Potential of Ni(II)-NTA-Modified Poly(ethylene imine) Glycopolymers as Carrier System for Future Dendritic Cell-Based Immunother

Purpose: Dendritic cell (DC)-based immunotherapy requires efficient delivery of antigens to immature DCs (iDCs) and maintenance of their immunostimulatory and migratory functions. Cationic PEI carriers are limited by dose-dependent toxicity and aggregation at endosomal pH, so a biocompatible, pH-responsive carrier is needed.

Hypothesis: If a Ni(II)-NTA-modified PEI glycopolymer (Ni(II)-NTA-DG) is used to bind His-tagged HIV Gp160 peptide, then peptide uptake into monocytes and iDCs will increase, the peptide will localize to endosomal/lysosomal compartments, iDCs will be activated, and mature DCs will retain their migration capacity.

Aims: Evaluate Ni(II)-NTA-DG as a carrier for His₆-Gp160 peptide in monocytes and iDCs. - Determine biocompatibility and peptide uptake efficiency at different polyplex ratios. - Analyze intracellular localization in early endosomal and lysosomal compartments. - Assess effects on iDC/mDC phenotype, cytokine secretion, and migration.

Delivery system: Platform: Ni(II)-NTA-modified hyperbranched PEI dendritic glycopolymer (Ni(II)-NTA-DG). - Polymer chemistry: Hyperbranched PEI (Lupasol WF, \(M_w\) 25,000 g/mol) surface-modified with maltose and NTA ligands; Ni(II) loaded onto NTA. NTA-DG molecular weight ~72,900 g/mol. - Payload: His-tagged HIV-derived peptide His₆-Gp160 (HR-2 region of gp41), FITC-labeled or unlabeled. - Binding mechanism: Ni(II)-NTA–His-tag affinity; up to ~6 His₆-Gp160 molecules per Ni(II)-NTA-DG; pH-sensitive release initiated below pH 6.2. - Targeting/adjuvant features: Maltose units may interact with mannose receptors on DCs; excess Ni(II)-NTA ligands may act as adjuvant via TLR-4 signaling.

Approach: In vitro human cell system: Monocytes isolated from healthy donor PBMCs; iDCs generated with GM-CSF/IL-4 for 7 days; mDCs obtained by LPS treatment. - Polyplex ratios: His₆-Gp160:Ni(II)-NTA-DG at 1:1, 1:2, and 1:4; controls included His₆-Gp160 alone and His₆-Gp160/NTA-DG without Ni(II). - Doses: His₆-Gp160 1, 2, or 4 µM; Ni(II)-NTA-DG 4, 8, or 16 µM. - No in vivo experiments.

Key methods: Flow cytometry for peptide uptake, viability (annexin V/PI), and surface markers CD86, CD80, HLA-DR, CCR7. - Confocal laser scanning microscopy for colocalization with EEA1 (early endosomes), LAMP1 (lysosomes), and RITC-labeled Ni(II)-NTA-DG. - ELISA for IL-8, IL-6, TNF-α, and IL-12(p70). - Transwell chemotaxis assay toward CCL19 and CCL21. - DLS-based stability data referenced for pH-dependent release.

Key results: Biocompatibility: 4 µM His₆-Gp160 and 20 µM Ni(II)-NTA-DG did not significantly reduce monocyte or iDC viability. Polyplex 1:4 at 4 µM peptide/16 µM polymer significantly reduced monocyte viability; iDC viability was not significantly affected. Highest working polyplex 1:4 concentration: 2 µM His₆-Gp160 for monocytes and 4 µM for iDCs. - Uptake: Monocytes showed >98% peptide-positive cells with polyplexes; polyplex 1:4 gave 2.8-fold higher uptake than His₆-Gp160 alone (1:2, 2.1-fold; 1:1, 1.6-fold). iDCs showed 6-fold higher uptake with 1:4 (1:2, 4-fold; 1:1, 2.5-fold). mDC uptake was low. - Localization: His₆-Gp160 colocalized with EEA1 (Pearson 0.70 monocytes, 0.72 iDCs) and LAMP1 (0.64 monocytes, 0.79 iDCs). His₆-Gp160 and Ni(II)-NTA-DG-RITC colocalized strongly (0.91 monocytes, 0.93 iDCs). - Phenotype/cytokines: Polyplexes up-regulated CD86 and CD80 on iDCs, with polyplex 1:4 showing highest CD86 up-regulation; HLA-DR was unchanged. IL-8 and IL-6 were increased by Ni(II)-NTA-DG and polyplexes. mDC cytokine secretion was not significantly influenced. - Migration: mDCs retained high CCR7 expression and migrated toward CCL19/CCL21 after polyplex treatment (e.g., ~68% migrated toward CCL19 with polyplex 1:4).

Interpretation: The authors conclude that Ni(II)-NTA-DG is a biocompatible carrier that increases His-tagged antigen uptake, delivers peptide to MHC class II–rich endosomal/lysosomal compartments, pre-stimulates iDCs via Ni(II)-NTA/TLR-4 signaling, and preserves mDC migration. It is proposed as a promising carrier/adjuvant system for future DC-based immunotherapy and HIV vaccine development.

Limitations: Purely in vitro study; no in vivo animal or human data. - Limited donor numbers (one to three donors depending on assay). - No direct antigen presentation or T-cell activation assays. - No HIV challenge or protective efficacy model. - mDC uptake was low; longer uptake times were not tested. - Some immunostimulatory effects may derive from free Ni(II)-NTA ligands, raising questions about specificity and long-term safety.