Potent Protein Delivery into Mammalian Cells via a Supercharged

Nov 6, 2018 - The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein de...
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Potent Protein Delivery into Mammalian Cells via a Supercharged Polypeptide Jun Yin,# Qun Wang,# Shan Hou, Lichen Bao, Wenbing Yao,* and Xiangdong Gao* Jiangsu Key Laboratory of Druggability of Biopharmaceuticals and State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China

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ABSTRACT: The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin β1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nucleartargeted drug delivery.

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INTRODUCTION Proteins have achieved popular success as research tools and therapeutics for a variety of diseases due to their high specificity and activity.1−4 The vast majority of these studies, however, have focused on the delivery to extracellular targets, and proteins located within the intracellular targets are relatively unexploited.5−7 Because most proteins are unable to spontaneously penetrate cell membranes, the development and utilization of intracellular protein delivery technologies could promote applications including transcription factordriven changes, intercellular enzyme replacement therapies, genome editing, and ex vivo cell-based therapies.8,9 Current approaches to enable exogenous proteins to bind intracellular targets is usually accomplished through the delivery of their DNA coding sequences.10,11 However, the intracellular delivery of exogenous DNA may raise the possibility of the potential disruption of endogenous genes and the permanent integration into the genome.6 To deliver mRNAs or mRNA analogues is another option without requiring the transport of an encoding DNA and is highly possible to reduce gene integration; however, RNA instability and immunogenicity restrict its application.12 Therefore, the intracellular delivery of proteins into the cell may offer higher safety, better specificity, and broader applicability.13 Over the past decades, several approaches for protein delivery have been developed, including cationic lipids, synthetic polymers, nanoparticles, oligonucleotides, cell-penetrating peptides, and highly charged natural or engneered proteins.14−18 While these and other approaches have promoted the field of intracellular protein delivery, challenges still exist, including low potency, significant cytotoxicity, lack of generality, and endosomal entrapment.19−21 Therefore, an © 2018 American Chemical Society

efficient and biocompatible vehicle that facilitates endosomal escape and enhances the cell uptake of proteins is highly desirable to advance protein-based basic research and therapeutics. In this work, we constructed and identified a novel supercharged polypeptide (SCP), which can effectively penetrate the cell membrane, then escape from the endosome and eventually nontoxically deliver the proteins into a subcellular compartment (Figure 1a).

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RESULTS AND DISCUSSION Design and Characterization of SCPs. In designing a chemically stable, soluble, and unstructured cationic SCP that provides an increased hydrodynamic radius to penetrate the cell membrane efficiently, we first eliminated hydrophobic amino acids (Y, W, V, M, L, I, and F) that could induce aggregation and might cause an immune reaction.22 Also excluded were the anionic amino acids (D and E), the cysteine (as a result of possible cross-linking), and the amide side residues of Q and N, which had a tendency to hydrolysis. In addition, subsequent attempts found that histidine-rich or arginine-rich supercharged polypeptides were hardly expressed in E. coli. Consequently, our original designs focused on the residual six amino acids, A, G, K, P, S, and T. To obtain a highly expressed and genetically stable supercharged polypeptide, we created a library of 12-aminoacid segments containing randomized sequences and screened ∼100 of these unique sequences for expression level (Table S1−2, Supporting Information (SI)). Highly expressed Received: September 28, 2018 Published: November 6, 2018 17234

DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

Article

Journal of the American Chemical Society

Figure 1. Engineered supercharged polypeptides and GFP fusion proteins and their ability to penetrate cells. (a) Schematic illustration of transport of SCP-GFPs across the nuclear membrane. (b) Flow cytometry analysis showing amounts of internalized SCP-GFPs in HeLa, BEL7402, A549, PC12, C6, and Jurkat cells. (c) Flow cytometry analysis showing amounts of internalized K4-GFP in HeLa cells compared with Tat-GFP or Arg10GFP treated cells. (d) Flow cytometry analysis of live HeLa cells after incubation with different concentrations of K4-GFP for 2 h (left panel) and with 2 μM K4-GFP for different time periods. Each control group is the untreated cells subjected to the same washing protocol and culture media.

different cell lines in vitro for 2 h at 37 °C, including HeLa cells, BEL-7402 hepatocellular carcinoma cells, human A549 lung adenocarcinoma cells, rat pheochromocytoma PC12 cells, rat C6 astroglial cells, and Jurkat T cells. After incubation, the cells were washed with heparin and analyzed by flow cytometry and fluorescence microscopy (Figure 1b and Figure S6−7, SI). The results demonstrated that the SCP-GFPs could penetrate all six types of cells, and their protein delivery efficiencies were associated with their theoretical net charge (K1-GFP, K2-GFP, and K3-GFP) and charge density (K1-GFP and K4-GFP). We observed that K4-GFP had the highest cellular uptake potency compared with the other three fusion proteins, indicating that the density of cationic groups in a supercharged polypeptide, and not just the magnitude of the positive charge, contributed to cellular uptake efficiency. Then, we evaluated the cytotoxicity of SCPs in vitro using the MTT assay. As shown in Figure S8, SI, SCPs had no significant cytotoxicity at a low concentration of 100 nM or even at a higher concentration of 10 μM against HeLa, BEL-7402, A549, PC12, C6, or Jurkat cells. Moreover, we investigated the cell penetrating ability of K4-GFP compared with that of Tat-GFP and Arg10-GFP on HeLa cells. The results of flow cytometry showed that the K4 polypeptide was able to achieve a more effective delivery of proteins into the cells than the classic cell-penetrating peptides, Tat and Arg10 (Figure 1c). The cellular uptake of K4-GFP appeared in a concentration- and time-dependent and dosesaturable manner (Figure 1d). Similar results were observed in several cell lines (Figure S9, SI). In addition, the delivery of K4-GFP was compatible with serum (Figure S10, SI). The K4GFP in aqueous solution had an average hydrodynamic diameter of 9.36 ± 0.56 nm (Figure S11, SI). The comparison clearly indicates that the K4 polypeptide is a highly efficient and serum-stable transporter for protein delivery.

sequences were iteratively ligated and further rescreened for maximal expression in E. coli. In other words, the genes of long supercharged polypeptides were obtained by the ligation of hybridized oligodeoxynucleotide building blocks for 12-aminoacid segments (Figure S1, SI). We obtained novel supercharged polypeptides with biophysical properties quite similar to those of PEG (Figure S1c, SI). We sought to illuminate (1) the relationship between net positive charge and cellular uptake efficiency and (2) the effect of charge density on intracellular delivery potency. Thus, a series of SCP-GFPs with different numbers of residues ranging from 120 to 360 and positive charges ranging from 20 to 60 (120 K20-GFP (K1GFP), 240 K40-GFP (K2-GFP), 360 K60-GFP (K3-GFP), and 120 K40-GFP(K4-GFP)) were selected in the library and were expressed in E. coli. The purity and molecular mass of the products were confirmed by SDS-PAGE, Western blot, reversed phase HPLC, SEC-HPLC, and MALDI-TOF (Figure S2, SI). SEC-HPLC indicated that the SCP-GFPs possessed much larger apparent molecular sizes (Figure S2e, SI). Circular dichroism spectroscopy of the SCPs showed a predominant negative minimum at 198 nm, characteristic of an unfolded polypeptide with random coils in the secondary structure (Figure S3, SI). The GFP fluorescence characteristics of the SCP-GFPs were very similar, which means that the polypeptides did not interfere with GFP function, enabling us to directly compare the cellular uptake of the SCP-GFPs by using fluorescence intensity (Figure S4, SI). Potent Cellular Delivery of SCPs. The evaluation of the ability of the SCP-GFPs to enter mammalian cells needs an approach to remove surface-bound, noninternalized SCPGFPs. Therefore, we confirmed the effectiveness of three washes with PBS containing heparin (20 U/mL) in removing surface-bound SCP-GFPs (Figure S5, SI). Next, we incubated 2 μM K1-GFP, K2-GFP, K3-GFP, and K4-GFP with several 17235

DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

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Journal of the American Chemical Society

Figure 2. Uptake mechanism and cellular fate of K4-GFP. (a) Cells were pretreated for 0.5 h with the indicated inhibitor to 2 h treatment with 2 μM K4-GFP in the continued presence of inhibitor, washed, and analyzed by flow cytometry. Treatment at 4 °C instead of 37 °C was included as a control (blue bars). (b) Confocal fluorescence images of HeLa cells stained by LysoTracker (red) after incubation with K4-GFP for 2 h (scale bars, 20 μm). (c) Confocal fluorescence images and line-scan profiles of fluorescence intensity for HeLa cells incubated for 2 h with K4-GFP. Nuclei were stained by Hoechst (blue) (scale bars, 10 μm). (d) Time-lapse confocal microscopy imaging revealing the direct nuclear delivery of K4-GFP (Movie 1, SI) (scale bars, 20 μm).

fluorescence overlapped with lysosome in the cytoplasm, which presented the effective endosomal escape of K4-GFP (Figure 2b and Figure S13, SI). Next, we carefully studied the subcellular distribution of K4-GFP after lysosomal escape. Confocal imaging showed that the green fluorescent signal of delivered K4-GFP seems to accumulate only in the nucleus (Figure 2c). The colocalization curve further supported the result that K4-GFP was localized specifically in the nucleus, rather than dispersed homogeneously in both the cytoplasm and the nucleus (Figure 2c). Using time-lapse confocal microscopy, we observed that K4-GFP penetrated the cell membrane quickly, reaching the nucleus after 60 s (Figure 2d and Movie 1, SI). Moreover, we observed that K4 had the highest nuclear-targeted delivery potency compared with the other three polypeptides (and the rate of nuclear transport followed K3 > K2 > K1) (Figure S14, SI). Although small proteins (molecular weights lower than 40 kDa) can passively diffuse through the nuclear pore, proteins should be distributed through-out the whole cell and not just in the nucleus.20 These results implied that the K4 polypeptide might be recognized by nuclear import receptors, thereby importing proteins of interest into the cell nucleus by nuclear transport. Nuclear Import Receptor-Driven Intracellular Distribution of SCP. Molecules larger than 40 kDa commonly require active transport through the nuclear membrane. Because nuclear transport of these molecules depends on their interaction with the importin α/β complex or importin β alone, we determined whether K4 associates with importin α or importin β. At least six importin αs and only one importin β have been reported. Among them, importin α6 is only present in tests.23,24 Therefore, we tested the association of GST-K4 with the other five importin αs and importin β1. The results showed that GST-K4 interacted with Myc-importin β1, suggesting that importin β1 could be responsible for the nuclear transport of K4 (Figure 3a). To confirm this observation, we transfected HeLa cells with K4-GFP and Myc-importin β1 and then performed a coimmunoprecipitation analysis. Overexpressed K4-GFP interacted with overex-

To elucidate the mechanism by which the K4 polypeptide penetrates cells, we repeated the protein delivery tests in HeLa cells under various conditions that block different components of the endocytosis pathway. When HeLa cells were cooled to 4 °C, intracellular K4-GFP was barely observed (Figure 2a). Similarly, HeLa cells treated with 0.5 mg/mL NaN3, an ATP synthesis inhibitor, resulted in a significant inhibition of cellular uptake of approximately 80% in comparison with the control experiments (Figure 2a). These results suggested that the uptake of K4-GFP requires an energy-dependent process. We next used a variety of known endocytic inhibitors to detect the roles of different endocytic pathways in the uptake of K4GFP. The concentrations of the inhibitors were chosen based on the MTT assay, and it was required that, at these concentrations, cell vitality was greater than 80% (Figure S12, SI). HeLa cells were treated with 20 U/mL heparin, a competitive inhibitor of heparan sulfate on the cell surface, which almost completely inhibited the delivery efficiency of K4-GFP, indicating that the internalization of K4-GFP requires interacting with anionic cell surface proteoglycans (Figure 2a). Both nystatin and filipin, small molecules known to inhibit caveolin-mediated endocytosis, resulted in a more than 40% decrease of K4-GFP uptake (Figure 2a). Treatment with 1 and 10 μg/mL chlorpromazine (CPZ), a clathrin-mediated endocytosis inhibitor, inhibited the cellular uptake of K4GFP by 68% and 74%, respectively (Figure 2a). Similarly, 5 and 20 μM dynasore, a small molecule dynamin II inhibitor, also reduced the delivery of K4-GFP by approximately 35% and 58%, respectively (Figure 2a). Treatments with amiloride and cytochalasin-D (CD), macropinocytosis inhibitors, reduced the uptake of K4-GFP to approximately 55% (Figure 2a). Taken together, the inhibitor study implied a multipathway mechanism for the cellular uptake of K4-GFP. Now that endosomal escape is broadly considered to be the major obstacle of protein delivery, we next performed a colocalization analysis of K4-GFP and lysosome (dyed with LysoTracker Red). Delivered K4-GFP showed a significant accumulation in the nucleus and with negligible punctate 17236

DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

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Journal of the American Chemical Society

Figure 3. Binding of importin proteins to K4. (a) Immunoblotting with Myc antibody in HeLa cells expressing either one of Myc-tagged importin αs or β (top left panel). Immunoblotting with GST antibody for GST-tagged K4 that was purified from E. coli (top right panel). A GST pull-down assay was performed with purified GST-K4 and immunoblotting with Myc antibody in HeLa lysates containing either one of Myc-tagged importin αs or β. A GST antibody immunoblotting confirmed equal amounts of GST-K4 (bottom left panel). A GST pull-down assay was performed with GST or GST-K4 and immunoblotting with Myc antibody in HeLa lysates containing importin β1. The amount of GST was detected by a GST antibody (bottom right panel). (b) HeLa cells were cotransfected with K4-GFP and Myc-tagged importin β1. Cell lysates were immunoprecipitated with a GFP antibody. Coimmunoprecipitated importin β1 was analyzed by Western blot with a Myc antibody. A GFP antibody immunoblotting confirmed that equal amounts of K4-GFP constructs were immunoprecipitated (left panel). Cells transfected as in the left panel were immunoprecipitated with a Myc antibody. Coimmunoprecipitated K4-GFP was blotted with a GFP antibody. The amount of importin β1 was detected by a Myc antibody (right panel). (c) Ivermectin or importazole was preincubated with HeLa cells for 30 min at 37 °C and then 2 μM K4GFP was added for 2 h. The intracellular distribution of K4-GFP was detected by confocal microscopy. Nuclei were stained by Hoechst (blue) (scale bars, 20 μm).

Figure 4. (a) Hemolytic activity of K4 at concentrations ranging from 0.1 to 10 μM. PBS and Triton X-100 (0.1%) was used as the negative and positive control, respectively. (b) Direct nuclear delivery of multiple K4-tagged proteins (FITC-labeled) with widely varying sizes and charges. Nuclei were stained by Hoechst (blue) (Scale bars, 20 μm). (c) Delivery of Cas9-K4: sgRNA complex to target CCR5 gene in HeLa cells resulted in efficient gene editing, as determined by T7 endonuclease I assays.

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DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

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Journal of the American Chemical Society pressed importin β1 (Figure 3b). Similar results were obtained with the use of ivermectin and importazole. Ivermectin,25 by inhibiting importin α/β but not importin β1 alone, had no influence on the nuclear localization of K4-GFP (Figure 3c). Treating HeLa cells with an importin β1 selective inhibitor, importazole,26 showed a decrease in the nuclear localization efficiency of K4-GFP (Figure 3c and Figure S15, SI). To further determine whether importin β1 was required for the nuclear localization of K4, HeLa cells were transfected with small interfering RNA (siRNA) against importin β1 (Figure S16, SI). The specific knockdown of importin β1 attenuated the effect of the nuclear localization of K4 in HeLa cells (Figure S16, SI). Thus, our results suggest that importin β1 is an important karyopherin of K4. Immunogenicity and Toxicity of SCP. To evaluate the immunogenicity of the K4 sequence, mice were injected with unmodified GFP and K4-GFP every other day for 15 days. The IgG antibody titers specific for the corresponding proteins were detected for each mouse, 2 weeks after the last injection. As expected, all native GFP-immunized mice generated a robust immune response independent of the coadministered adjuvant (Figure S17, SI). By comparison, K4-GFP displayed a significantly reduced immunogenicity (Figure S17, SI). After repeated immunization with the adjuvant, a strong immune reaction to the GFP portion of K4-GFP, and only a weak or no IgG titer specific response to the K4 portion of K4-GFP were detected (Figure S17, SI). Furthermore, production of early innate cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were measured in primary human peripheral blood mononuclear cells (PBMCs) and mice after administration of K4. Unlike lipopolysaccharide (LPS), K4 could not trigger cytokine responses (Figure S18, SI). Collectively, these data reveal that K4 is not strongly immunogenic. In addition, K4 showed minimal hemolytic activity (Figure 4a and Figure S19, SI) and negligible hematologic, hepatic and renal toxicity in vivo (Figure S20− 21, SI). Generality of Protein Delivery. Targeted intracellular drug delivery systems, which are expected to reduce the toxic side effects and simultaneously improve the therapeutic efficiency, have attracted great attention in the past several years.27−29 Most of these studies, however, have concerned the intracellular delivery of drugs mainly in the cytoplasm and rarely in the cell nucleus.30,31 To further confirm that functional proteins can be delivered into the cell nucleus by fusion with K4, we chose several different proteins with a series of isoelectric points (pI), molecular weights and bioactivities: prothymosin-α (MW = 11.8 kDa, pI = 3.71, chromatin remodelling protein);32 GFP (MW = 27.0 kDa, pI = 5.9, fluorescent protein); Cas9 (MW = 158 kDa, pI = 8.98, genome-editing protein);33 HOXB4 (MW = 27.6 kDa, pI = 9.8, stimulator of hematopoietic stem cell Expan-sion);34 and histone 2A (MW = 13.5 kDa, pI = 10.6, DNA packaging protein)35(Figure 4b). We attached the K4 polypeptide to these proteins using a similar method for attaching to GFP (Figure S22, SI). These fusion products (except K4-GFP) were labeled with fluorescein isothiocyanate (FITC) for imaging studies. The molecules were incubated with HeLa cells for 2 h at 37 °C. All K4 fusion proteins were mainly distributed in the nucleus, establishing the universality of the approach (Figure 4b and Figure S23, SI). The development of approaches for delivering the Cas9 protein along with a single-guide RNA (sgRNA) directly into

cells could offer a transient way of editing genes.36−38 After achieving successful delivery of Cas9-K4, we detected its function and activity in the delivered cells. We first determined whether Cas9-K4 was functional by using an in vitro cleavage assay, which showed that a target sequence was efficiently cleaved by Cas9-K4 only in the presence of the sgRNA (Figure S24, SI). An electrophoretic mobility shift assay showed that Cas9-K4 completely retarded the sgRNA at weight ratios of 4 and higher (Figure S24, SI). We assembled Cas9-K4 with the sgRNA targeting human CCR5 gene and delivered these complexes into the HeLa cells. As shown in Figure 4c, targeting the CCR5 gene resulted in up to 15.2% indel efficiency. No cleavage was observed with Cas9-K4 alone, or at 4 °C. The genome editing was further validated in HEK-293T and Jurkat cells (Figure S25, SI). These results collectively showed that K4-mediated Cas9 delivery may provide an efficient genome editing capability.

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CONCLUSIONS In conclusion, we present here a universal method for the nuclear-targeted protein delivery through K4-tagged proteins. It was shown that K4 achieved enhanced cellular up-take, benefiting from its high density of positive charges, and could combine with importin β1 after cellular internalization. This unusual potency was complemented by low toxicity and immunogenicity, stability in mammalian serum, generality across various mammalian cell types, and simple use by fusing with a protein of interest. The versatility of our approach was confirmed by delivering several proteins with diverse charges, sizes, and bioactivities. This method could offer unique advantages for the delivery to the cell nucleus and the ex vivo manipulation of cells, and its in vivo application need further develop. Another future direction for this research could be to incorporate the therapeutic payload onto the cysteines in the SCP, creating a chemical conjugation to facilitate the widespread use of our method.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b10299. Materials and Methods, experimental details, and supporting data (PDF) Movie S1 as mentioned in the text (MPG)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] (X.G.). *[email protected] (W.Y.). ORCID

Xiangdong Gao: 0000-0002-4979-5110 Author Contributions #

J.Y. and Q.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 81430082, 81703402, 81473216), the National Science and Technology Major Project (2018ZX09201001-003-002), the Project funded by China 17238

DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

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Journal of the American Chemical Society

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Postdoctoral Science Foundation (2017M611957), the Fundamental Research Funds for the Central Universities (2015XPT02), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China (No. 111-2-07), the Innovation Team of the “Double-First Class” Disciplines (CPU2018GF08).

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DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240

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Journal of the American Chemical Society Delivery of Cas9 Protein and Guide RNA. Genome Res. 2014, 24, 1020. (38) Wang, M.; Zuris, J. A.; Meng, F.; Rees, H.; Sun, S.; Deng, P.; Han, Y.; Gao, X.; Pouli, D.; Wu, Q.; et al. Efficient Delivery of Genome-editing Proteins using Bioreducible Lipid Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 2868.

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DOI: 10.1021/jacs.8b10299 J. Am. Chem. Soc. 2018, 140, 17234−17240