells/well, 96-well); 24 h transfection; washed; PHA stimulated for 24-48 h

Parameter: Proliferation Measurement; Details: LTT: ³H-thymidine (1 μCi/well, 20 μCi/mL); 16-18 h incorporation; β-radiation measured (Wallac 1450 MicroBeta TriLux); results as CCPM

Parameter: Cell Cycle Analysis; Details: APC BrdU Flow kit (BD Biosciences); BrdU (10 μM) added for up to 6 days; BrdU/7-AAD staining; flow cytometry; G0/G1 (BrdU⁻/7-AAD⁻), S (BrdU⁺), G2/M (BrdU⁻/7-AAD⁺)

Parameter: Gene Expression; Details: RT-qPCR (QuantStudio Flex); Ki67 and IL-2; Rplp0 housekeeping; ΔΔCt method; N=4 donors

Parameter: Uptake Measurement; Details: Flow cytometry; FAM-labeled AON or Alexa488-labeled siRNA; median fluorescence intensity

Parameter: Viability; Details: Trypan blue exclusion (necrosis only)

Parameter: In Vivo GvHD Model; Details: Splenocytes (2×10⁷) transfected ex vivo with PEI (50 μg) + AON (10 μg) or Ibafect (30 μg) + MA Enhancer (10 μg) + AON; 4 h; washed; mixed 1:1 with BM cells; transplanted i.v. into irradiated BALB/c

Parameter: Groups (GvHD); Details: • PEI-Co (PEI-transfected; n=16)<br>• Iba-Co (Ibafect-transfected; n=3)<br>• GvHD-Co (untransfected; n=16)<br>• NaCl-Co (irradiation control, no cells; n=4)

Parameter: Clinical Scoring; Details: Daily: weight, mobility, posture, skin, fur (0-10 cumulative); endpoint: score >6 or weight loss >25%

Parameter: Statistical Tests; Details: Student's t-test; Log-rank (Mantel-Cox) for survival

Analysis Category: Proliferation; Methods: ³H-thymidine incorporation (LTT); CCPM (cell counts per minute)

Analysis Category: Cell Cycle; Methods: BrdU incorporation + 7-AAD staining; flow cytometry (BD FACSCanto); G0/G1, S, G2/M phases

Analysis Category: Gene Expression; Methods: RT-qPCR: Ki67 (proliferation marker), IL-2 (T cell proliferation cytokine), Rplp0 (housekeeping); ΔΔCt method

Analysis Category: Uptake; Methods: Flow cytometry: FAM (AON) or Alexa488 (siRNA); median fluorescence intensity

Analysis Category: Viability; Methods: Trypan blue exclusion

Analysis Category: In Vivo Monitoring; Methods: Daily clinical score (weight, mobility, posture, skin, fur); peripheral blood flow cytometry (CD4⁺, CD8⁺, CD3⁺, CD19⁺, CD25⁺) every 2 weeks

Analysis Category: Statistical Analysis; Methods: Student's t-test; Log-rank (Mantel-Cox) for survival (SPSS v22)

Reagent: Ibafect (AON); Magnetic Enhancement: -; Proliferation Change (vs. Control): +89.1% (stimulation); Significance: p = 0.0013

Reagent: Lipofectamine (AON); Magnetic Enhancement: -; Proliferation Change (vs. Control): +96.4% (stimulation); Significance: p = 4×10⁻⁵

Reagent: PEI (AON); Magnetic Enhancement: -; Proliferation Change (vs. Control): No significant change; Significance: NS

Reagent: PEI (siRNA); Magnetic Enhancement: -; Proliferation Change (vs. Control): Reduced; Significance: -

Reagent: PEI (siRNA); Magnetic Enhancement: + FluidMag; Proliferation Change (vs. Control): Reduced; Significance: p = 2.5×10⁻⁴

Reagent: PEI + FluidMag (siRNA); Cell Type: naPBMCs; % Proliferation Decrease: 85.8%; Significance: p = 2.5×10⁻⁴

Reagent: PEI + FluidMag (siRNA); Cell Type: PBMCs; % Proliferation Decrease: 39.9%; Significance: Significant

Reagent: Ibafect + MA Enhancer; Cell Type: PBMCs/naPBMCs; % Proliferation Decrease: Reduced; Significance: Trend (not all significant)

Condition: PEI + magnetic beads (PBMCs); Effect: Increased G0/G1 (resting), reduced mitosis (days 1-6)

Condition: PEI + magnetic beads (Jurkat); Effect: Increased G0/G1, reduced mitosis (up to day 2)

Condition: Ibafect ± magnetic beads; Effect: No significant cell cycle changes (despite anti-proliferative effect)

Condition: Matra-A beads alone; Effect: Induced cell cycle arrest in Jurkat and RLD1 cells

Gene: IL-2; Condition: PEI (no beads); Expression Change: Increased; Significance: p = 0.003

Gene: IL-2; Condition: PEI + beads; Expression Change: Decreased; Significance: p = 0.002

Gene: IL-2; Condition: Ibafect + beads; Expression Change: Decreased; Significance: p = 0.007

Gene: Ki67; Condition: PEI + beads; Expression Change: Decreased; Significance: p = 0.002

Gene: Ki67; Condition: Ibafect ± beads; Expression Change: No significant change; Significance: NS

Parameter: Uptake rate (all methods); Result: ~30-50%

Parameter: Oligonucleotide amount per cell; Result: Increased with magnetic beads (significant for Ibafect; trend for PEI)

Parameter: Viability (PEI); Result: Significantly reduced vs. control (p < 0.0001)

Parameter: Viability (with vs. without magnetic beads); Result: No significant difference

Group: NaCl-Control (irradiation only); Median Survival: 12 days; vs. GvHD-Control: -; Significance: -

Group: GvHD-Control (untransfected); Median Survival: ~35 days; vs. GvHD-Control: -; Significance: -

Group: PEI-Co (PEI-transfected); Median Survival: Significantly prolonged; vs. GvHD-Control: Improved; Significance: p = 0.002

Group: Iba-Co (Ibafect-transfected); Median Survival: Prolonged; vs. GvHD-Control: Improved; Significance: p = 0.060 (n=3)

1. In vitro observations with non-therapeutic nucleic acids: The study used nonsense (non-functional) AON and siRNA, not therapeutic gene payloads; effects may differ with functional nucleic acids.

2. Correlative mechanism: While the study shows association between proliferation inhibition and cell cycle arrest/gene expression changes, the causative link between magnetofection, inhibited proliferation, and arrested cell cycle is "still unclear." The authors hypothesize about membrane tension/endocytosis but do not definitively prove the mechanism.

3. Small Ibafect group in vivo: The Ibafect-treated GvHD group had only n=3 animals, limiting statistical power and interpretation (p = 0.060).

4. Sudden deaths in irradiation controls: The authors note that four NaCl-treated irradiated control animals died at day 12 due to hematopoietic insufficiency—a known issue in the model—but this complicates survival interpretation.

5. In vitro cell viability measured only by Trypan blue: Trypan blue only detects necrotic cells, not apoptosis; the authors acknowledge that "most [methods] did not induce apoptosis at standard dosages" but did not present detailed apoptosis data.

6. BrdU incorporation may underestimate proliferation: The cell cycle analysis with BrdU relies on S-phase labeling; cells that divided after BrdU was added may have diluted the label and been misclassified.

7. Limited mechanistic exploration: The study did not investigate the signaling pathways (e.g., PI3K/AKT, MAPK) that might mediate the observed proliferation effects.

8. Potential confounding from AON/siRNA sequences: The use of specific nonsense sequences may have had off-target effects or sequence-specific immune stimulation (e.g., TLR activation) not controlled for.

9. No comparison to viral transduction: The study focuses solely on non-viral methods; the findings may not generalize to viral gene delivery systems.

10. Clinical translation uncertainty: While the authors suggest possible therapeutic applications, the study does not demonstrate active therapeutic gene delivery; the observed effects are on the transfection reagents themselves, not on the therapeutic payload.

Report prepared based on the published PLOS ONE article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.