Nanoparticle Targeting Strategies for Lipid and Polymer-Based Gene Delivery to Immune Cells In Vivo

Abstract

Lipid nanoparticles and polymeric nanoparticles are promising biomaterial platforms for robust intracellular DNA and mRNA delivery, highlighted by the widespread use of nanoparticle- (NP) based mRNA vaccines to help end the COVID-19 pandemic. Recent research has sought to adapt this nanotechnology to transfect and engineer immune cells in vivo. The immune system is an especially appealing target due to its involvement in many different diseases, and ex vivo-engineered immune cell therapies like chimeric antigen receptor (CAR) T therapy have already demonstrated remarkable clinical success in certain blood cancers. Although gene delivery can potentially address some of the cost and manufacturing concerns associated with current autologous immune cell therapies, transfecting immune cells in vivo is challenging. Not only is extrahepatic NP delivery to lymphoid organs difficult, but immune cells like T cells have demonstrated particular resistance to transfection. Despite these challenges, the modular nature of NPs allows researchers to examine critical structure-function relationships between a particle's properties and its ability to specifically engineer immune cells in vivo. Herein, several nanomaterial components are outlined, including targeting ligands, nucleic acid cargo, chemical properties, physical properties, and the route of administration to specifically target NPs to immune cells for optimal in vivo transfection.

Keywords: biomaterials; biotechnology; gene delivery; immunoengineering; nanotechnology.

Figure 1

NP synthesis and screening is a modular process. A) Different nanomaterial building blocks, including many kinds of lipids (for LNPs) and polymers (for PNPs) can self‐assemble with many nucleic acid cargos encoding different therapeutic constructs to create NPs with diverse chemical and physical properties. These NPs can be further engineered through surface modification or ligand conjugation. This combinatorial synthesis process gives rise to a library of NPs with diverse structural features. B) These diverse NP libraries can be screened in vitro to identify top‐performing formulations and characterize NP structure–function relationships. Finally, top‐performing formulations identified from in vitro screening can be delivered in vivo to evaluate their ability to transfect endogenous immune cells and engineer therapeutic function. Figure made with BioRender.

Figure 2

1) (A) Overview of antigen‐presenting NP (APN) synthesis process whereby pMHC monomers are loaded via UV‐mediated peptide folding and conjugated to LNP surface and (B) can then be delivered to mice to assess antigen‐specific transfection. 2) (A) APNs were synthesized using either traditional peptide folding or UV‐mediated peptide exchange and (B) delivered to PR8‐infected mice, (C) where they demonstrated robust antigen‐specific T cell transfection and minimal off‐target T cell transfection. (D) A multiplexed library of APNs specific to the top three epitopes associated with PR8 was then formed using UV peptide exchange and delivered to mice, (E) where it demonstrated robust transfection of each of the three antigen‐specific populations. Reproduced (Adapted) with permission.^{[ 50 ]} Copyright 2022, Science Advances.

Figure 3

Overview of chemical targeting approaches employed for NP splenic tropism and T cell transfection in vivo. NPs with anionic surface charge have generally been found to preferentially traffic to the spleen, potentially due to the formation of a protein corona containing serum proteins like β2‐glycoprotein I (β2‐GPI). Additionally, NPs with an apparent pKa between 5 and 6 have been demonstrated to show enhanced endosomal escape and transfection in T cells. These findings can be leveraged to specifically deliver nucleic acid cargo to splenic T cells. Figure made with BioRender.

Figure 4

1) Overview of combinatorial PBAE library synthesis whereby a linear backbone monomer is reacted with lipophilic side‐chain monomers of variable lengths and different end‐capping monomers. 2) (A,B) Transfection efficiency of different lipophilic PBAEs was screened in DCs in vitro, with NPs possessing longer lipophilic side chains demonstrating generally higher transfection. (C) The top‐performing PBAE formulation, R18D, was also compared to Lipofectamine, where it demonstrated higher efficacy across many doses. 3) (A) R18D PBAE NPs delivering OVA‐encoding mRNA were used for in vivo cancer vaccination in a B16‐OVA melanoma model, where they demonstrated decreased tumor burden, (B) increased survival, and (C–E) robust antigen‐specific T cell expansion. (F,G) R18D NPs demonstrated similar efficacy in a cancer vaccination approach with a B16‐F10 melanoma model. Reproduced (Adapted) with permission.^{[ 92 ]} Copyright 2023, Proceedings of the National Academy of Sciences.

Figure 5

NP size influences the uptake mechanism by different immune cells. Almost all immune cells (and most cells in general) are capable of caveolae‐mediated endocytosis, clathrin‐mediated endocytosis, and macropinocytosis. Although there are exceptions, NPs in the 50–100 nm range are generally taken up via caveolae‐ or clathrin‐mediated endocytosis and may also be taken up by phagocytosis. NPs in the 100–200 nm range are primarily taken up by clathrin‐mediated endocytosis or macropinocytosis and may also be taken up by phagocytosis. Macrophages and neutrophils are capable of endocytosing larger particles (≈400 nm) via phagocytosis. Upon cellular uptake, all particles must escape endosomes in order for their nucleic acid cargo to be translated.^{[ 47} , ^{126 ]} Importantly, NP uptake is a complex process, with other parameters besides size, such as charge and targeting ligand, playing a key role in determining if the immune cell employs phagocytosis, macropinocytosis, or endocytosis. Figure made with BioRender.

Figure 6

1) (A) Overview of elastic PEG aAPC synthesis whereby crosslinker amount was varied to modulate PEG microparticle stiffness. (B) Microparticles (MP) were characterized with transmission electron microscopy and (C) conjugated to T cell‐activating molecules to form aAPCs. 2) Binding and uptake of aAPCs with varying stiffnesses were evaluated in vitro. (A) The intermediate (850 kPa) MPs demonstrated higher specific T cell binding at low doses, with interactions between T cells (green) and MPs (pink) being demonstrated in microscopy images in (B). (C,D) When administered in vivo, the softest MPs (50 kPa) demonstrated the longest half‐life and (E) enhanced delivery to the lung and spleen, while the stiffest particles (5000 kPa) demonstrated enhanced delivery to the liver. (F) Additionally, the stiffest 5000 kPa MPs demonstrated the highest macrophage uptake and (G) binding in vitro. Reproduced (Adapted) with permission.^{[ 148 ]} Copyright 2024, Nano Research.

Figure 7

1) Overview of PNP with CD68‐promoter pDNA design whereby CRISPR‐Cas9 enhanced green flourescent protein (EGFP) plasmid will only be translated in macrophages and monocytes, even if NPs are taken up by other cell types. 2) (A,B) PNPs delivering Cy5‐labeled nucleic acid were delivered systemically, where they demonstrated robust uptake in neutrophils, macrophages, and monocytes across different organs. (C) However, only NPs with the pM458 plasmid containing the CD68 promoter demonstrated robust EGFP transfection in macrophages and monocytes in vivo, while NPs with the control pX458 plasmid also transfected neutrophils. Reproduced (Adapted) with permission.^{[ 211 ]} Copyright 2018, ACS Nano.

Figure 8

1) Overview of CD3‐mPEG‐PCL‐PEI/pCAR hydrogel design where supramolecular hydrogel could be implanted proximal to the tumor site to transfect endogenous T cells to generate antitumor CAR‐T cells. 2) (A) Humanized mice were inoculated with tumors and treated with PBS, nontransduced T cells, hydrogel with no plasmid, ex vivo generated CAR‐T cells, PEI/plasmid NPs, NP‐hydrogel with plasmid but no CD3, or NP‐hydrogel with plasmid and CD3. (B–E) The CD3‐mPEG‐PCL‐PEI/pCAR supramolecular hydrogel treatment resulted in the greatest reduction of solid tumor burden. Reproduced (Adapted) with permission.^{[ 196 ]} Copyright 2023, Advanced Materials.