Strategies to deliver RNA by nanoparticles for therapeutic potential

Abstract

The use of a variety of RNA molecules, including messenger RNA, small interfering RNA, and microRNA, has shown great potential for prevention and therapy of many pathologies. However, this therapeutic promise has historically been limited by short in vivo half-life, lack of targeted delivery, and safety issues. Nanoparticle (NP)-mediated delivery has been a successful platform to overcome these limitations, with multiple formulations already in clinical trials and approved by the FDA. Although there is a diversity of NPs in terms of material formulation, size, shape, and charge that have been proposed for biomedical applications, specific modifications are required to facilitate sufficient RNA delivery and adequate therapeutic effect. This includes optimization of (i) RNA incorporation into NPs, (ii) specific cell targeting, (iii) cellular uptake and (iv) endosomal escape ability. In this review, we summarize the methods by which NPs can be modified for RNA delivery to achieve optimal therapeutic effects.

Keywords: Cellular uptake; Drug delivery; Endosomal escape; Nanomedicine; RNA therapy; Targeting.

Figure 1.

Barriers to RNA delivery for therapeutic use. ECM: extracellular matrix, MW: molecular weight.

Figure 2.

Schematic of RNA that is encapsulated within NPs (left) vs. adsorbed on the surface of NPs (right), as shown with the example of liposomes. These methods can be used with NPs consisting of a variety of materials, including lipids and polymers. Adapted from (Blakney et al., 2019) under the terms of the Creative Commons CC BY license.

Figure 3.

Schemes showing common methods of RNA encapsulation in lipid- and/or polymer-based NPs. a) Thin lipid film hydration. Lipids are suspended in an organic solvent, which is then evaporated under nitrogen flow. Further evaporation can be carried out under vacuum to ensure solvent evaporation. The resulting lipid film is rehydrated with an aqueous solution containing RNA, and then vortexed and sonicated to produce NPs containing RNA in their core. b) Nanoprecipitation. An organic solvent containing NP components (lipids and/or polymers) is added dropwise to an aqueous solution containing RNA while stirring. The solvent is evaporated under nitrogen or by using a Rotavapor, leaving an aqueous solution with NPs containing RNA in their core.

Figure 4.

Scheme indicating the layer-by-layer formation of NPs for siRNA delivery, adapted with permission from (Elbakry et al., 2009). Copyright 2009 American Chemical Society. Gold NPs (AuNP) were coated with 11-mercaptoundecanoic acid (MUA) to facilitate binding of the subsequent layers. Poly(ethylene imine) (PEI; molecular weight = 25 kDa) was then added to create a positive surface for siRNA to be attached. Finally, an outer layer of PEI was added to protect the siRNA from premature release or degradation in the serum. The hydrodynamic diameter of the NPs at some steps are indicated in nanometers. 1. indicates the compound and its concentration when added to form the next layer, and 2. indicates the centrifugation settings used to remove any unbound material after each 30 min incubation.

Figure 5.

Scheme showing specific targeting of cancer cells by NPs, adapted with permission from (Liyanage et al., 2019). Cancer cells can be specifically targeted as they overexpress receptors such as the estrogen receptor and progesterone receptor (ER/PR) and human epidermal growth factor receptor (HER2) compared to healthy cells. Ligands for these receptors can be attached to NPs containing RNA, facilitating their preferential uptake and therapeutic effects in cancer cells vs. healthy cells.

Figure 6:

Scheme showing NP modifications to enhance endosomal escape, modified with permission from (Donahue et al., 2019). a) Endosomal membrane-disrupting modifications on the NP can form openings by which the NP can 1) escape with its RNA cargo or 2) promote NP fusion to the endosomal membrane and release RNA directly into the cytoplasm. b) pH-sensitive modifications. The acidic environment in endosomes weakens the bonds that bind the NPs to the RNA cargo, thus allowing the RNA to escape the endosome

Figure 7.

Scheme displaying different types of membrane destabilization that can lead to endosomal escape, reprinted with permission from (Martens et al., 2014). a) Pore formation on the endosomal membrane due to cationic molecules or fusogenic peptides. b) Endosomal rupture due to osmotic swelling caused by an increased cationic charge. c) Fusion of the endosomal membrane and the NP (e.g., positively charged liposome) allowing for RNA release from the NP into the cytoplasm.