Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing

Abstract

Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy via cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate via dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome C, saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.

---

Xun Liu

Xun Liu obtained his PhD degree at the College of Chemical and Biological Engineering, Zhejiang University, in 2017 and worked as a postdoctoral fellow at the Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, from 2018 to 2021. He is currently an associate professor in The Second Affiliated Hospital of Soochow University. His research interests focus on the development of polymeric nanocarriers and pro-protein engineering strategies for the cytosolic delivery of therapeutic proteins/peptides.

---

1. Introduction

The delivery of cargo proteins into cancer cells holds great potential for manipulating intracellular biochemical processes and advancing cancer treatment;¹ examples include the use of intrabodies to block or activate definite signaling pathways^2–4 and Cas9 for CRISPR-Cas9-based gene editing to rectify genetic disorders.^5–11 However, proteins are generally cell membrane-impermeable owing to the large molecular size and hydrophilicity. Therefore, clinically available protein drugs are developed toward extracellular domains, and the lack of efficient strategy to deliver proteins into cells limits the expansion of protein drugs.^12,13 As a result, the development of novel approaches for efficient intracellular protein delivery has attracted considerable interest.

For nucleic acid delivery, cationic materials have been widely developed to condense negatively charged nucleic acids via electrostatic attraction.^14–18 However, this strategy is not appropriate for cytosolic protein delivery because different proteins can be positive, negative, or neutral under physiological conditions. A well-established strategy is enhancing the negative charge of proteins by conjugating them with anionic species, such as carboxylic acid,^19,20 phosphoric acid,²¹ boronic acid,^22,23 oligonucleotides,²⁴ anionic polymers²⁵ or other proteins.²⁶ Nonetheless, such modifications often involve complex synthesis processes.²⁷ Moreover, nanocomplexes (NCs) formed from cationic materials and anionic proproteins via a single electrostatic interaction may be unstable in salt solutions.²¹ In addition to effective protein loading, NCs should be efficiently delivered into the cytosol to ensure that the protein performs its intended function. Commonly used cationic materials internalize into cells mainly via endocytosis, where they are entrapped in the endolysosomes.^28–33 If NCs are unable to escape from the endolysosome quickly, the protein's activity can be significantly compromised.^34,35 Cell penetrating peptides (CPPs) are relatively short cationic peptides that adopt an α-helix conformation and have been used to facilitate cellular internalization and endolysosomal escape, thereby enabling efficient intracellular protein delivery.^36–38 However, CPPs generally lack sufficient chain length, which limits their ability to effectively deliver large proteins.³⁹ In comparison, the guanidyl ligand-rich linear polypeptide (LPP), which adopts an α-helical confirmation, exhibits a structure similar to that of CPPs.^40,41 In particular, LPP possesses a sufficient backbone length and cationic charge density, enabling enhanced cell internalization and endolysosomal escape, thus facilitating the effective delivery of large proteins.

In this study, we first engineered an anionic proprotein via facile dynamic covalent chemistry in NaHCO₃ buffer. The native protein was decorated with adenosine triphosphate (ATP) on the lysine residues using 2-acetylbenzeneboronic acid (ABA) as the linker to obtain a phosphate group-rich proprotein, termed A-protein (Scheme 1A). Then, a guanidyl ligand-rich α-helical LPP was developed to efficiently mediate cytosolic A-protein delivery. In the acidic endolysosomes, the native protein was recovered due to the cleavage of reversible boronate ester and imine groups in the A-protein. Moreover, the guanidyl ligand-rich LPP with a sufficient backbone length and rigid α-helical conformation could induce quick endolysosomal escape via membrane disruption (Scheme 1B). Therefore, this cytosolic protein delivery strategy features various advantages, such as a facile anionic proprotein modification, stable protein encapsulation, quick endolysosomal escape, and complete restoration of the native protein. It was observed in HeLa cells that LPP could mediate the efficient transduction of various proteins with different isoelectric points (pIs) and molecular weights (MWs), which could maintain their bioactivity. Notably, it was observed that this strategy enabled the effective transduction of CRISPR-Cas9 ribonucleoproteins (RNPs) and provoked robust polo-like kinase 1 (PLK1) genome editing to inhibit cancer cell growth.

Scheme 1 Schematic of the A-protein synthesis (A) and LPP-mediated cytosolic A-protein delivery (B). A-protein was synthesized via facile dynamic covalent chemistry in NaHCO₃ buffer. LPP could efficiently bind A-protein to form stable NCs via electrostatic attraction and hydrogen bonding. After endocytosis, the rigid α-helical LPP induced quick endolysosomal escape, while the pH-sensitivity of the boronate ester and imine enabled complete restoration of the native protein in the acidic endolysosomes.

2. Results and discussion

2.1. Preparation and characterization of LPP/A-protein NCs

The protein was engineered to obtain A-protein according to a previous report.²¹ Briefly, native protein was first conjugated with ABA via the formation of a “Schiff base” structure in NaHCO₃ buffer solution (pH 8.5), followed by reaction with ATP via the formation of a diol-boronate linkage to yield A-protein. The MWs of bovine serum albumin (BSA, negatively charged protein model) and cytochrome C (Cyt C, positively charged protein model) were increased from 65 331 Da to 67 620 Da and from 12 030 Da to 14 034 Da, respectively, as detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, indicating the successful syntheses of A-BSA and A-Cyt C (Fig. 1A and B). A-Cyt C was not one product, due to the variation of conjugated ATP molecules on each individual Cyt C molecule. The secondary structures of both A-BSA and A-Cyt C were not changed after ATP modification, as determined by the circular dichroism (CD) spectra (Fig. S1, ESI†). After HCl treatment (pH 6.0, 2 h), the MWs of the A-BSA and A-Cyt C recovered to 65 331 and 12 030 Da, respectively, suggesting the pH-triggered complete restoration of native proteins. Guanidyl ligand-rich α-helical LPP was also synthesized by a ring-opening polymerization of N-carboxyanhydride (NCA) and followed a click reaction between an alkyne and azide according to the reported procedures,^41,42 and the chemical structure was confirmed by ¹H NMR (Fig. S2, ESI†). FITC-labeled BSA (BSA-FITC) was used as a model protein to evaluate the cellular uptake of LPP/A-BSA-FITC NCs by flow cytometric analysis. As shown in Fig. 1C and Fig. S3 (ESI†), at a LPP/A-BSA-FITC weight ratio of 5, the NCs showed remarkable cellular internalization in HeLa cells, significantly higher than that at the weight ratio of 1. However, at the weight ratio of 10, the cellular uptake level of NCs was not notably increased. Therefore, the LPP/A-protein weight ratio of 5 was adopted in the following study, whereupon the LPP/A-BSA NCs adopted a uniform spherical morphology with an average diameter of 96 nm and positive zeta potential of 4.5 mV under this ratio (Fig. 1D). Using BSA as the model protein, we probed the protein encapsulation efficiency of the LPP/A-protein NCs in PBS (pH 7.4). As shown in Fig. S4 (ESI†), the BSA encapsulation efficiency was determined as 89.2% in LPP/A-BSA NCs, significantly higher than that in the LPP/BSA NCs (46.6%). We further characterized the stability of the NCs in PBS (pH 7.4) by measuring the particle size after incubation for different times (1, 2, 4, 6, 8, and 12 h). The size of LPP/A-BSA NCs slightly increased from 99.9 nm to 199.5 nm after incubation for up to 12 h, indicating the desired stability of the NCs in physiological conditions (Fig. S5, ESI†).

Fig. 1 Characterization of A-proteins and LPP/A-BSA NCs. MALDI-TOF mass spectra of A-BSA (A) and A-Cyt C (B) with or without HCl treatment (pH 6.0, 2 h). (C) Mean fluorescence intensity per cell (MFI per cell) of HeLa cells following 4-h incubation with LPP/A-BSA-FITC NCs in a serum-free medium at different LPP/A-BSA-FITC weight ratios (2 μg A-BSA-FITC per mL, n = 3). (D) Size and morphology of LPP/A-BSA NCs at an LPP/A-BSA weight ratio of 5, as determined by dynamic laser scanning (DLS) and transmission electron microscopy (TEM), respectively (scale bar: 200 nm).

2.2. LPP-mediated cytosolic A-BSA delivery

LPP/A-BSA-FITC NCs (2 μg A-BSA-FITC per mL) were incubated with HeLa cells for 4 h in serum-free DMEM and the intracellular delivery efficiency was monitored by confocal laser scanning microscopy (CLSM). Extensive and permeated green fluorescence was observed in the LPP/A-BSA-FITC NCs-treated cells but not in the LPP/BSA-FITC NCs-treated cells, indicating LPP-mediated efficient A-BSA delivery (Fig. 2A). The mean fluorescence intensity per cell (MFI per cell) of HeLa cells incubated with LPP/A-BSA-FITC NCs was 73-fold higher than that of HeLa cells incubated with LPP/BSA-FITC NCs, as determined by flow cytometric analysis (Fig. 2B and Fig. S6, ESI†). Meanwhile, LPP showed minimal toxicity on the tested HeLa cells (Fig. S7, ESI†). To clarify the endocytosis mechanism of LPP/A-BSA-FITC NCs, HeLa cells were incubated at 4 °C or pre-incubated with different endocytosis inhibitors, including wortmannin (WTM), to inhibit macropinocytosis, methyl-β-cyclodextrin (mβCD) to inhibit lipid raft-mediated endocytosis, chlorpromazine (CPZ) to inhibit clathrin-mediated endocytosis, and genistein (GNT) to inhibit caveolae-mediated endocytosis. A temperature of 4 °C and addition of CPZ decreased the cellular internalization by 53.5% and 69.3%, respectively, while WTM and GNT showed negligible effects, indicating that the NCs were internalized via energy-dependent endocytosis and mainly via clathrin-mediated endocytosis (Fig. 2C). Moreover, LPP/A-BSA-FITC NCs showed quick endolysosomal escape after 4-h incubation, as determined by the minimal overlap of the LysoTracker deep red (LDR)-stained endolysosomes (red fluorescence) and NCs (green fluorescence), which was due to LPP-induced membrane disruption (Fig. 2D).

Fig. 2 LPP-mediated cytosolic A-BSA delivery. Confocal images ((A) scale bar: 50 μm) and flow cytometric analysis ((B) n = 3) of HeLa cells after 4-h treatment with LPP/BSA-FITC NCs, LPP/A-BSA-FITC NCs, BSA-FITC, or A-BSA-FITC at a concentration of 2 μg BSA-FITC per mL. (C) Relative cellular uptake level of LPP/A-BSA-FITC NCs in HeLa cells at 4 °C or in the presence of various endocytic inhibitors (n = 3). (D) Confocal images of HeLa cells treated with LPP/A-BSA-FITC NCs at a concentration of 2 μg BSA-FITC per mL for 2 h or 4 h (scale bar: 20 μm). Cell nuclei were stained with Hoechst (5 μg mL⁻¹), while endolysosomes were stained with LDR (200 nM).

2.3. Generality of LPP-mediated intracellular A-protein and A-peptide delivery

FITC-labeled various proteins with distinct pIs and MWs, including ovalbumin (OVA), Cyt C, bovine pancreatic ribonuclease A (RNase A), and α-chymotrypsin (α-Chyt), were used to prepare A-protein-FITC and evaluate the universality of LPP-mediated cytosolic A-protein delivery (Fig. 3A). At an LPP/A-protein-FITC weight ratio of 5, LPP/A-protein-FITC NCs were incubated with HeLa cells for 4 h in serum-free DMEM and the delivery efficiency was determined by CLSM and flow cytometric analysis. As shown in Fig. 3B, the CLSM images revealed that the LPP/A-protein-FITC NCs-treated cells but not LPP/protein-FITC NCs-treated cells showed significant green fluorescence. The MFI per cell of LPP/A-protein-FITC NCs-treated cells showed values 5–47 folds higher than that of the LPP/protein-FITC NCs-treated cells, as determined by flow cytometric analysis (Fig. 3C and Fig. S8, ESI†). Apart from proteins, FITC-labeled peptide P1 (RRVKKKKKC) and P2 (DDKKKHHTM) with extremely low membrane permeability were also used to evaluate the capability of the LPP-mediated cytosolic peptide delivery strategy. As shown in Fig. S9 and S10 (ESI†), both LPP/A-P1-FITC NCs and LPP/A-P2-FITC NCs could enter HeLa cells, as indicated by the extensive intracellular green fluorescence, showing higher delivery efficiency than PULSin/P1-FITC NCs and PULSin/P2-FITC NCs, respectively. Moreover, using BSA-FITC as the model protein, we explored the delivery efficiency of LPP/A-protein NCs in various cancer cells (A549, CT26, PANC-1, and B16F10) and non-cancerous cells (293T, HL-1, HRMEC, and MPVEC). As shown in Fig. S11 (ESI†), all the cells after LPP/A-BSA-FITC NCs treatment revealed extensive and robust green fluorescence distribution in the whole cytosol, outperforming the PULSin/BSA-FITC NCs-treated cells. These results confirmed the high generality of the LPP-mediated cytosolic A-protein and A-peptide delivery strategy.

Fig. 3 LPP-mediated intracellular delivery of various A-proteins. (A) MWs and pIs of various proteins, including OVA, α-Chyt, RNase A, and Cyt C. Confocal images ((B) scale bar: 50 μm) and flow cytometric analysis ((C) n = 3) of HeLa cells after 4-h treatment with LPP/protein-FITC NCs or LPP/A-protein-FITC NCs at the concentration of 2 μg protein-FITC per mL. Cell nuclei were stained with Hoechst (5 μg mL⁻¹).

2.4. Cytosolic enzymes delivery

Besides a high delivery efficiency, it was critical to maintain the biological activity of the proteins after intracellular delivery. β-Galactosidase (β-gal) and horseradish peroxidase (HRP) were used as two model enzymes to explore their bioactivity after intracellular delivery. Following 4-h incubation with LPP/A-β-gal NCs, the HeLa cells revealed a remarkable blue pigment accumulation after 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) staining, while following incubation with β-gal or LPP/β-gal NCs, the HeLa cells had negligible blue dye accumulation (Fig. 4A). Notably, the LPP/A-β-gal NCs-treated cells revealed remarkably higher β-gal activity than the commercial protein transduction reagent PULSin/β-gal NCs-treated cells. Quantitative measurement of the β-gal activity revealed that LPP/A-β-gal NCs maintained 73.8% of the relative enzyme activity, remarkably outperforming PULSin (Fig. 4B). Similarly, after LPP/A-HRP NCs treatment for 4 h, a remarkable blue color was observed in cells in the tetramethylbenzidine (TMB) assay (Fig. 4C). The quantitative analysis revealed that nearly ∼85% of the HRP activity was maintained in the LPP/A-HRP NCs-treated cells, which was also higher than that of the PULSin/HRP NCs-treated cells (Fig. 4D). These results therefore confirmed that the NCs could effectively deliver enzymes into cells and maintain their bioactivity.

Fig. 4 LPP-mediated intracellular enzymes delivery. X-gal staining ((A) scale bar: 50 μm) and relative β-gal activity ((B) n = 3) of HeLa cells following 4-h treatment with LPP/A-β-gal NCs, PULSin/β-gal NCs, LPP/β-gal NCs, or β-gal at a concentration of 2 μg β-gal per mL. TMB staining (C) and relative HRP activity ((D) n = 3) of HeLa cells incubated with LPP/A-HRP NCs, PULSin/HRP NCs, LPP/HRP NCs, or HRP for 4 h at a concentration of 2 μg HRP per mL.

2.5. Cytosolic delivery of toxins and antibodies to inhibit cancer cells growth

Two toxins, namely RNase A and saporin, were employed to evaluate LPP-mediated cytosolic delivery and its cancer cell-killing efficacy. After formation of the LPP/A-RNase A NCs, the activity of RNase A was reduced by 46.3%, but almost recovered following HCl treatment (pH 6.0, 2 h), suggesting a pH-triggered native protein release (Fig. S12, ESI†). Native RNase A revealed minimal toxicity in HeLa cells at a concentration up to 20 μg mL⁻¹ owing to its poor ability to cross cell membranes, while the LPP/A-RNase A NCs elicited a remarkable cell killing effect in a concentration-dependent manner, conferring an IC₅₀ value of 2.7 μg mL⁻¹ (Fig. 5A). Similarly, the LPP/A-saporin NCs caused significant toxicity, with an IC₅₀ value of 0.4 μg mL⁻¹ (Fig. 5B). Moreover, we further investigated LPP-mediated saporin delivery to induce cell killing in various cancer and non-cancerous cells. As shown in Fig. S13 (ESI†), the LPP/A-saporin NCs provoked significant cytotoxicity in both cancer and non-cancerous cells, conferring a >80% cell-killing efficacy at a saporin concentration up to 1 μg mL⁻¹. For cancer therapy, it would lead to remarkable side effect after saporin delivery. Based on this, the NCs could be further coated on the surface with hyaluronic acid (HA) or the tumor cell membrane via electrostatic interaction to enhance tumor targeting. Alternatively, the LPP/A-protein delivery system can be used to deliver tumor-specific target proteins to ensure tumor selectivity.

Fig. 5 LPP-mediated cytosolic toxins and antibody delivery against cancer cell growth. Viability of HeLa cells following 4-h treatment with LPP/A-RNase A NCs (A) or LPP/A-saporin NCs (B) at different toxin concentrations in a serum-free medium and with an additional 44-h incubation in an FBS-containing medium (n = 3). (C) Western blot analysis showing PARP cleavage in HeLa cells following 4-h treatment with LPP/A-anti-pAkt NCs at the anti-pAkt concentration of 2 μg mL⁻¹ in a serum-free medium and with an additional 20-h incubation in an FBS-containing medium. Uncropped western blot images are shown in Fig. S20 (ESI†). (D) Viability of HeLa cells following 4-h treatment with LPP/A-anti-pAkt NCs at different concentrations in a serum-free medium and with an additional 44-h incubation in an FBS-containing medium (n = 3).

For intracellular antibody delivery, FITC-labeled human immunoglobulin G (IgG) was first used as a model antibody to evaluate the delivery efficiency. CLSM images and flow cytometric analysis showed that LPP could effectively deliver A-IgG into HeLa cells (Fig. S14, ESI†). Then, an antibody against phospho-Akt (anti-pAkt) was employed to investigate the LPP-mediated cytosolic antibody-delivery efficiency, which could block the Akt-related pathway and reactivate the apoptotic pathway to inhibit cancer cell growth. After LPP/A-anti-pAkt NCs treatment, extensive intracellular green fluorescence was observed in HeLa cells after staining with the caspase3/7 assay kit (Fig. S15, ESI†), indicating the activation of caspase3/7. Accordingly, the downstream poly (ADP-ribose) polymerase (PARP) was cleaved from 116 kDa into 89 kDa and 24 kDa, as determined by western blot analysis (Fig. 5C). As a consequence, LPP/A-anti-pAkt NCs but not anti-pAkt provoked significant toxicity in HeLa cells in a concentration-dependent manner, conferring an IC₅₀ value of 3.3 μg mL⁻¹ (Fig. 5D). These data demonstrate the effectiveness of LPP-mediated toxins and antibody delivery to induce a potent anticancer effect.

2.6. Intracellular delivery of CRISPR-Cas9 ribonucleoproteins

Next, we explored the capability of LPP-mediated cytosolic RNP delivery to elicit genome editing. A-Cas9 was synthesized according to the same method as for A-BSA and allowed to form A-RNP with single-guide RNA (sgRNA) before incubation with LPP to prepare LPP/A-RNP NCs. Herein, we first investigated the capability of the LPP/A-RNP_GFP NCs (sgRNA targeting GFP) for gene knockout in HeLa cells stably expressing GFP (HeLa-GFP). As shown in Fig. 6A, the LPP/A-RNP_GFP NCs-treated HeLa-GFP cells revealed a significantly decreased fluorescence intensity, while the RNP_GFP-treated cells revealed a similar fluorescence intensity to that of untreated cells. Flow cytometric analysis further revealed that the GFP gene knockout efficiency of LPP/A-RNP_GFP NCs was 33% (Fig. 6B). Next, we explored the potential of polo-like kinase 1 (PLK1) gene deletion for anticancer therapy. After cytosolic LPP/A-RNP_PLK1 NCs delivery, HeLa cells revealed obvious indels (26.8%) at the target site, as determined by the T7 endonuclease I (T7E1) assay (Fig. 6C). Accordingly, the levels of mRNA (73.0%) and protein (82.5%) were remarkably decreased in the LPP/A-RNP_PLK1 NCs-treated HeLa cells (Fig. 6D and E). As a result of PLK1 disruption, LPP/A-RNP_PLK1 NCs inhibited the proliferation of HeLa cells by 50.8% (Fig. 6F). Notably, the genome editing efficiency of LPP/A-RNP NCs was significantly higher than that of the commercial Lipofectamine CMAX transfection reagent. These findings demonstrate the potency of LPP-mediated cytosolic A-RNP delivery for genome editing.

Fig. 6 LPP-mediated intracellular delivery of A-RNPs for genome editing. Confocal images ((A) scale bar: 50 μm) and flow cytometric analysis ((B) n = 3) of HeLa-GFP cells incubated with RNP_GFP, CMAX-RNP_GFP NCs, or LPP/A-RNP_GFP NCs (2 μg Cas9 per mL and 1 μg sgRNA_GFP per mL) for 4 h in a serum-free medium and with an additional 44-h incubation in an FBS-containing medium. Cell nuclei were stained with Hoechst (5 μg mL⁻¹). (C) T7E1 assay to evaluate the indels of the PLK1 gene in HeLa cells following 4-h incubation with RNP_PLK1, CMAX-RNP_PLK1 NCs, or LPP/A-RNP_PLK1 NCs (2 μg Cas9 per mL and 1 μg sgRNA per mL) in a serum-free medium and with an additional 44-h incubation in an FBS-containing medium. Relative PLK1 mRNA ((D) n = 3) and protein (E), n = 3) levels in HeLa cells. Uncropped western blot images are shown in Fig. S21 (ESI†). (F) Viability of HeLa cells following 4-h incubation with RNP_PLK1, CMAX-RNP_PLK1 NCs, and LPP/A-RNP_PLK1 NCs and with an additional 44-h incubation (n = 3).

2.7. In vivo antitumor efficiency of LPP/A-saporin NCs

We further explored the capability of LPP-mediated A-protein delivery in vivo. As a proof of concept, LPP/A-saporin NCs were administrated by intratumoral injection in HeLa xenograft tumor-bearing mice and their antitumor efficacy was evaluated. When the tumor volume reached ∼50 mm³, the mice were randomly divided into three groups (6 mice per group) and intratumorally injected with PBS, free saporin, or LPP/A-saporin NCs at 0.1 mg saporin per kg on days 0 and 2. As shown in Fig. 7A–C, LPP/A-saporin NCs remarkably suppressed the tumor growth within the 11-day observation period, with a tumor inhibition rate of 83.3%. In contrast, the mice treated with PBS or saporin revealed significant tumor volume increases. The hematoxylin and eosin (H&E) staining, TUNEL staining, and Ki-67 staining of tumor tissues harvested from LPP/A-saporin NCs-treated mice exhibited remarkable apoptotic profiles, indicating their robust anticancer efficacy (Fig. 7D). During the 11-day observation period, the body weight of the mice gradually increased and the H&E-stained major organs, including heart, liver, spleen, lung, and kidney, revealed negligible histological abnormality (Fig. S16 and S17, ESI†). In addition, the mice injected with LPP/A-saporin NCs revealed minimal abnormality compared to those treated with PBS in terms of the hematological and blood biochemical analyses. These results collectively indicated that LPP/A-saporin NCs provoked negligible systemic toxicity after intratumoral injection (Fig. S18 and S19, ESI†).

Fig. 7 In vivo antitumor efficacy of LPP/A-saporin against HeLa xenograft tumors. PBS, saporin, or LPP/A-saporin NCs (0.1 mg saporin per kg) were intratumorally injected into HeLa tumor-bearing mice on days 0 and 2. (A) Evolution of the tumor volume (n = 6). Representative images (B) and weights (C) of tumors harvested on day 10. (D) H&E, TUNEL, and Ki-67 staining images of tumor sections harvested on day 10 (scale bars = 50 μm).

3. Conclusion

In summary, we developed a guanidyl ligand-rich α-helical LPP-mediated cytosolic A-protein delivery system. This proprotein delivery platform possessed various advantages, as following: (1) facile A-protein preparation via dynamic covalent chemistry; (2) pH-triggered complete restoration of the native protein; (3) strong binding between LPP and A-protein via electrostatic attraction and hydrogen bonding; (4) α-helical LPP-induced membrane disruption, allowing quick endolysosomal escape. Thus, this strategy was able to efficiently deliver functional enzymes, antibody, toxins, and RNPs into cells and maintain their bioactivity. This study is an effective complement to existing protein delivery systems and it holds great potential for protein-based cancer therapy.

4. Experimental section

4.1. Synthesis of A-proteins

All proteins were allowed to react with ABA and ATP at the amino group at a protein/ABA/ATP molar ratio of 1/1.5/1.5. For example, BSA (1 mg, 0.96 μmol amino group) was dissolved in NaHCO₃ buffer solution (20 mM, pH 8.5, 902 μL). Next, ABA (10 mg mL⁻¹ in NaHCO₃ buffer solution, pH 8.5, 24 μL) was slowly added to the protein solution and incubated for 30 min at room temperature. Then, ATP dissolved in NaHCO₃ buffer solution (10 mg mL⁻¹, pH 8.5, 74 μL, ABA/ATP = 1/1, mol mol⁻¹) was added to the above mixture, which was stirred at room temperature for another 2.5 h, followed by ultrafiltration (MWCO = 3 kDa) to obtain A-BSA. The modification degrees of ATP in the A-proteins (with BSA and Cyt C as representatives) were determined by MALDI-TOF-MS.

4.2. Preparation and characterization of LPP/A-protein NCs

First, LPP was dissolved in phosphate buffer (PBS, 10 mM, pH 7.4) at 2 mg mL⁻¹ to make a stock solution, and A-proteins were dissolved in PBS (10 mM, pH 7.4) at 1 mg mL⁻¹ to make a stock solution. Before use, the protein stock solutions were diluted with PBS (pH 7.4) to the concentration of 100 μg mL⁻¹, and the LPP stock solutions were diluted with PBS (pH 7.4) to the concentrations of 100, 500, or 1000 μg mL⁻¹. The protein solution was mixed with the polymer solution in an equal volume, vortexed for 10 s, and then incubated at room temperature for 30 min to allow NCs formation. The size and zeta potential of LPP/A-BSA NCs were evaluated using a Zetasizer Nano system (Malvern, ZS). The morphology of the NCs was observed by transmission electron microscopy (TEM, FEI Tecnai F20, acceleration voltage = 200 kV).

4.3. Cytosolic delivery of the LPP/A-protein NCs

HeLa cells were seeded on a glass-bottomed culture dish (d = 15 mm) at 1 × 10⁴ cells per dish and cultured for 24 h. After replacement with fresh serum-free DMEM, BSA-FITC, A-BSA-FITC, LPP/BSA-FITC NCs, or LPP/A-BSA-FITC NCs (LPP/BSA-FITC = 5/1, w/w) in PBS were added at a final concentration of 2 μg BSA-FITC per mL. After incubation at 37 °C for 4 h, the cells were washed three times with cold PBS and incubated with trypan blue (0.4%) for 10 min to quench the fluorescence of NCs physically attached to the cell surface. Cell nuclei were stained with Hoechst 33 258 (5 μg mL⁻¹, 15 min) before observation by CLSM. In a parallel experiment, HeLa cells were seeded on 24-well plates at 1 × 10⁴ cells per well and cultured for 24 h. The cells were treated with BSA-FITC, A-BSA-FITC, LPP/BSA-FITC NCs, or LPP/A-BSA-FITC NCs as described above. Cells were washed three times with PBS and subjected to flow cytometric analysis (Beckton Dickinson, USA). Data were analyzed using Cell Quest software, with the cellular internalization level represented by the mean fluorescence intensity per cell.

To explore the internalization pathway of LPP/A-BSA-FITC NCs, HeLa cells were seeded on 96-well plates at 1.5 × 10⁴ cells per well and cultured for 24 h. Cells were first pre-treated with various endocytic inhibitors, including mβCD (5 mM), GNT (100 μg mL⁻¹), WTM (10 μg mL⁻¹), and CPZ (10 μg mL⁻¹), for 0.5 h to inhibit the potential endocytic pathway. After replacement with fresh serum-free DMEM, LPP/A-BSA-FITC NCs were added at a final concentration of 2 μg BSA-FITC per mL. After incubation at 37 °C for 4 h, the cells were washed three times with cold PBS and then incubated with trypan blue (0.4%) for 10 min. In addition, cells were treated with LPP/A-BSA-FITC NCs at 4 °C for 4 h. After washing three times with PBS, the cells were lysed by a passive lysis buffer (100 μL per well). The amount of internalized LPP/A-BSA-FITC NCs was determined by spectrofluorimetry (λ_ex = 488 nm, λ_em = 528 nm). The uptake level was represented as ng BSA-FITC per mg cellular protein, and the results were denoted as the percentage uptake level of NCs at 37 °C in the absence of endocytic inhibitors.

To evaluate the endolysosomal escape efficiency of the LPP/A-BSA-FITC NCs, HeLa cells were treated according to the same method as described above, and stained with LDR (200 nM, 30 min for endolysosomes) and Hoechst 33 258 (5 μg mL⁻¹, 15 min for nuclei). The cells were washed with PBS three times and then observed by CLSM.

4.4. Cytosolic delivery of LPP/A-RNP NCs for genome editing

A-RNP was prepared by mixing A-Cas9 proprotein and sgRNA (sequence listed in Table S1, ESI†) in a weight ratio of 2 : 1, and allowed to form LPP/A-RNP NCs using the same method as described above. HeLa-GFP cells were seeded on a glass-bottomed culture dish (d = 15 mm) at 1 × 10⁴ cells per dish and cultured for 24 h. After replacement with serum-free DMEM, RNP_GFP, CMAX-RNP_GFP NCs, or LPP/A-RNP_GFP NCs (2 μg Cas9 per mL and 1 μg sgRNA_GFP per mL) were added into each well. After incubation at 37 °C for 4 h, the cells were washed three times with PBS and incubated with 10% FBS-containing medium for another 44 h. The cells were then stained with Hoechst 33 258 (5 μg mL⁻¹) for 10 min before being observed by CLSM. In a parallel experiment, the mean fluorescence intensity of HeLa-GFP cells was determined by flow cytometry. The commercial reagent CMAX served as the positive control and was used according to the manufacturer's protocol.

To evaluate PLK1 genome editing, HeLa cells were seeded in 6-well plates at 1 × 10⁵ cells per well and cultured for 24 h. After replacement with serum-free DMEM, RNP_PLK1, CMAX-RNP_PLK1 NCs, or LPP/A-RNP_PLK1 NCs (2 μg Cas9 per mL, and 1 μg sgRNA_PLK1 per mL) were added into each well. After incubation at 37 °C for 4 h, the cells were washed three times with PBS and incubated for another 44 h in 10% FBS-containing DMEM. The genomic DNA was then isolated using the Cell/Tissue Genomic DNA Extraction Kit (GENERAY BIOTECH, China) and subjected to a polymerase chain reaction (PCR) using specific primers (Table S2, ESI†) according to the following condition, 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min. Afterward, the PCR product (200 ng) was dissolved in 1 × NEBuffer2 (20 μL), and further underwent a denature/annealing process to form a heteroduplex (95 °C for 5 min, −2 °C s⁻¹ to 85 °C, and −0.1 °C s⁻¹ to 25 °C). The reannealed PCR products (200 ng) were mixed with T7E1 (0.3 μL) and incubated at 37 °C for 30 min prior to analysis by electrophoresis in 2% agarose gel. The band intensity was quantified using ImageJ software, and the indel percentage was defined as follows: 100 × (1 − (1 − fraction cleaved)^1/2), where the fraction cleaved = intensity of each digested band/(intensity of each digested band + intensity of undigested band).

In a parallel study, total RNA was isolated from LPP/A-RNP_PLK1 NCs-treated HeLa cells using the Trizol reagent (Biosharp). The PLK1 mRNA level was determined by real-time PCR with specific primers (Table S3, ESI†). Also, the PLK1 protein level was determined by western blot using the anti-PLK1 rabbit monoclonal primary antibody (1 : 1000 dilution) and HRP-conjugated goat anti-rabbit IgG secondary antibody (1 : 10 000 dilution). The in vitro cytotoxicity of HeLa cells was determined by MTT assay.

4.5. In vivo antitumor efficacy

A HeLa xenograft tumor model was established via the subcutaneous inoculation of HeLa cells (1 × 10⁷, in 100 μL PBS) in the right flank of BALB/c nude mice. When the tumor volume reached ∼50 mm³, the mice were randomly divided into three groups (6 mice per group), and were intratumorally injected with PBS, free saporin, or LPP/A-saporin NCs (0.1 mg saporin per kg) on days 0 and 2. The tumor volume and body weight were monitored every other day within the 11-day observation period. The tumor volume (V, cm³) was calculated as (tumor length) × (tumor width)²/2. On day 10, the mice were sacrificed, and the tumors as well as major organs were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness. The tissue sections were stained with hematoxylin and eosin (H&E) before histological examination. In parallel, to observe the apoptotic level of tumor cells, tumors were embedded in OCT, and the frozen tissues were sectioned at 5 μm in thickness and stained using the One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, China) before CLSM observation. Alternatively, cryosections of tumor tissues were stained with the anti-Ki-67 rabbit polyclonal primary antibody (0.5 μg mL⁻¹, Abcam, USA) and FITC-conjugated goat anti-rabbit IgG secondary antibody (1 : 500 dilution, Beyotime, China) followed by CLSM observation.

4.6. Statistical analysis

All data are presented herein as the mean ± standard deviation (SD). The two-tailed Student's t-test was performed for two-group comparisons, while one-way analysis of variance (ANOVA) with Tukey's correction was used for the comparison of more than two groups. Differences were considered to be significant at *p < 0.05 and very significant at **p < 0.01 and ***p < 0.001.

Data availability

All relevant data are within the manuscript and its additional files.

Conflicts of interest

The authors declare that they have no competing financial interest.

Acknowledgements

We acknowledge the financial support from the National Natural Science Foundation of China (52303200, 82241008, and 52325305), the Natural Science Foundation of Jiangsu Province (BK20220245), Suzhou Science and Technology Development Project (SKY2023052), Pre-research Fund Project of the Second Affiliated Hospital of Soochow University (SDFEYJBS2111), Collaborative Innovation Center of Suzhou Nano Science & Technology, the 111 project, Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, and Suzhou Key Laboratory of Nanotechnology and Biomedicine.

References

1. X. Liu, F. Wu, Y. Ji and L. C. Yin, Bioconjugate Chem., 2019, 30, 305–324.
2. K. Dutta, P. Kanjilal, R. Das and S. Thayumanavan, Angew. Chem., Int. Ed., 2021, 60, 1821–1830.
3. P. Y. Yuan, F. Yang, S. S. Liew, J. C. Yan, X. Dong, J. F. Wang, S. B. Du, X. Mao, L. Q. Gao and S. Q. Yao, Biomaterials, 2022, 281, 121376.
4. S. B. Du, S. S. Liew, C. W. Zhang, W. Du, W. J. Lang, C. C. Y. Yao, L. Li, J. Y. Ge and S. Q. Yao, ACS Cent. Sci., 2020, 6, 2362–2376.
5. T. Wan, Q. Pan, C. Y. Liu, J. J. Guo, B. W. Li, X. J. Yan, Y. Y. Cheng and Y. Ping, Nano Lett., 2021, 21, 9761–9771.
6. X. Q. Wang, Y. H. Li, X. Wang, D. M. Sandoval, Z. L. He, A. Sigen, I. L. Sáez and W. X. Wang, ACS Nano, 2023, 17, 17799–17810.
7. E. C. Tan, T. Wan, C. L. Yu, Q. Q. Fan, W. B. Liu, H. Chang, J. Lv, H. Wang, D. L. Li, Y. Ping and Y. Y. Cheng, Nano Today, 2022, 46, 101617.
8. Y. Ji, L. S. Fan, S. C. Qu and X. Han, J. Mater. Chem. B, 2022, 10, 7694–7707.
9. M. Z. Zhang, S. Y. Sun, X. Liang, Z. G. Liu, J. X. Yin, Q. S. Li and S. C. Yang, Biomater. Sci., 2024, 12, 1197–1210.
10. Z. Iqbal, K. Rehman, J. Xia, M. Shabbir, M. Zaman, Y. Liang and L. Duan, Biomater. Sci., 2023, 11, 3762–3783.
11. Z. Le, Q. Pan, Z. He, H. Liu, Y. Shi, L. Liu, Z. Liu, Y. Ping and Y. Chen, ACS Cent. Sci., 2023, 9, 1313–1326.
12. N. Pathak, C. A. Patino, N. Ramani, P. Mukherjee, D. Samanta, S. B. Ebrahimi, C. A. Mirkin and H. D. Espinosa, Nano Lett., 2023, 23, 3653–3660.
13. A. Jash, A. Ubeyitogullari and S. S. H. Rizvi, J. Mater. Chem. B, 2021, 9, 4773–4792.
14. P. Q. Ma, Q. Wang, X. Luo, L. Z. Mao, Z. X. Wang, E. Y. Ye, X. J. Loh, Z. B. Li and Y. L. Wu, Biomater. Sci., 2023, 11, 5078–5094.
15. C. J. Yan, J. L. Zhang, M. L. Huang, J. J. Xiao, N. L. Li, T. Wang and R. Ling, J. Mater. Chem. B, 2023, 11, 8096–8116.
16. C. Liu, Q. Q. Shi, X. A. Huang, S. Koo, N. Kong and W. Tao, Nat. Rev. Cancer, 2023, 23, 526–543.
17. L.-X. Shi, X.-R. Liu, L.-Y. Zhou, Z.-Q. Zhu, Q. Yuan and T. Zou, Biomater. Sci., 2023, 11, 7709–7729.
18. L. Cui, L. Kudsiova, F. Campbell, D. J. Barlow, H. C. Hailes, A. B. Tabor and M. J. Lawrence, Biomater. Sci., 2023, 11, 3335–3353.
19. M. Wang, K. Alberti, S. Sun, C. L. Arellano and Q. B. Xu, Angew. Chem., Int. Ed., 2014, 53, 2893–2898.
20. Y. Lee, T. Ishii, H. J. Kim, N. Nishiyama, Y. Hayakawa, K. Itaka and K. Kataoka, Angew. Chem., Int. Ed., 2010, 49, 2552–2555.
21. X. Liu, Z. Y. Zhao, W. Li, Y. J. Li, Q. Yang, N. Y. Liu, Y. B. Chen and L. C. Yin, Angew. Chem., Int. Ed., 2023, 62, e202307664.
22. M. Wang, S. Sun, C. I. Neufeld, B. Perez-Ramirez and Q. B. Xu, Angew. Chem., Int. Ed., 2014, 53, 13444–13448.
23. Z. Y. Zhao, X. Liu, M. Y. Hou, R. X. Zhou, F. Wu, J. Yan, W. Li, Y. J. Zheng, Q. M. Zhong, Y. B. Chen and L. C. Yin, Adv. Mater., 2022, 34, 2110560.
24. A. A. Eltoukhy, D. L. Chen, O. Veiseh, J. M. Pelet, H. Yin, Y. Z. Dong and D. G. Anderson, Biomaterials, 2014, 35, 6454–6461.
25. V. Postupalenko, D. Desplancq, I. Orlov, Y. Arntz, D. Spehner, Y. Mely, B. P. Klaholz, P. Schultz, E. Weiss and G. Zuber, Angew. Chem., Int. Ed., 2015, 54, 10583–10586.
26. J. A. Zuris, D. B. Thompson, Y. Shu, J. P. Guilinger, J. L. Bessen, J. H. Hu, M. L. Maeder, J. K. Joung, Z. Y. Chen and D. R. Liu, Nat. Biotechnol., 2015, 33, 73–80.
27. X. Liu, Z. Y. Zhao, F. Wu, Y. B. Chen and L. C. Yin, Adv. Mater., 2022, 34, 2108116.
28. J. Liu, T. L. Luo, Y. F. Xue, L. Q. Mao, P. J. Stang and M. Wang, Angew. Chem., Int. Ed., 2021, 60, 5429–5435.
29. J. A. Kretzmann, D. C. Luther, C. W. Evans, T. Jeon, W. Jerome, S. Gopalakrishnan, Y. W. Lee, M. Norret, K. S. Iyer and V. M. Rotello, J. Am. Chem. Soc., 2021, 143, 4758–4765.
30. X. F. Qin, C. M. Yu, J. Wei, L. Li, C. W. Zhang, Q. Wu, J. H. Liu, S. Q. Yao and W. Huang, Adv. Mater., 2019, 31, 1902791.
31. P. Y. Yuan, X. Mao, X. F. Wu, S. S. Liew, L. Li and S. Q. Yao, Angew. Chem., Int. Ed., 2019, 58, 7657–7661.
32. G. Y. Rong, L. J. Chen, F. Zhu, E. C. Tan and Y. Y. Cheng, Nano Lett., 2022, 22, 6245–6253.
33. Y. Rui, D. R. Wilson, J. Choi, M. Varanasi, K. Sanders, J. Karlsson, M. Lim and J. J. Green, Sci. Adv., 2019, 5, eaay3255.
34. Y. W. Lee, D. C. Luther, R. Goswami, T. Jeon, V. Clark, J. Elia, S. Gopalakrishnan and V. M. Rotello, J. Am. Chem. Soc., 2020, 142, 4349–4355.
35. S. B. Du, S. S. Liew, L. Li and S. Q. Yao, J. Am. Chem. Soc., 2018, 140, 15986–15996.
36. O. Tietz, F. Cortezon-Tamarit, R. Chalk, S. Able and K. A. Vallis, Nat. Chem., 2022, 14, 284–293.
37. G. Lättig-Tünnemann, M. Prinz, D. Hoffmann, J. Behlke, C. Palm-Apergi, I. Morano, H. D. Herce and M. C. Cardoso, Nat. Commun., 2011, 2, 453.
38. M. Sánchez-Navarro, Adv. Drug Delivery Rev., 2021, 171, 187–198.
39. J. Diaz, M. Pietsch, M. Davila, G. Jaimes, A. Hudson and J. P. Pellois, Bioconjugate Chem., 2023, 34, 1861–1872.
40. C. Ge, J. Zhu, H. Ye, Y. Wei, Y. Lei, R. Zhou, Z. Song and L. Yin, J. Am. Chem. Soc., 2023, 145, 11206–11214.
41. F. F. Li, Y. J. Li, Z. C. Zhou, S. X. Lv, Q. R. Deng, X. Xu and L. C. Yin, ACS Appl. Mater. Interface, 2017, 9, 23586–23601.
42. R. J. Zhang, N. Zheng, Z. Y. Song, L. C. Yin and J. J. Cheng, Biomaterials, 2014, 35, 3443–3454.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02009j

‡ These authors contributed equally to this work.

---