Changes in Generations of PAMAM Dendrimers and Compositions of Nucleic Acid Nanoparticles Govern Delivery and Immune Recognition

Abstract

Nucleic acid nanoparticles (NANPs) are promising immune modulators due to their well-established structural properties and distinct structure-activity relationship with the immune system. We previously identified that NANPs' size, shape, composition, and type of delivery vehicle define their uptake by immune cells and subsequently induced cytokine profile. In this work, we examined the delivery efficiencies and immunological impacts of two representative NANPs─DNA cubes and RNA cubes─complexed with a benchmark delivery vehicle, Lipofectamine 2000 vs. different generations of amine-terminated poly(amidoamine) dendrimers. Using molecular dynamics simulations, we modeled dendrimer interactions with nucleic acid cargos. Next, we used traditional 2D and more recently established 3D cell cultures to assess dendrimers' influence on NANPs uptake. Immune activation was evaluated in several cell lines engineered with reporter genes driven by key immune signaling pathways. Specifically, HEK-lucia reporter cells were used to evaluate RIG-I activation, while THP1-Dual cells provided quantitative readouts for both IRF and NF-κB transcription factor activity. Our findings demonstrate that both dendrimer generation and NANP composition influence cellular uptake and immune responses. This study underscores the importance of formulation in shaping NANPs' biological properties and further advances the understanding of their immunological properties critical for the development of NANPs-based adjuvants.

Keywords: NANPs; PAMAM dendrimers; delivery; immune response; interferons; spheroids.

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Graphical summary of the current study. Representative NANPs are complexed with PAMAM dendrimers of increasing generation, which are then characterized by both their physicochemical properties and in vitro behavior.

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AFM images of RNA and DNA cubes used in this work.

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Computational and experimental assessment of nucleic acid duplexes binding with each dendrimer generation. Complex formations of (A) DNA and (B) RNA with dendrimers. Each structure is the MD snapshot at 100 ns. The DNA and the RNA are represented by blue and red helices, respectively. Red, blue, and white regions on the electrostatic potential surfaces represent negatively charged, positively charged, and neutral areas, respectively. (C) Electromobility shift assays of DNA and RNA duplexes binding with each dendrimer generation and description of each dendrimer generation to include their molecular weight, diameter, and number of available amines that can bind with duplexes. Parameters for the summary table are used from Dendritech, Inc.

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Evaluation of cellular uptake and cell death in PANC-1 and HEK-293FT using 3D model spheroids. (A) Merged microscopy images of PANC-1 spheroids treated with varying NANP-dendrimer complexes. (B) OVERTON flow cytometry analysis of cellular uptake and cell death in PANC-1 spheroids following treatment with NANP-dendrimer complexes. (C) Merged microscopy images of HEK-293FT spheroids treated with NANP-dendrimer complexes. (D) OVERTON flow cytometry analysis of cellular uptake and cell death in HEK-293FT spheroids following treatment with NANP-dendrimer complexes. In (A and C), scale bar = 100 μM. In (B and D), N = 3, Mean ± SEM.

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TEER analysis and microscopy images of Caco-2 cultures transfected with dendrimer-nucleic acid complexes. (A) Layout of a 3L64 OrganoReady Colon Caco-2 chip, and 3D drawing showing the Caco-2 tubule seeded against the collagen-I. (B) Representative microscopy images of OrganoPlate cultures at 4× magnification showing fluorescent uptake of dendrimer-NANP complexes for 24 h. The microscopy images show the uptake of NANPs within the Caco-2 3D tubule, where the uptake increases as the generation of dendrimers increases from G3-G7. The intensity of the Al488 signal was quantified and normalized to the cells-only control, presented as mean ± SD, (n = 3) (C) Transepithelial electrical resistance (TEER) measurements following treatment with DNA or RNA cubes complexed with different generations of dendrimers. Control conditions include vehicles alone and positive control that disrupts the barrier integrity. TEER values are normalized against t = 0, and presented as mean ± SEM, (n = 3).

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Assessment of immune activation and cell viability to DNA cubes, RNA cubes, and DNA duplexes complexed with dendrimers. Normalized fold induction of IRF activation using (A) HEK-Lucia RIG-I and (B) THP1-Dual cells. Note that PC in (A) is the same as L2K/RNA cube sample. Within each graph, there is a subset of the normalized cell viability for each cell line after transfected with each sample. (N = 3, Mean ± SEM). THP-1 Dual cells were also tested for NF-κB activation in each sample (data shown in the SI).