Delivery Systems for Nucleic Acids and Proteins: Barriers, Cell Capture Pathways and Nanocarriers

Abstract

Gene therapy has been used as a potential approach to address the diagnosis and treatment of genetic diseases and inherited disorders. In this line, non-viral systems have been exploited as promising alternatives for delivering therapeutic transgenes and proteins. In this review, we explored how biological barriers are effectively overcome by non-viral systems, usually nanoparticles, to reach an efficient delivery of cargoes. Furthermore, this review contributes to the understanding of several mechanisms of cellular internalization taken by nanoparticles. Because a critical factor for nanoparticles to do this relies on the ability to escape endosomes, researchers have dedicated much effort to address this issue using different nanocarriers. Here, we present an overview of the diversity of nanovehicles explored to reach an efficient and effective delivery of both nucleic acids and proteins. Finally, we introduced recent advances in the development of successful strategies to deliver cargoes.

Keywords: delivery; gene therapy; internalization; nanovehicles.

Figure 1

The biological barriers for non-viral gene delivery systems. Nucleic acids (e.g., DNA, mRNA, siRNA, miRNA, and oligonucleotides (ONs)) are usually incorporated into nanoparticles (NPs) to modulate protein expression levels. Extracellular (EC) barriers include: (i) the clearance through the kidney, liver, and spleen; (ii) the mononuclear phagocyte system (MPS), activated through the opsonization of the nanoparticles, with nucleases degrading the genetic cargo and finally, (iii) extravasation. Once EC barriers are circumvented, NPs must face intracellular (IC) barriers: Firstly, the plasma membrane must be crossed, usually by endocytic pathways, to ensure cellular entry. Next, NPs are encapsulated into an endosomal vesicle, which keeps them physically separated from the cytosol. Endosomal routes must be suppressed to avoid the lysosomal action responsible for degrading the internalized cargo and consequently its inability to gain access to the cytosol. Another challenge includes avoiding being recycled back to the EC environment. Once the cytosol is reached, the internalized NPs face autophagy and cytoplasmic degradation. Finally, pDNA must overcome the nuclear envelope to be delivered into the nucleus. Figure based on the work of Vermeulen et al. and Okholm et al. [12,13]. Created with BioRender.com.

Figure 2

Mechanisms of internalization in living cells. (A) Phagocytosis; (B) Macropinocytosis; (C) Clathrin-dependent endocytosis; (D) Clathrin-independent endocytosis; (E) Caveolae-mediated endocytosis; (F) Direct translocation. Other conventions: IgG, Immunoglobulin G; Fcγ Rec, Fcγ receptor; TfR, Transferrin receptor; Folate-Rec, Folate receptor; LDL-Rec, low-density lipoprotein receptor; EGF-Rec, Epidermal growth factor receptor; ER, Endoplasmatic reticulum. This figure was based on Yameen et al. and Hillaireau et al. [48,49]. Created with BioRender.com.

Figure 3

Schematic of different types of nanocarriers for drug and gene delivery. (A) Polymeric nanoparticle; (B) Liposome; (C) Inorganic nanoparticle. Adapted from Weng et al. [90], Molecular Therapy Nucleic Acids, 2020.

Figure 4

Strategies to improve the endosomal escape ability of some nanocarriers. This schematic illustrates the association between the more typically employed surface modifications and the endosomal escape mechanisms (EEMs): (A) Proton-Sponge and osmotic lysis; (B) Membrane fusion; (C) Particle swelling, and (D) Membrane translocation and destabilization. The color legend below the EEMs corresponds to the surface modifications that likely trigger the corresponding EEM. Created with BioRender.com.

Figure 5

Schematic of the photochemical internalization followed by UCNPs to escape from endosomal entrapment. UCNPs and photosensitizers (PS) are taken up by the cell via endocytosis and co-localized with endosomes. PS intercalate within endosomal membranes due to their amphiphilic properties. After NIR irradiation, the energy absorbed by sensitizer ions (S) is transferred to activator ions (A), then emitting radiation, either in the UV or Vis range. Next, the PS absorbs the activator ion’s energy and transfers it to molecular oxygen to produce highly toxic singlet oxygen, which causes severe damage to the endosomal membrane due to its oxidative effects on amino acids (e.g., tryptophan, cysteine, histidine, methionine, and phenylalanine), unsaturated fatty acids and cholesterol. Ultimately, membrane disruption is achieved, and UCNPs escape from endosomes. H: host matrix. This schematic was based on Rueda-Gensini et al. [73]. Created with BioRender.com.