Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics

Materials

HBpep peptide, resins and Fmoc-protected amino acids used in solid-phase peptide synthesis were purchased from GL Biochem. N-hydroxysuccinimide, tetrahydrofuran, triphosgene, sodium azide, triphosgene and benzoic acid were purchased from Tokyo Chemical Industry (TCI). N,N′-diisopropylcarbodiimide, acetic acid, 2-hydroxyethyl disulfide, N,N-diisopropylethylamine, piperidine, trifluoroacetic acid, triisopropylsilane, 2,4,6-trinitrobenzenesulfonic acid, glutathione, bovine serum albumin, lysozyme, saporin, β-galactosidase, R-phycoerythrin, methylthiazolyldiphenyl-tetrazolium bromide, Hoechst 33342, methyl-β-cyclodextrin, chlorpromazine hydrochloride, amiloride chloride, monoclonal anti-β-actin-peroxidase antibody l-buthionine-sulfoximine, reduced l-glutathione (GSH) and the Pur-A-Lyzer Maxi Dialysis Kit Maxi 50000 were obtained from Sigma-Aldrich. Dichloromethane, N,N-dimethylformamide, LysoTracker Red DND-99, Opti-MEM, RNase A, Pierce protein transfection reagent (Pro-Ject), Ni-NTA His Bind resin and 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside were purchased from Thermo Fisher Scientific. Xfect protein transfection reagent was purchased from Takara Bio. Organic solvents, including ethyl acetate, hexane and diethyl ether were purchased from Aik Moh Paints & Chemicals Pte Ltd. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS) and antibiotic-antimycotic (100X) liquid were purchased from Gibco. The Nano-Glo Dual-Luciferase kit used for luciferase detection and the CytoTox 96 non-radioactive cytotoxicity assay kit were purchased from Promega. HyClone McCoy’s 5A medium was purchased from Cytiva. Trans-Blot Turbo 0.2-μm nitrocellulose transfer packs, 4–20% Criterion TGX stain-free protein gel and Clarity and Clarity Max Western ECL substrate were purchased from Bio-Rad. Mouse monoclonal p21 (F-5) and p53 (DO-1) horseradish peroxidase (HRP) antibodies were purchased from Santa Cruz Biotechnology. Polyclonal rabbit anti-mouse immunoglobulin HRP was purchased from Dako. EGFP was expressed in Escherichia coli BL21 strain and purified with Ni-NTA His Bind resin. Luciferase-encoding mRNA and EGFP-encoding mRNA used for mRNA transfection experiments were obtained from Trilink. HepG2, HEK293, A549, NIH 3T3, H1299 and HCT116 cell lines were obtained from ATCC. T22 and ARN8 cell lines were established by following previous works^55,56.

Self-immolative moiety synthesis

The self-immolative moieties conjugated to HBpep-K peptide were designed based on the literature²⁹ (the synthesis routes for the amine-reactive species are shown in Supplementary Fig. 8). First, for the synthesis of the side-blocked intermediate product, HO-SS-R, 2-hydroxyethyl disulfide (1 equiv., 10 mmol) was dissolved in 15 ml of tetrahydrofuran (THF), then another 15 ml of THF containing a carboxylic acid reactant such as acetic acid or benzoic acid (0.9 equiv., 9 mmol) was added. Then, under an ice bath, 15 mmol of N,N′-diisopropylcarbodiimide (DIC) was slowly added into the reaction mixture. The reaction was kept at 0 °C for another 0.5 h and then increased to room temperature. After overnight reaction, the mixture was filtered and the supernatant evaporated under reduced pressure. The raw products were purified using silica gel chromatography with ethyl acetate/hexane (1/4) as elute. The purified products were isolated by rotary evaporation (R-215 Rotavapor, BUCHI).

Intermediate products HO-SS-R and N-hydroxysuccinimide (NHS) were coupled using triphosgene. Specifically, HO-SS-R (1 equiv., 5 mmol) and 4-dimethylaminopyridine (DMAP, 0.1 equiv., 0.5 mmol) were dissolved in 10 ml of THF. Triphosgene (0.37 equiv., 1.85 mmol) in 10 ml of THF was added into the previous solution dropwise under an ice bath. After another 0.5 h on the ice bath, the reactions were continued at 40 °C for 4 h, followed by evaporation under reduced pressure to remove excess phosgene. NHS (1.5 equiv., 7.5 mmol) in 20 ml of THF, and N,N-diisopropylethylamine (DIPEA, 1.5 equiv., 7.5 mmol) were then pipetted into the mixtures. The reactions were kept at 40 °C for 24 h before evaporation. The raw products were purified using silica gel chromatography with ethyl acetate/hexane (1/3) as elute. The purified products were isolated by rotary evaporation. The amine-reactive products NHS-SS-Ac and NHS-SS-Ph were synthesized from acetic acid and benzoic acid. The chemical structures of the HO-SS-R and NHS-SS-R were verified by ¹H NMR spectroscopy, as shown in Supplementary Fig. 9. The NMR spectra were collected on a Bruker Advance 400 spectrometer.

Peptide synthesis and purification

The peptides used in this study were synthesized by the classical Merrifield solid-phase peptide synthesis (SPPS) technique⁵⁷. Wang resin (1.0 g, 0.56 mmol) was first swollen in 15 ml of dichloromethane (DCM) for 0.5 h with bubbling nitrogen flow. The DCM was drained with increased pressure, and the resin was washed three times with N,N-dimethylformamide (DMF).

For N-terminal protected amino acid (Fmoc-AA-OH) coupling, Fmoc-AA-OH (2 equiv., 1.12 mmol) was dissolved in 5 ml of DMF, then 5 ml of DMF with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 1.9 equiv., 1.064 mmol) and DIPEA (5 equiv., 2.80 mmol) was added into the solution. The mixture was reacted for 2 min at room temperature before being added onto the resin for 1 h of coupling reaction with bubbling nitrogen flow. The resin was washed with DCM and then DMF three times each after the coupling reaction. The coupling efficiency was evaluated using 2,4,6-trinitrobenzenesulfonic acid (TNBS).

For deprotection of the N-terminal amine, 15 ml of 20% piperidine in DMF (volume ratio) was added onto the resin. The deprotection continued for 0.5 h at room temperature with bubbling nitrogen flow, then the resin was washed with DCM then DMF three times, and the deprotection efficiency was evaluated using TNBS.

After all amino acids in the peptide sequence were coupled onto the resin by performing coupling/deprotection cycles in the C-terminal to N-terminal direction, peptides were cleaved from the resins using a cocktail containing 95% of trifluoroacetic acid (TFA), 2.5% of H₂O and 2.5% of triisopropylsilane (TIPS). After 2 h of cleavage, the reaction mixtures were filtered. The supernatants were concentrated using a nitrogen flow and precipitated into 50 ml of cold diethyl ether. After centrifugation, the pellets were dried under vacuum and re-dissolved using 90% of 10 mM acetic acid and 10% acetonitrile for purification by HPLC (1260 Infinity, Agilent Technologies) equipped with a C8 column (Zorbax 300SB-C8, Agilent Technologies). The purified peptides were isolated by lyophilization (FreeZone 4.5 Plus, Labconco) from HPLC elutes.

Peptide modification

The redox-responsive peptides were synthesized by reacting the ε-amine of the single Lys residue of the N-terminal protected peptide (Fmoc-HBpep-K, Fmoc-GHGVY-GHGVY-GHGPY-K-GHGPY-GHGLYW) with the amine-reactive species NHS-SS-R, followed by deprotection. First, the Fmoc-HBpep-K peptide (1 equiv., 15 μmol) was dissolved in 5 ml of DMF containing DIPEA (15 equiv., 225 μmol). After 30 min of deprotonation, NHS-SS-R (1.5 equiv., 22.5 μmol) in 0.5 ml of DMF was added into the solution. The mixture solutions were allowed to react at room temperature for 24 h before precipitation by adding 50 ml of cold diethyl ether. The raw products were collected from the pellets by centrifugation and dried under reduced pressure. The purification of modified peptides was conducted on an HPLC system equipped with a C8 column. The purified Fmoc-protected peptides were isolated by lyophilization from the HPLC fractions.

The purified Fmoc-protected peptides were dissolved in 5 ml of DMF containing 20% piperidine. The mixture was stirred at room temperature for 2 h of N-terminal deprotection. The raw products were collected from the precipitates after adding 50 ml of cold diethyl ether into the reaction mixtures and purified by HPLC. The final products were isolated by lyophilization as white solids. Two modified peptides were synthesized, HBpep-SA from NHS-SS-Ac and HBpep-SP from NHS-SS-Ph. The molecular weights of Fmoc-HBpep-K and the modified peptides were verified by MALDI-TOF mass spectrometry using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix (Supplementary Fig. 10). Both molecular weights were consistent with the expected molecular weights of the peptides. The MALDI-TOF spectra were collected on an AXIMA Performance spectrometer (Shimadzu Corporation). The modified peptides HBpep-SA and HBpep-SP were dissolved in 10 mM acetic acid solution at 10 mg ml⁻¹ as stock solution.

Coacervation of peptide and therapeutic recruitment

The phase separation behaviour of the HBpep-K and HBpep-SR peptides at various values of pH was monitored by turbidity measurements using a UV–vis spectrometer (UV-2501PC, Shimadzu). The absorbance at 600 nm (A₆₀₀) was used to calculate the relative turbidity as²²

The recruitment of the macromolecules within the peptide coacervates was conducted during the coacervation process at the optimal pH (7.5 for HBpep and 6.5 for HBpep-SR). The therapeutics were dissolved or diluted in 10 mM phosphate buffers (pH 7.5 or 6.5, ionic strength of 100 mM) to achieve the target concentrations. The peptide stock solutions were then mixed with the therapeutics containing the buffer at a 1:9 volume ratio to induce coacervation and recruitment of the therapeutics. As shown in Extended Data Fig. 6, the recruitment efficiency of HBpep-SP coacervates was calculated by comparing the supernatant fluorescence in the buffer solution before and after coacervation and centrifugation using microplate reader (Infinite M200 Pro, Tecan). The fluorescence of EGFP (or FITC), R-PE and Cy5 was detected using 488 nm/519 nm, 532 nm/584 nm and 640 nm/680 nm for the excitation/emission wavelengths, respectively.

Confocal microscopy of EGFP delivery mediated by HBpep coacervates

For a three-dimensional (3D) view of coacervate-treated cells, cells treated overnight with EGFP-loaded coacervates (0.01 mg ml⁻¹ of EGFP, 0.2 mg ml⁻¹ of HBpep) were first stained with a plasma membrane stain and fixed before image acquisition. Briefly, cells were rinsed with HBSS buffer and stained with either 1× CellTracker CM-DiI (C7000; Thermo Fisher) for 5 min at 37 °C followed by 15 min at 4 °C or 1× CellMask Deep Red Plasma membrane stain (Thermo Fisher) for 10 min at 37 °C. Cells stained with membrane dyes were rinsed once with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After fixing, the cells were washed three times with PBS and finally resuspended in PBS. Confocal Z-stack images were collected on an Olympus FV1000 inverted scanning confocal microscope using a ×40 oil immersion objective (NA 1.3). The Z-stacks were reconstructed into 3D images or animations with the aid of Imaris software 3D View and Animation modes.

For live-cell imaging, T22 cells treated with EGFP-loaded DgHBP-2 coacervates were split and seeded on days 3 and 7 to achieve 50–60% confluency, and images were acquired after cells adhered ~4 h after seeding. Z-stack images (differential interference contrast or fluorescence) were acquired on a Nikon Eclipse Ti inverted fluorescence microscope, using a ×40 oil immersion objective (NA 1.3) and sum slices projection was applied to all the stack images using ImageJ software.

Characterization of redox-responsive peptide coacervates

Optical and fluorescence microscopy images of HBpep-SP coacervates and fluorescence images of macromolecules-loaded HBpep-SP coacervates were taken using an inverted fluorescence microscope (AxioObserver.Z1, Zeiss). A dynamic light scattering (DLS, ZetaPALS) system was used to measure the size and zeta potential of pristine HBpep-SR coacervates and macromolecules-loaded HBpep-SR coacervates. The freshly prepared pristine or macromolecules-loaded coacervates (with or without 0.1 mg ml⁻¹ macromolecules, 1 mg ml⁻¹ modified peptides) were diluted into PBS or PBS containing various percentages of FBS with a volume ratio of 1:9 before the DLS test. The redox-responsivities of HBpep-SA and HBpep-SP were evaluated by measuring the decrease in concentration in the presence of GSH. The freshly prepared HBpep-SA or HBpep-SP coacervates (50 μl, 1 mg ml⁻¹ peptide) were diluted in 450 μl of PBS containing 1 mM of GSH. The mixtures were incubated at 37 °C before adding 25 μl of acetic acid to dissolve all the unreacted peptides, and their concentrations were measured by HPLC. The HBpep-SP coacervates incubated in PBS containing various concentrations of GSH (0, 1 and 10 mM) at 37 °C for 24 h were injected into an HPLC system equipped with a C8 column. Fractions were collected and measured by MALDI-TOF for their molecular weights using CHCA as matrix. The redox-triggered EGFP release was conducted using a dialysis tube (molecular weight cutoff of 50 kDa). The freshly prepared EGFP-loaded HBpep-SP coacervates (200 μl, 1 mg ml⁻¹ HBpep-SP, 0.1 mg ml⁻¹ EGFP) were diluted in 1.8 ml of PBS (pH 7.4, ionic strength of 0.15 M) and dialysed against 20 ml of PBS (pH 7.4, ionic strength of 0.15 M) containing various concentrations of GSH (0, 0.1 and 1 mM). A 0.3 ml sample was collected from each group and replaced with 0.3 ml of fresh PBS every 2 h to measure the release of EGFP by using a microplate reader using 488 nm/519 nm for the excitation/emission wavelengths.

Delivery of proteins and peptides

For protein delivery into cells, 1 × 10⁵ cells were suspended in 1 ml of DMEM supplemented with 10% FBS, 100 U ml⁻¹ of penicillin and 100 μg ml⁻¹ of streptomycin, and then transferred into 35 cm² culture dishes. After 24 h of incubation at 37 °C with 5% CO₂, the medium was replaced with 900 μl of Opti-MEM, then 100 μl of freshly prepared peptide or protein-loaded HBpep-SR coacervate suspensions (0.05 mg ml⁻¹ peptide or 0.1 mg ml⁻¹ protein, 1 mg ml⁻¹ HBpep-SR) were prepared by adding the HBpep-SR peptide stocks into cargos containing buffer, then added into the medium. After 4 h of incubation, the coacervates-containing medium was removed and the cells were washed with PBS twice before adding 1 ml of fresh medium (DMEM, 10% FBS, antibiotics). The cells were incubated for another 20 h and then washed twice at pH 5.0 in phosphate buffer to remove any coacervates that had not entered the cells. The release of proteins was determined by the distribution of fluorescence signals inside the cells using fluorescence microscopy (AxioObserver.Z1, Zeiss) or confocal microscopy (LSM 780, Zeiss). For comparison with commercially available protein transfection reagents, Pro-Ject (Thermo Fisher Scientific) and Xfect (Takara Bio) were used according to protocols from the manufacturers to evaluate the delivery efficiency. The EGFP- and R-PE-transfected cells were imaged under a fluorescence microscope (AxioObserver.Z1, Zeiss) and analysed by FACS (LSR Fortessa X20, BD Biosciences).

Delivery of p53-activating peptides

HCT116 p53(+/+) cells suspended in HyClone McCoy’s 5A medium supplemented with 10% FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin were seeded at a density of 8.0 × 10⁴ cells per well in 24-well culture dishes. After 24 h of incubation at 37 °C with 5% CO₂, the medium was replaced with 450 μl of Opti-MEM, then 50 μl of freshly prepared free peptide (32.81 μM), peptide-loaded HBpep-SP coacervate suspensions (32.81 μM cargo peptide, 1 mg ml⁻¹ HBpep-SP) or HPpep-SP-only coacervate suspensions (1 mg ml⁻¹) was added into the medium. After 4 h of incubation, the medium was removed and replaced with 0.5 ml of fresh medium. For Nutlin-3a treatment, 10 μM Nutlin-3a was added to the medium. After an additional 20 h of incubation, 50 μl of medium was removed for lactate dehydrogenase release assay and cells collected for western blots. Western blots were performed by loading equal amounts of total protein (30 μg) from whole-cell lysates on 4–20% Criterion TGX stain-free protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes and blotted with p53 (D0-1) HRP, anti-β-actin-peroxidase or p21(F-5) antibody followed with anti-mouse immunoglobulin HRP antibodies. Immunoblots were developed using Clarity Western ECL substrate or Clarity Max Western ECL substrate for weaker signals and detected with a Bio-Rad ChemiDoc imaging system. For the LDH release assay, 50 μl of culture medium from treated cells was transferred to 96-well microplates and assayed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Lysis solution was added to lytic control wells for maximum LDH release 45 min before the culture medium was transferred for the assay.

EGFP delivery into the cell with GSH depletion

To verify that the cargo release of HBpep-SR coacervates was triggered by the endogenous reducing agent GSH, HepG2 cells were pretreated with 0.5 mM l-buthionine-sulfoximine (BSO) for 12 h (ref. ³¹). The medium was then replaced with 900 μl of Opti-MEM and 100 μl of freshly prepared EGFP-loaded HBpep-SP coacervate suspensions (0.1 mg ml⁻¹ EGFP, 1 mg ml⁻¹ HBpep-SP). After 4 h of uptake, the medium was removed and the cells were washed with PBS twice before adding 1 ml of BSO containing full medium (DMEM, 10% FBS, antibiotics, 0.5 mM BSO). The cells were incubated for another 20 h, then washed twice at pH 5.0 in phosphate buffer to remove any coacervates that had not entered the cells, before being imaged under the fluorescence microscope (AxioObserver.Z1, Zeiss).

Delivery and transfection of mRNA

Two reporter genes including luciferase and EGFP were used to evaluate the mRNA transfection efficiency of the HBpep-SR coacervates. Before transfection, HepG2 or HEK293 cells were incubated in 96-well plates with a density of 1 × 10⁴ cells per well for 24 h. The medium was replaced with 90 μl of Opti-MEM, followed by the addition of 10 μl of freshly prepared mRNA-loaded coacervate suspensions (1 or 2 mg ml⁻¹ of modified peptides). The final concentration of luciferase-encoding mRNA used in the transfection was 3.3 μg ml⁻¹. After 4 h of incubation, the medium was removed and the cells were washed with PBS twice before adding 100 μl of medium (DMEM, 10% FBS, antibiotics). Transfection was then continued for another 20 h before testing the luminescence using the Nano-Glo Dual-Luciferase kit and a microplate reader. For EGFP-encoding mRNA (Cy5-labelled) transfection, the cultures were conducted in 35-cm² dishes into which 100 μl of mRNA-loaded HBpep-SP coacervates (1 mg ml⁻¹ HBpep-SP) was added to achieve the final mRNA concentration of 1 μg ml⁻¹. The transfection was conducted for 4 h of uptake and 20 h of expression before imaging the cells under a fluorescence microscope and testing the transfection efficiency by FACS (LSR Fortessa X20, BD Biosciences).

Protection of mRNA from RNase A

To test whether the coacervates could protect recruited mRNA from enzymatic degradation, 10 μl of the freshly prepared mRNA (luciferase)-loaded HBpep-SP coacervate suspension (1 mg ml⁻¹ of HBpep-SP, 0.1 μg ml⁻¹ mRNA) was diluted into 30 μl of PBS before adding 4 μl of RNase A (10 mg ml⁻¹). The mixture was incubated at 37 °C for 2 h, then 2 μl of 2-mercaptoethanol was added into the mixture and the temperature was raised to 70 °C for 30 min to deactivate the RNase A and release mRNA from the coacervates. Two control groups, including untreated mRNA and free mRNA treated with RNase A, were also used. The integrity of the mRNA in the three groups was determined by 1% agarose gel electrophoresis.

Cytotoxicity study

The cytotoxicity of the therapeutics-loaded or pristine peptide coacervates was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Following literature protocols⁵⁸, 1 × 10⁴ HepG2 or HEK293 cells in 100 μl of medium (DMEM, 10% FBS, antibiotics) were transferred into 96-well plates and incubated for 24 h. The medium was then replaced with 100 μl of Opti-MEM containing therapeutics-loaded coacervates (various concentration of therapeutics, 0.1 mg ml⁻¹ HBpep-SP) or various concentrations of pristine coacervate suspensions. After 4 h of uptake, the medium was removed and the cells were washed by PBS twice before adding 100 μl of medium (DMEM, 10% FBS, antibiotics). The cells were incubated for another 20 h before 10 μl of 5 mg ml⁻¹ MTT dissolved in PBS was added. The medium was removed after 4 h of incubation with MTT, and the cells were washed by PBS twice. Next, 100 μl of DMSO was added per well for absorbance measurements at 570 nm using a microplate reader (Infinite M200 Pro, Tecan). The relative cell viability was calculated as

where A_t, A_b and A_c represent the absorbance of tested cells, no cells and untreated cells, respectively.

Internalization mechanism study

LysoTracker staining was conducted by following the manual from the manufacturer. Similar to protein delivery, 1 × 10⁵ of HepG2 cells were incubated in 35-cm² dishes with DMEM for 24 h, then the medium was replaced with 900 μl of Opti-MEM and 100 μl of EGFP-loaded or AF-BSA-loaded HBpep-SP coacervates (0.1 mg ml⁻¹ EGFP, 1 mg ml⁻¹ HBpep-SP). The cells were cultured for another 2 h before being washed twice with phosphate buffer (pH 5.0) to remove any coacervates that had not entered the cells. After that, 1 ml of Opti-MEM containing 50 nM LysoTracker was added for 30 min of staining under cell culture conditions. The treated HepG2 cells were washed by PBS twice and fixed with 4% formaldehyde solution. Before being imaged by confocal microscopy (LSM 780, Zeiss), the cells were treated with 1 μg ml⁻¹ of Hoechst 33342 for 10 min to stain the nucleus.

Based on the literature^13,50,51, various inhibitors were used to study the pathway of coacervates internalization. HepG2 cells were treated with chlorpromazine (CPM, 30 μM), amiloride chloride (AM, 20 μM), sodium azide (NaN₃, 100 mM) or methyl-β-cyclodextrin (MβCD, 2.5 mM) separately for 1 h, then 100 μl of EGFP-loaded HBpep-SP coacervates (0.1 mg ml⁻¹ EGFP, 1 mg ml⁻¹ HBpep-SP) was added. After another 4 h of incubation, the cells were washed twice with pH 5.0 phosphate buffer followed by PBS twice. The treated cells were imaged by fluorescence microscopy or dissociated by trypsin for FACS. For the 4 °C treated group, the HepG2 cells were pre-incubated for 1 h and kept at low temperature during the 4 h uptake process. Two control groups—totally untreated cells (negative control, NC) and cells treated by EGFP-loaded coacervates without any inhibitors (blank)—were also examined.

Statistics and reproducibility

All experiments were repeated three times. The data are presented as mean ± standard deviation (s.d.). Statistical significance (P < 0.01) was evaluated using a two-sided Student’s t-test when only two groups were compared. All microscopy experiments were repeated independently three times and showed no differences.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.