Abstract
Biomacromolecules are highly promising therapeutic modalities to treat various diseases. However, they suffer from poor cellular membrane permeability, limiting their access to intracellular targets. Strategies to overcome this challenge often employ nanoscale carriers that can get trapped in endosomal compartments. Here we report conjugated peptides that form pH- and redox-responsive coacervate microdroplets by liquid–liquid phase separation that readily cross the cell membrane. A wide range of macromolecules can be quickly recruited within the microdroplets, including small peptides, enzymes as large as 430 kDa and messenger RNAs (mRNAs). The therapeutic-loaded coacervates bypass classical endocytic pathways to enter the cytosol, where they undergo glutathione-mediated release of payload, the bioactivity of which is retained in the cell, while mRNAs exhibit a high transfection efficiency. These peptide coacervates represent a promising platform for the intracellular delivery of a large palette of macromolecular therapeutics that have potential for treating various pathologies (for example, cancers and metabolic diseases) or as carriers for mRNA-based vaccines.
Main
Biomacromolecules, including peptides1, proteins2,3 and RNAs4,5,6, are promising therapeutic modalities for the treatment of various diseases owing to key advantages such as high potency, specificity and safety7. However, their therapeutic potential has not yet been fully realized due to their poor cell membrane permeability and/or endosomal entrapment that limits their intracellular exposure8. Hence, there is substantial interest in developing safe vehicles that can deliver therapeutic cargo to the cytosol. Ideally, endosomal escape can be chemically encoded in the carrier to facilitate the release of the therapeutic payload9,10. Alternatively, approaches to use non-endocytic entry mechanisms could also enhance delivery efficiency7,8. In addition, it is important that the encapsulation method does not affect cargo bioactivity and that the carriers exhibit negligible cytotoxicity.
Current strategies to tackle these issues rely on nanoscale carriers such as inorganic nanoparticles11, synthetic polymers12 or nanoscale hybrid assemblies that can mediate cell membrane fusion13,14. In alternative approaches, the macromolecular drugs are conjugated or complexed with cell-penetrating peptides9,15 to enhance endosomal escape. Although these methods are promising and increasingly considered for clinical translation, they also have pitfalls7. Specifically, fabrication methods can be complex and often use organic solvents that can decrease cargo bioactivity16,17. Some carriers are limited to a specific type of biomacromolecule, whereas others are restricted to the release of payloads with relatively small molecular weights18,19. Safety concerns have also been raised for some carriers, such as inorganic and lipid nanoparticles17,20,21. Whether the carriers are inorganic- or organic-based (polymers, lipids, peptides or fusions thereof), it is generally considered that they must have dimensions below ~200 nm to cross the cell membrane8,18. Recent studies in our laboratory have challenged this notion. Specifically, we have found that micrometre-sized peptide coacervates obtained by liquid–liquid phase separation (LLPS), within which both proteins22 and low-molecular-weight compounds23 can be recruited, are also capable of crossing the cell membrane through an endocytosis-independent pathway23, potentially opening new avenues for intracellular delivery of therapeutics. Peptide coacervates inspired by the self-coacervating histidine (His)-rich beak proteins (HBPs)22 exhibit several advantages over traditional nanoscale delivery vehicles24,25, including (1) fast (within seconds) and efficient recruitment of therapeutic cargo within the coacervate microdroplets; (2) bioactivity-preserving aqueous-based recruitment conditions and (3) negligible cytotoxicity of the peptide building blocks. Furthermore, the physicochemical conditions to induce coacervation can be precisely tuned by single amino acid mutations26,27.
Based on these benefits, we hypothesized that peptide coacervates could be used for the intracellular delivery of a broad palette of macromolecular therapeutics featuring a wide range of molecular weights and isoelectric points (IEPs). To achieve this, we developed short His-rich, pH-responsive beak peptide (HBpep) coacervates conjugated with disulfide bond-containing self-immolative moieties that trigger disassembly of the droplets, facilitating payload delivery within the intracellular reducing environment. We show that these coacervate microdroplets bypass classical endocytic pathways and are capable of direct and efficient cytosolic delivery of a wide range of macromolecules, from therapeutic peptides as small as 726 Da to large enzymes as large as 430 kDa. They can also deliver messenger RNAs (mRNAs) with a high transfection efficiency while also preventing their premature degradation by RNase. Overall, these robust conjugated peptide coacervates can be used as general intracellular delivery vehicles for a broad range of macromolecular therapeutics.
Results and discussion
Preparation and characterization of redox-responsive peptide coacervates
As an initial self-coacervating peptide we selected the histidine-rich beak peptide HBpep, because of advantages including its biological origin, its ability to recruit client molecules with high efficiency (above 95%) and its low toxicity22. Notably, HBpep coacervates can cross the cell membrane via an endocytosis-free pathway23, suggesting their potential for the intracellular delivery of therapeutics. HBpep is characterized by low sequence complexity consisting of five copies of the tandem repeat sequence GHGXY (where X could be leucine (L), proline (P) or valine (V) amino acids) and a single C-terminus tryptophan (W) residue, providing opportunities to further tune its phase separation behaviour. A key feature of the HBpep are the five His residues that confer pH-responsive LLPS behaviour26. Specifically, HBpep adopts a monomeric state at low pH, but then quickly phase-separates into coacervate microdroplets at neutral pH, concomitantly recruiting various macromolecules from the solution in the process (Fig. 1). Preliminary attempts to use HBpep coacervates to recruit and deliver enhanced green fluorescence protein (EGFP) resulted in successful cellular uptake in various cell lines (Supplementary Fig. 1 and Supplementary Videos 1 and 2). However, the EGFP-containing microdroplets formed organelle-like structures within the cells that remained stable for at least three days in HepG2 cells and up to seven days in a T22 cell line without releasing their cargo (Supplementary Figs. 2 and 3).
By single amino acid level manipulation, the pH at which the HBpep phase-separates was dramatically altered. Specifically, the insertion of a single lysine (Lys) residue at position 16 (HBpep-K) shifted the phase separation from approximately pH 7.5 (ref. 22) to 9.0 (Fig. 2a and Supplementary Table 1). Next, a disulfide-containing moiety was conjugated to the amine group of the inserted Lys residue to neutralize the extra positive charge and increase the hydrophobicity of the peptide (Fig. 1). The conjugated moiety is self-immolative, eventually restoring the amine group of the Lys residue through a series of autocatalytic reactions, starting with the reduction of the disulfide bond and followed by a series of side-group rearrangements (Fig. 1)28,29. After the modification, both conjugated peptides with acetyl (HBpep-SA) and phenyl (HBpep-SP) groups at the extremity of the self-immolative moiety were able to phase-separate at the lower pH of 6.5, forming stable microdroplets (Fig. 2a,b and Supplementary Table 1). This design allowed the modified peptides (HBpep-SA and HBpep-SP, collectively referred to as HBpep-SR) to form coacervate microdroplets with a diameter of ~1 μm and a relatively narrow size distribution (Extended Data Fig. 1a,b). Critically, HBpep-SR peptides were able to recruit a wide range of macromolecules during the self-coacervation process at pH 6.5, including proteins, small peptides and mRNAs (Extended Data Fig. 1a–c), without a significant change in their size and zeta potential. The cargo-loaded coacervates were stable at near-physiological and serum conditions until internalization by the cells (Extended Data Fig. 2a,b). Owing to the self-immolative nature of the flanking moiety, reducing agents such as glutathione (GSH), which is abundant in the cytosol, could trigger the reduction and subsequent cleavage of the modified side chain, to convert HBpep-SR back to HBpep-K (Fig. 1). As verified by HPLC and matrix-assisted desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF) (Extended Data Fig. 3), most of the HBpep-SP coacervates were reduced in the presence of GSH for 24 h, producing a thiol-containing intermediate that was eventually converted into HBpep-K. Because HBpep-K reverts to the single phase at neutral pH (that is, monomeric peptide in solution, Fig. 2a), we postulated that GSH-triggered reduction would cause disassembly of coacervate microdroplets in the cytosol, thus releasing the cargo. Therefore, as shown in Fig. 1, by combining pH- and redox-responsivity of the peptides, it should be possible to design an intracellular delivery platform. It is noteworthy that a simple modification at the end of the flanking moiety of HBpep-SR (HBpep-SA versus HBpep-SP) resulted in significant variation in the rate of peptide reduction (Fig. 2c). The concentration decay of HBpep-SA and HBpep-SP in the early stage (2–10 h) could be well fitted by a first-order reaction (Supplementary Fig. 4), with the reaction rate of HBpep-SP about twofold higher than that of HBpep-SA, thus potentially providing a way to tune the kinetics of therapeutic release….
