Intracellular Protein Delivery: Approaches, Challenges, and Clinical Applications

Abstract

Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions. However, their clinical use has been limited to extracellular applications due to their inability to cross plasma membranes. Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases. In this review, we highlight progress made toward achieving cytosolic delivery of recombinant proteins. We start by first considering intracellular protein delivery as a drug modality compared to existing Food and Drug Administration-approved drug modalities. Then, we summarize strategies that have been reported to achieve protein internalization. These techniques can be broadly classified into 3 categories: physical methods, direct protein engineering, and nanocarrier-mediated delivery. Finally, we highlight existing challenges for cytosolic protein delivery and offer an outlook for future advances.

Fig.  1.

Overview of common intracellular protein delivery approaches. Physical approaches involve mechanical disruption of the cell membrane and includes techniques such as electroporation, sonoporation, and microinjection. Direct protein engineering approaches rely on fusion/bioconjugation to transduction domains originally derived from viral and bacterial proteins. Nanocarrier systems have been developed for protein delivery using diverse materials from synthetic lipid nanoparticles and polymeric supramolecular assemblies to bioinspired VLPs.

Fig. 2.

Harnessing CPPs for bioPROTAC delivery and undruggable target degradation. (A) Design of BCL11A-targeting bioPROTACs fused to a helical ZF5.3 cationic CPP. (B) Western blots demonstrate intracellular uptake of bioPROTACs and reveal ZF5.3-2D9-tSPOP as an active BCL11A degrader. (C) Time course of BCL11A degradation in HUDEP-2 cells when treated with 10 μM CPP-fused bioPROTACs. (D) Degradation is nanobody (2D9)-dependent. (E) The proteasome inhibitor MG-132 rescues BCL11A degradation, validating the bioPROTAC mechanism of action. This figure was reproduced under a CC BY 4.0 license and is attributed to Shen et al. [135].

Fig. 3.

LNPs for the intracellular delivery of ApP-fused protein cargo. Therapeutic DARPins are fused with a negatively charged ApP sequence as well as a split GFP S11 tag. The protein cargo is encapsulated within LNPs via microfluid mixing with lipids, cholesterol, and polyethylene glycol (PEG). Formulated LNPs efficiently transport protein cargo across cell membranes, facilitate endosomal escape, and enable specific inhibition of undruggable targets. Cytosolic delivery can be detected with a split GFP complementation assay in reporter cells expressing the GFP(1–10) fragment. Reprinted (adapted) with permission from Haley et al. [179]. Copyright 2023 American Chemical Society.

Fig. 4.

Fluoroalkyl-grafted polymers demonstrate enhanced protein uptake. (A) Schematic for grafting functional chains onto branched PEI for protein co-assembly. (B) Structures of alkanes (A1 to A4), cycloalkanes (C1 to C4), and fluoroalkanes (F1 to F4) grafted onto PEI polymer backbones. (C) Intracellular delivery of FITC-labeled BSA with a library of conjugated PEI polymers and controls. The number after each dash represents the degree of PEI functionalization with 1 being the lowest and 4 being the highest. This figure was reproduced under a CC BY license and is attributed to Zhang et al. [197].

Fig. 5.

EXPLORs for coupling exosome cargo loading and biogenesis. (A) Fusion protein constructs used for optogenetic recruitment of cargo to exosomes. (B) Blue light illumination induces recruitment of mCherry-CRY2 to membrane-bound CIBN-CD9. (C) Representative fluorescent images of HEK293T cells expressing EXPLOR components with or without 488-nm laser stimulation. Cargo co-localizes to the inner leaflet upon illumination. Scale bars are 20 μm and 5 μm for whole and inset images, respectively. (D) Time course images of mCherry-CRY2 signal in cells pulsed with blue light. (E) Quantitation of mCherry-CRY2 signal in both cytosolic and membrane subcellular compartments following blue light stimulation. This figure was reproduced under a CC BY license and is attributed to Yim et al. [224].

Fig. 6.

In vivo delivery of exosomes manufactured by the EXPLORs method. (A) Differentiated neurosphere-derived cells expressing loxP-STOP-loxP-EGFP were treated with Cre:EXPLORs or transfected with pCMV-Cre vectors. Fixed cells were stained with antibodies against a neuron-specific class III beta-tubulin marker, Tuj1, GFP, and Hoechst 33342. Scale bars are 100 μm. (B) Percentage of EGFP-expressing cells following treatment. (C) Schematic for the administration of Cre:EXPLORs in loxp-stop-loxp-eNpHR3.0-EYFP transgenic mice by ventrolateral injection. (D) Fluorescent images of fixed brain slices following EXPLOR injection in transgenic mice. Green fluorescence indicates eNpHR3.0-EYFP protein expression and blue fluorescence indicates cell nuclei. Insets show high-magnification detail in neurons. Scale bars are 500 μm and 50 μm for whole and inset images, respectively. Hip, hippocampus; Th, thalamus. (E) Representative image of NeuN/GFAP immunohistochemistry of the brain following Cre:EXPLORs treatment. Recombination occurs mainly in (NeuN)-positive neurons rather than red, glial fibrillary acidic protein (GFAP)-positive astrocyte cells. Scale bars are 20 μm. This figure was reproduced under a CC BY license and is attributed to Yim et al. [224].

Fig. 7.

Intracellular delivery of LNP:protein in vivo. (A) Mice transfected with an LgBiT transposon reporter were injected I.V. with either LNP:DARPinK27-D30-HiBiT or free DARPinK27-D30-HiBiT. Bioluminescent imaging of organs ex vivo detected NanoLuc activity in the LNP treatment group only. (B) Quantitation of NanoLuc activity in livers from mice left untreated, injected with free protein, or injected with LNP:protein. (C) Ex vivo bioluminescent images of livers quantified in B. Reprinted (adapted) with permission from Haley et al. [179]. Copyright 2023 American Chemical Society.