Self-Assembled Nanoparticles Prepared from Low-Molecular-Weight PEI and Low-Generation PAMAM for EGFRvIII-Chimeric Antigen Receptor Gene Loading and T-Cell Transient Modification

Purpose: CAR-T cell therapy is limited by complex manufacturing, viral vector use, high cost, and severe toxicities. A nonviral, transient CAR modification approach using self-assembled nanoparticles could provide a simpler, safer, and potentially broadly applicable alternative for T-cell engineering.

Hypothesis: If plasmid DNA is loaded into self-assembled nanoparticles prepared from low-molecular-weight PEI and low-generation PAMAM, then the optimal formulation pDNA@SNP G1/800 will efficiently transfect Jurkat T cells with low cytotoxicity, enable transient EGFRvIII-CAR expression, and allow CAR-positive T cells to specifically recognize EGFRvIII-positive tumor cells.

Aims: Prepare and screen a series of pDNA@SNPs from Ad-PAMAM G1/G5 and CD-PEI 800/2000/25000 with Ad-PEG. - Select the best formulation based on Jurkat T-cell transfection efficiency and cytotoxicity. - Characterize the selected pDNA@SNP G1/800 for size, zeta potential, morphology, stability, cellular binding, and endosomal escape. - Construct pEGFRvIII-CAR, encapsulate it into SNP G1/800, and evaluate transient CAR expression in Jurkat cells. - Assess whether EGFRvIII-CAR-positive Jurkat cells specifically interact with EGFRvIII-positive tumor cells.

Delivery system: Platform: Supramolecular self-assembled nanoparticles (SNPs) formed by adamantane–cyclodextrin host–guest recognition. - Components: Adamantane-grafted PAMAM dendrimer (Ad-PAMAM G1 or G5); cyclodextrin-grafted branched PEI (CD-PEI 800, 2000, or 25,000 Da); adamantane-grafted PEG (Ad-PEG). - Selected formulation: pDNA@SNP G1/800, prepared from Ad-PAMAM G1, CD-PEI800, and Ad-PEG. - Payload: Plasmid DNA: pGL3 luciferase, gWIZ-GFP, and pEGFRvIII-CAR. - CAR construct: Third-generation EGFRvIII-specific CAR with humanized EGFRvIII scFv, CD8α hinge/transmembrane region, and CD28/4-1BB/CD3ζ signaling domains. - Targeting ligand: None; delivery relies on nanoparticle incubation with Jurkat T cells.

Approach: In vitro only: Jurkat T-cell line for transfection, cytotoxicity, uptake, and CAR expression. HuH7 hepatocarcinoma cells engineered to overexpress EGFRvIII were used as target tumor cells. - Formulation screening: Six pDNA@SNP formulations tested for pGL3 transfection in Jurkat cells. - Selected formulation: pDNA@SNP G1/800 further characterized and used for pEGFRvIII-CAR delivery. - Controls: Naked pDNA, PEI800/pDNA complexes, and Lipofectamine 2000/pDNA complexes where relevant. - No in vivo experiments.

Key methods: Gel electrophoresis for pDNA loading. - DLS and TEM for size, zeta potential, and morphology. - Luciferase assay for transfection efficiency. - Fluorescence microscopy for GFP expression. - CCK-8 assay for cytotoxicity. - Confocal microscopy with dual-labeled pDNA@SNP G1/800 (Cy5.5-CD-PEI800 and YOYO-1-pDNA) and LysoTracker Red for cellular binding and endosomal escape. - Flow cytometry with FITC-protein L for surface EGFRvIII-CAR expression. - Western blot for EGFRvIII-CAR expression. - Confocal microscopy for interaction between DiI-labeled CAR-positive Jurkat cells and CFSE-labeled EGFRvIII-positive HuH7 cells.

Key results: Formulation screening: pDNA@SNP G1/800 gave the highest luciferase expression in Jurkat cells. Its activity was not significantly different from pDNA@SNP G1/2000 but was higher than other formulations, including up to ~40-fold higher than pDNA@SNP G5/25000. - Physicochemical properties: pDNA@SNP G1/800 had a mean hydrodynamic diameter of ~120 nm, zeta potential ~+20 mV, uniform morphology by TEM, and stable size in PBS over 24 h. - Cytotoxicity: pDNA@SNP G1/800 showed lower cytotoxicity than PEI800/pDNA complexes at increasing pDNA doses, especially at 3.0 and 6.0 µg. - Transfection: pGL3@SNP G1/800 produced ~10-fold higher luciferase activity than Lipofectamine 2000/pGL3. pGFP@SNP G1/800 yielded high GFP expression, whereas PEI800/pGFP gave no detectable GFP. - Cellular trafficking: Dual-labeled nanoparticles bound to Jurkat cells within 15–30 min, and YOYO-1-labeled pDNA separated from LysoTracker Red by 4 h, indicating endosomal escape. - CAR expression: FACS with protein L and Western blot confirmed EGFRvIII-CAR expression on pEGFRvIII-CAR@SNP G1/800-transfected Jurkat cells. - Tumor cell recognition: EGFRvIII-CAR-positive Jurkat cells specifically recognized and bound EGFRvIII-positive HuH7 cells; non-CAR Jurkat cells did not.

Interpretation: The authors conclude that pEGFRvIII-CAR@SNP G1/800 can efficiently deliver CAR-encoding plasmid into Jurkat T cells, achieve transient EGFRvIII-CAR surface expression, and enable specific recognition of EGFRvIII-positive tumor cells. They propose this nonviral self-assembled nanoparticle system as a promising approach for transient T-cell CAR modification and CAR-T cancer therapy.

Limitations: Purely in vitro; no primary human T cells, no in vivo antitumor efficacy, and no toxicity/biodistribution data. - Jurkat is a leukemic T-cell line, not primary T cells; its CD4/CD8 properties may differ from patient T cells. - No functional cytotoxicity or tumor-killing assay was performed; only recognition/binding was shown. - Transient CAR expression duration, stability, and repeated-dosing potential were not fully characterized. - No active T-cell targeting ligand; delivery depends on direct incubation. - No direct comparison with viral vectors or mRNA electroporation for CAR-T generation. - Authors state future studies are needed in primary T cells and in vivo models.