Intracellular Delivery of Native Proteins by BioReversible Arginine Modification (BioRAM) on Amino Groups

Abstract

Protein-based tools are emerging as innovative solutions to interfere with biological pathways in molecular biology and medicine. They offer advantages over traditional small molecules due to their adaptable structural diversity and their ability to engage previously inaccessible cellular targets. However, most proteins do not penetrate the lipid bilayer of mammalian cells and are therefore restricted to extracellular targets. Despite recent advances, a general method for the delivery of functional proteins into human cells remains a significant challenge. In this study, we present a bioreversible protein modification strategy of amines using short arginine-containing peptides (termed BioRAM) that enables cytosolic delivery starting from genetically non-engineered proteins. We optimized the bioconjugation strategy to achieve fast intracellular cleavage and complete recovery of the native protein. In combination with our previously established cell-penetrating peptide (CPP)-additive protocol, we show superior delivery of fluorescent protein and functional RNase A into the cytosol, achieving physiological response. Moreover, we are able to demonstrate the excellent performance of BioRAM in the presence of serum, thereby broadening the scope for intracellular applications of functional proteins.

Keywords: Bioconjugation; Bioreversible modification; Cell‐penetrating peptides; Intracellular protein delivery; RNase A.

Figure 1

a) Selection of previous approaches using CPP‐conjugates for protein delivery in comparison to this work. Advantages and challenges are listed in green and red, respectively. CPP = oligoarginine peptide, mostly R_8–10 (cyclic and linear); for the structure of the CPP‐additive, see Figure S1 and Arafiles et al, JACS 2023.^{[ 21 ]} b) Concept of Bioreversible Arginine Modification (BioRAM) for protein delivery following the steps: selective bioconjugation on aliphatic amines (lysine side chains and N‐terminus) (1), cellular delivery (2), cleavage of the CPP from protein by reduction of the disulfide bond (3), and traceless immolation of the residual linker back to the native protein (4).

Figure 2

a) Synthesis of disulfide linker with α‐substitution. Compound 1a is commercially available; compounds 2b‐c were synthesized following published procedures (Figure S2). i) 2,2′‐dithiobis(5‐nitropyridine), DCM, MeOH, 20 °C, 24 h; ii) 4‐nitrophenyl chloroformate, Et₃N, MeCN, 0→20 °C, 24 h; iii) H₂O, pH 4.35, 20 °C, 16 h; iv) H₂O, pH 8.25, 20 °C, 16 h; v) H₂O, pH 7.4, 37 °C. Reduction and immolation of 3a‐b (0.1 mmol) is monitored using analytical HPLC (Figure S5). Half‐lives (t_1/2) based on integrated peak intensity at 220 or 280 nm. Dimethylated disulfides 2c were unreactive towards thiols. The respective mixed disulfide 6 could only be accessed via 5, using thiourea as the activating group. vi) thiourea, formamidine, DMF, 20 °C, 1 h. vii) MeCN/H₂O (1:1), pH 8.25, 20 °C, 30 min; viii) MeCN/H₂O (1:1), pH 8.25, 20 °C, 16 h; ix) H₂O, pH 7.4, 20 °C. Half‐lives based on integrated peak intensity of starting material measured by UPLC‐MS (Figure S4). b) Synthesis of polyarginine peptide R₄‐R₁₀ followed by selective disulfide exchange at pH 4.35 yielding 8a/b‐R_n, which can be further reacted with native protein to produce BioRAM bioconjugates. x) MeCN/H₂O (1:1), pH 4.35, 20 °C, 2 h; xi) H₂O (1:1), pH 8.25, 20 °C, 4 h. c) Bioconjugation of superfolded 50 µM GFP (a) with 5–30 equivalents of 8a‐R₁₀ in 200 mM HEPES yielding GFPs B‐D. QTof HR‐MS analysis of GFPs (b‐d) indicated that an increase in equivalents of 8a‐R₁₀ resulted in a higher average modification of the GFPs, showing a linear correlation at the concentrations tested. d) Reducing and non‐reducing SDS‐PAGE gel analysis of GFPs A‐D reacted with different equivalents of 8a‐R₁₀. e) Reduction and immolation of 1 µM sfGFP D (reacted with 8a‐R₁₀) upon treatment with 10 mM GSH. f) Reduction and immolation of 1 µM sfGFP E ‐8b‐R₁₀ upon treatment with 10 mM GSH.

Figure 3

a) General workflow for the modification and purification using BioRAM. b) apm calculated from QTof‐HR‐MS analysis (refer to Figure S8). Added arginine residues are calculated based on the peptide length multiplied by the average modification. The calculated added charge considers the modification of one free lysine (positively charged) for the attachment of one polyarginine. c) Fluorescent SDS‐PAGE gel analysis of NLS‐mCherry‐BioRAM in DMEM‐high glucose medium supplemented with 10% FCS over 1, 4, and 24 h. The fluorescent 25 kDa band of the ladder is marked (*) d) Schematic treatment protocol for cell viability assessment. e) Cell viability assay using WST‐1 for protein treatment at 0% serum (left) and 10% serum (right) in the presence of 5 µM CPP‐additive. Data is represented as the mean of three biological replicates (N = 3).

Figure 4

a) Schematic workflow assessing the cellular uptake of fluorescent BioRAM‐proteins into cells. b) Visualization of uptake, nuclear translocation, and colocalization to the nucleus. c) Representative confocal microscopy images from the automated acquisition during quantification measurements in a 96‐well plate using HeLa cells (Nikon 40× Air). Microscope settings and image contrast are kept constant for all images. RFP‐mask was generated using nuclear segmentation and endosome exclusion script (Figure S12B). Scale bar = 20 µm. (*incubation for 1 h, 5 µM CPP‐additive, 0% serum) d), e) Quantification results displaying integrated relative fluorescence intensity. Data are reported as the mean of three biological replicates (N = 3, black line), with each biological replicate (dot) representing the mean of 150–200 individual cells. Fold change of relative integrated fluorescence intensity is displayed for selected conditions.

Figure 5

Transiently transfected HeLa cells expressing cytosolic galectin 3‐EGFP fusion protein for detection of endosomal escape. Nuclear mCherry signal indicates successful cytosolic delivery. Punctuated EGFP‐signal is indicative of endosomal rupture as induced by the positive control LLOMe. Microscopy images (Nikon 60× oil), scale bar = 50 µM.

Figure 6

a) Schematic representation of RNase A‐BioRAM delivery and intracellular function. b) SDS‐PAGE gel of generated RNase A‐BioRAM conjugates. c) Characterization of generated RNase A‐BioRAM conjugates and results of cell viability assays after treatment of protein in presence of 5 µM CPP‐additive. (The data is presented as the mean of three replicates (N = 3)). d) Cell viability in different cell lines after treatment with the indicated concentrations of RNase A‐B in the presence of 5 µM CPP‐additive. (The data is presented as the mean ± SD of three replicates (N = 3)). e) Cell viability of HeLa cells after 24 h of treatment with RNase A‐B compared to RNase A‐A in the presence of 5 µM CPP‐additive (The data is presented as the mean ± SD of three biological replicates (N = 3)). f) Activation of caspase‐3/7 was measured using a luminogenic caspase‐3/7 substrate producing a luminescent signal by luciferase after caspase activation. Supernatant was analyzed with a plate reader. (The data is presented as the mean ± SD of three biological replicates (N = 3). The indicated P‐values are calculated using one‐way ANOVA followed by Tukey's posthoc analysis). g) PS exposure was assessed using fluorochrome‐conjugated Annexin V, which binds specifically to extracellularly exposed PS on the cell membrane. Stained cells were analyzed by flow cytometry to quantify apoptotic populations based on fluorescence intensity (the data is presented as mean ± SD of three biological replicates (N = 3)). Representative pictures of cell populations can be found in Figure S17.