Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins

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Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins Shubo Du, Si Si Liew, Lin Li, and Shao Q. Yao J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06584 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Journal of the American Chemical Society

Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins Shubo Du,†§ Si Si Liew,† Lin Li,*‡ and Shao Q. Yao*† †

Department of Chemistry, National University of Singapore, 117543, Singapore

§

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 117456, Singapore ‡

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 211800, P.R. China ABSTRACT: Therapeutic proteins have increased dramatically in both number and frequency of use in recent years, primarily owing to advances in protein engineering. Protein therapy provides the advantages of high potency and specificity, as well as low oncogenic risks. To date, due to their inability to cross the plasma membrane into the intracellular space of mammalian cells, most therapeutic proteins can only target secreted modulators or extracellular receptors. The full potential of protein therapy is however being gradually realized by the development of various strategies capable of intracellular protein delivery. Notwithstanding, most of these strategies suffer from severe endosomal trapping, resulting in very low protein delivery efficiency. In this perspective, we discuss various methods to directly transport proteins into the cell cytoplasm, thus bypassing the problems associated with endocytosis.

1. INTRODUCTION Why protein therapeutics? Proteins are the fundamental carriers of cellular activities. Their dynamic and diverse roles in cellular functions pose both challenges and exciting opportunities in biology and medicine. Malfunctioning, mutation, reduced expression or any other abnormalities in a key cellular protein may result in diseases. Viewed from this perspective, intracellular delivery of functional proteins to 1) directly replace deficient or dysfunctional proteins or 2) agonize or antagonize critical intracellular pathways, can be the most unambiguous method to combat diseases.1,2 Protein-based therapy offers the advantages of high specificity and potency over smallmolecule drugs, due to their structural complexity that is often required for specificity. Moreover, some proteins that are essential players in signaling pathways are inaccessible to small-molecule inhibitors, rendering them “undruggable” in this context. In many cases, such proteins function by primarily facilitating protein-protein interactions (PPIs). Most PPI interfaces are hydrophobic and relatively flat, making it difficult for small molecules to anchor and block such large surfaces.3 Macromolecules such as proteins or peptides could however efficiently inhibit PPIs. This is where protein-based therapy becomes relevant, bridging the gap between small molecule inhibitors and large protein targets. In recent years, drugs based on antibodies (a special class of proteins) have emerged as the largest and fastest growing class of protein therapeutics, comprising several of the ten top-selling global prescription drugs.4 However, the vast majority of antibody-based drugs can only target cell surface or secreted proteins, sig-

nificantly limiting their potential applications.5 Since antibodies can bind to virtually any immunogenic targets with exquisite specificity and high affinity, they have long been considered the ideal “magic bullets” against an untapped reservoir of potential therapeutic targets if they can cross the cell membrane and reach the sites of action.6 Challenges. Despite apparent benefits of proteinbased biologics, there are several major challenges in delivering therapeutic proteins into the mammalian cells. Firstly, the plasma membrane can effectively block most macromolecules from entering the cells, which necessitates protein cargos to be modified in order to make them cell-permeable.2 Secondly, once inside the cell, there is a greater challenge of endosomal escape. In most cases, since proteins are delivered by endocytosis, they are often trapped in endolysosomal vesicles. The majority of these trapped proteins eventually undergo protease-mediated degradation and exocytosis. As a result, the overall therapeutic efficacy is limited because only a very small fraction of successfully delivered proteins can interact with their cytosolic targets.7,8 Thirdly, to transduce protein into cells, significant genetic or chemical modifications to the proteins are usually necessary, or the cargo proteins need to be exposed to undesirable conditions (e.g., temperature, pH, chemicals, and organic solvents). The modification process may impair the stability of proteins, and their functionality may be altered with the appendant.8 Progress has been made in developing strategies to bring proteins (especially antibodies and their derivatives) to engage intracellular targets.9-18 One method is to express antibodies inside cells through either viral vectors or plasmids, hence bypassing the transportation of proteins across the plasma membrane entirely. Intracellular antibodies (intrabodies) are recombinant antibody fragments

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Challenges •

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exosome nanomotor stabilized nanocapsules

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Figure 1. Schematic illustration of challenges in intracellular protein delivery and currently available systems for non-endocytic delivery. Delivery methods based on endocytic pathways usually suffer from severe endosomal entrapment and low overall delivery efficiency. Direct cytosolic delivery of proteins allows cargo transport to the site of action and maximizes delivery.

that are produced intracellularly and bind an antigen within the same cell.9 Intrabodies are usually expressed in the form of a single-chain variable fragment (scFv). The direct expression of antibody fragments in the cytoplasm is challenging because the intracellular reducing environment can impair the formation of disulfide bonds and specialized chaperones needed for proper antibody folding are lacking. Despite the challenges, high efficacies in targeting cytoplasmic proteins leading to their functional knockdown have been successfully demonstrated by numerous intrabodies. Nanobodies are single-domain antigen-binding fragments derived from camelid’s single-chain IgG2.10,12 They have a binding affinity and specificity similar to conventional antibodies but are much smaller in size (~14 kD) and exhibit higher stability. When conjugated with fluorescent proteins, the fluorescent nanobodies (or chromobodies) become nanoprobes that can trace the dynamics of endogenous cellular processes in living cells.11 Nanobodies have drawn great attention as useful in vitro and intracellular reagents in a variety of biological applications.12 Similar to intrabodies, however, the intracellular application of nanobodies is limited due to the need to introduce them genetically, therefore suffering from some key disadvantages that are often observed in gene therapy, for example, permanent integration of genetic materials into the host cell’s genome. It can also induce stress responses, carcinogenesis and immunogenicity.9 Alternatively, with protein delivery methods that make use of endocytosis pathways, an endosomolytic agent can be used to disrupt endosomes and release the trapped proteins into the cytosol.13,14 Endosome rupture

can be induced by membrane-destabilizing agents, including pH-sensitive membrane-perturbing peptides and polymers. Such compounds feature the ability to switch their structures inside the acidic endosomal compartments, resulting in successful lysis of endosomal membranes. An excellent example was recently reported by Futaki et al. in which commonly used monoclonal antibodies were successfully delivered to mammalian cells and subsequently liberated from endosomes by using pH-sensitive peptides.13 The authors designed new endosomolytic peptides by introducing one or two glutamic acid residues on the hydrophobic face of an amphipathic helical cationic peptide. Upon endocytosis, the acidic residues became protonated due to the pH decrease in the endosome, and ensuing perturbation of negatively charged endosome membranes led to the release of the endosomal contents. One of the peptides L17E was shown to enable a marked cytosolic liberation of cargos. In addition to antibodies, successful cytosolic delivery of a ribosome-inactivation protein (saporin) and Cre recombinase was achieved. While some progress has been made to address the roadblocks of cytosolic protein delivery, efficiency remains a key challenge due to endosomal entrapment. Since the endocytosis-dependent routes often result in very low cytosolic release of the delivered cargo (< 10%), methods capable of endocytosis-independent protein delivery have emerged as valuable alternatives and hold promise for future therapeutic applications. Although they are still relatively understudied when compared to the more popular endocytosis-based delivery strategies, there have been some innovative and insightful examples recently.15-18 In


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this perspective, we will discuss approaches of direct cytosolic protein delivery that dodge endosomes completely, by focusing on the following five broad categories (Figure 1): physical methods, nanocarriers, bioreversible esterification, cell-penetrating peptides (CPPs) and cell-penetrating poly(disulfide)s (CPDs).

2. PHYSICAL METHODS Physical methods are considered the most straightforward and traditional approaches to achieve direct cytosolic delivery of proteins. Several techniques have been developed such as microinjection, electroporation, sonoporation, and more recently mechanical deformation and microﬂuidics electroporation.7,19 These membrane disruption-based techniques have found success in numerous in vitro applications because they are able to deliver proteins directly to the cytosol with immediate bioavailability.20-22 For example, a recent study reported a novel method called Trim-Away which enables rapid degradation of endogenous proteins in mammalian cells.21 Trim-Away harnessed the cell’s very own protein degradation machinery that uses an antibody-binding protein named TRIM21 (an E3 ubiquitin ligase that binds with high affinity to the Fc domain of antibodies). By introducing an antibody against the protein-of-interest (POI) into mammalian cells via microinjection or electroporation, the antibody-bound POI was subsequently recognized by TRIM21, leading to eventual TRIM21-mediated ubiquitination and degradation of the POI by the proteasome. These physical methods for protein delivery, however, are low-throughput, disruptive and require specialized instrumentation to mechanically/physically puncture membranes, limiting their utility for large-scale and pharmaceutical applications. Furthermore, the volume of tissue that could be physically accessed is very limited, and the transient cell permeabilization induced by such methods may also cause influx of other proteins and biomolecules into the cells, thus generating potential side effects.8

A Fusogenic liposome

Nanoparticleprotein complex

Protein

Fusogenic proteoliposome

Fusogenic liposome

NPSCs

Exosome

Nanomotor

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Template D generation

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Figure 2. Protein delivery system based on nanocarriers: (A) fusogenic liposomes, (B) exosomes, (C) nanomotors and (D) nanoparticle-stabilized nanocapsules (NPSCs). Reproduced from references 27, 29, 36 and 39 with permission from American Chemical Society.

3. NANOCARRIERS Fusogenic liposomes. Direct delivery of therapeutic proteins, antibodies, enzymes, and cytokines in living mammalian cells has been accomplished by using liposomal carriers.23,24 The lipid bilayers help maintain protein stability by shielding the cargo proteins from extracellular and endosomal proteases. Such liposomes are usually taken up via endocytosis, and once in the endocytic pathway, the cargo molecules may eventually reach the cytosol by destabilization and disruption of the endosomal membrane.25 Fusogenic liposomes (FLs) are a unique group of liposomes (Figure 2A). These unilamellar liposomes can rapidly fuse with the cellular plasma membrane and deliver the liposomal content directly to the cell cytosol.26 Csiszár and colleagues reported an efficient approach to deliver proteins using FLs.27 Attractive electrostatic interactions between the positively charged carriers and the negatively charged cargo resulted in effective proteoliposome formation. The liposomal membrane efficiently fused with the cellular plasma membrane upon contact and released the cargo to the cytoplasm without degradation. Proteins such as EGFP, Dendra2, and R-phycoerythrin were successfully transduced into mammalian cells with high efficiency. It should be noted that positively charged protein cargos cannot be delivered via this system because the repulsive interactions between the cargos and the similarly charged liposomes completely prevented the complex formation. Later, Kros and coworkers designed a system which minimized the interaction between the protein cargo and fusogenic lipid bilayer, thus overcoming the requirement for cargo charges.28 Cuboidal mesoporous silica nanoparticles (MSNs) encapsulated with cytochrome-C (cytC) were coated with a fusogenic lipid bilayer decorated with coiled-coil lipopeptides. Delivery of MSNs in the presence of various endocytosis inhibitors suggested that endocytosis was minimized and membrane fusion was the dominant mechanism of cellular uptake. Enhanced level of apoptosis demonstrated the intracellular release of functional cytC. However, delivery of large proteins such as antibodies may be difficult in such a system. Exosomes. Exosomes are natural cell-derived vesicles secreted by a variety of cell types and tissues, originated from internal endocytic compartments and multi-vesicular bodies (Figure 2B). They participate in intercellular communication by transporting macromolecules from one cell to another.29 The concept of using exosomes to encapsulate and transport exogenous macromolecules into the cytosol of living cells has been demonstrated.30-32 For example, Yim and coworkers recently reported a new approach (exosomes for protein loading via optically reversible proteinprotein interactions, or EXPLORs) to load cargo proteins into newly generated exosomes, followed by subsequently transporting them in recipient cells where the cargo release occurred upon exposure to blue light.32 Functional proteins including mCherry, Bax, super-repressor IkB protein and Cre enzyme were successfully delivered into the target cells (in vitro) and brain parenchymal cells (in vivo). Impressively, Cre recombinase-loaded EXPLORs showed



efficient and functional neuronal protein delivery following brain injection. Currently, however, the protein loading efficiency of the method is very limited, with an average of one or two cargo molecules contained in each particle of EXPLOR. Furthermore, the transfection efficiency also requires significant optimization before it can be applied to other intracellular proteins, such as transcription factors, signal transducers, and enzymes. Nanomotor. Nano/micromotors are nano/microscale devices which mimic natural biological motors (Figure 2C). They are built from a few nano/microscale components and designed to perform different types of mechanical movements in response to specific stimuli. Nano/microvehicles have demonstrated considerable promise for the delivery of a wide variety of cargoes (including proteins), to both intra- and extra-cellular spaces.33 In an earlier work, Fan and co-workers reported that protein-functionalized gold nanowires (AuNWs) could be propelled electrically to deliver and release conjugated cytokines such as tumor-necrosis factor-alpha (TNFa) to specific mammalian cells with subcellular resolution, leading to concomitant activation of canonical nuclear factor-kappaB (NF-kB) signaling.34 More recently, Wang’s group demonstrated an ultrasound (US)-propelled AuNW for oligonucleotide delivery.35 The nanomotor rapidly pierced and penetrated through cellular membranes and directly delivered interfering RNA (siRNA) to the cytosol, leading to an impressive 94% silencing of the target protein within a few minutes. Inspired by this, Wang and colleagues further applied US-propelled nanomotors to deliver a functional therapeutic protein, caspase-3 (CASP-3), into human gastric adenocarcinoma cells.36 US-propelled AuNWs were coated with a CASP-3-loaded pH-responsive polymer. The polymer coating protected the enzyme from premature release and inactivation in extracellular acidic microenvironment. The active and fast movement of these AuNWs under a US field facilitated their rapid cell internalization, and upon exposure to the neutral cytosolic environment, the polymer quickly dissolved, and CASP-3 was released, achieving 80% apoptosis of cancer cells within minutes. The same nanomotor approach was then readily applied to a Cas9-sgRNA complex in a GFP knockout experiment.37 The Cas9-sgRNA-loaded nanomotors displayed high geneediting efficiency with more than 80% GFP-knockout within 2 h of cell incubation. Nanoparticle-stabilized nanocapsules (NPSCs). In recent years, the Rotello group has developed a highly efficient strategy, nanoparticle-stabilized capsules (NPSCs), for direct delivery of functional proteins to the cytosol (Figure 2D).38-44 These nanocapsules were shown to be non-toxic to mammalian cells even after 24-h incubation. By forming a membrane fusion-like hydrophobic interaction with the cell membrane, the NPSCs facilitate the direct release of protein payload into the cytoplasm, thus avoiding endosomal sequestration. In 2013, Rotello and colleagues demonstrated the first example of using NPSCs to deliver GFP and CASP-3 intracellularly.39 Fully functional CASP-3 was transduced to HeLa cells and effectively induced apoptosis. Delivery of GFP demonstrated rapid release into the cytosol of HeLa

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cells by NPSCs. Further proof of delivery versatility was demonstrated through efficient subcellular targeting of a GFP fusion protein to the peroxisome. This report demonstrated the potential of the system to be used for both therapeutic and imaging applications. High-molecular-weight proteins can be transduced efficiently into cells via a NPSC complex of protein-GIPA (1-guanidino-2-(4-imidazole) propionic acid).41 GIPA was used as the terminal group of the AuNP ligand for optimum cytosolic delivery of payload proteins and their subsequent dissociation from the NPSC. Both dsRed (112 kD) and β-galactosidase (464 kD) showed effective cellular uptake while retaining their biological functions. These methods, however, are limited to proteins whose pI values are below 7. To further explore the potential of this protein-particle co-engineering strategy and minimize the optimization process needed for each protein cargo, the same group reported a general method featuring universal protein-nanoparticle complex formation.42 Proteins genetically tagged with an oligo-glutamic acid (E-tagged proteins) were allowed to self-assemble with arginine-functionalized AuNPs, generating hierarchical spherical nanoassemblies capable of intracellular protein delivery. The versatility of this method was demonstrated by using five proteins spanning a wide range of sizes, charges and functions, as well as through transduction to multiple cell types. It is noteworthy that such engineered proteins remained active after intracellular delivery, as demonstrated through the delivery of active Cre recombinase and Granzyme A. After the protein was trafficked into the cytosol, further subcellular translocation was achieved by the attached organelle-targeting moiety in the payload. For example, nuclear delivery of GFP through attachment of a nuclear localization signal (NLS) sequence40 or a benzyl boronate tag,44 has been achieved. In 2017, the same group reported the direct cytoplasmic/nuclear delivery of Cas9 protein and a guide RNA (sgRNA) through the co-engineering of Cas9 protein (with an N-terminal E-tag and a C-terminal NLS) and argininefunctionalized AuNPs.43 The nanoassemblies delivered both the protein and the RNA efficiently to the cytoplasm, where the NLS sequence further facilitated transportation of the cargos to the nucleus. The approach exhibited remarkable efficiencies in both protein delivery (∼90%) and concomitant gene-editing (∼30%).

4. BIOREVERSIBLE ESTERIFICATION Esterification is a common strategy employed in the design of prodrugs, in which the negatively charged carboxylic acid of an otherwise cell-impermenat drug is masked in the form of an ester, thereby decreasing the drug’s polarity and improving its cellular uptake.45 Built on a previous discovery that a diazo compound could be used to efficiently convert carboxylic acids to esters in an aqueous environment near neutral pH,46 Raines and co-workers extended the concept of esterification to proteins in order to improve their cell uptake efficiency.47 In a process referred to as protein bioesterification, the authors chemically modified surface-exposed aspartic acids and glutamic


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Figure 3. Intracellular protein delivery by bioesterification of GFP with diazo compounds 1-6. Reproduced from reference 47 with permission from American Chemical Society.

acids of a model protein, GFP, and subsequently showed its successful cytosolic delivery without the use of any other delivery agent. As shown in Figure 3, the cloaking of the negative charges on the solvent-exposed surface of GFP was performed with one of the diazo compounds (1-6); due to the increase in the hydrophobicity of the modified GFP, it was readily taken up by mammalian cells. In the cytosol, spontaneous ester hydrolysis catalyzed by endogenous esterases occurred, leading to regeneration of the original GFP. Remarkably, based on images of confocal laser scanning microscopy (CLSM) obtained from the intracellularly delivered GFP, the authors found the protein was distributed uniformly throughout the entire cell, with no sign of endolysosomal trapping. This bioreversible esterification method thus provides a novel means for direct cytosolic delivery of native proteins. Notwithstanding, its generality for efficient intracellular delivery of other proteins, e.g. functional enzymes and high-molecular-weight antibodies (~150 kDa), has yet to be investigated. Its exact cell-uptake mechanism remains to be further elucidated. Other issues that are commonly associated with chemical modifications of proteins, including protein denaturation and inactivation, will also have to be carefully addressed in future studies.

5. CELL-PENETRATING PEPTIDES Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are generally short peptides with a net positive charge at physiological pH. They have the ability to cross the cell membrane at low concentrations, thereby taking their cargos, bound electrostatically or covalently, with them into cells.48 Amongst the many types of CPPs that have been designed and synthesized, arginine-rich CPPs, which include HIV-1 Tat peptide and polyarginines, are the most extensively employed and studied.49 Since the two independent reports in 1988, that the human immunodeficiency virus type I (HIV-1) TAT protein readily entered cells,50,51 and later studies showing the cell-penetrating property of this protein was conferred by its cation-rich region (Tat49-57), many more extensive studies have been carried out to understand how the hydrophilic, polycationic CPPs could translocate across the

hydrophobic membrane.49,52-55 The mechanisms of CPPmediated intracellular cargo delivery are currently still under heated debate. Many lines of evidence supporting both energy-independent processes (direct permeation) and endocytosis-based internalization of CPPs have been reported, indicating CPPs are capable of entering mammalian cells via multiple mechanisms.49 Because cell internalization greatly depends on not only CPP itself (e.g. its peptide sequence and physicochemical properties), but also its cell-uptake conditions (e.g. local peptide concentration, local lipid composition and membrane potential),56 variation in any of these factors might influence the relative contribution of different uptake mechanisms. Furthermore, different from CPP itself, the CPP-cargo conjugate should be considered a unique molecule and therefore its cell-entry mechanism(s) also strongly depends on the physicochemical properties of the cargo.57,58 Hence, the overall efficiency of a specific CPP-cargo conjugate remains hard to predict.59 The mechanism and applications of CPPs have been extensively reviewed elsewhere.49,52,53,55-60 Here we will focus on discussion of direct membrane penetration and application in protein delivery. Direct membrane penetration of arginine-rich peptides. Arginine-rich peptides (RRPs) have been found to possess the ability to traverse biological membranes in a non-endocytic mode with immediate bioavailability throughout the cytosol and nucleus, if a peptide-specific threshold concentration was met.61 Early studies of CPPs have already provided experimental evidence for direct membrane transduction, since TAT peptide internalization occurred at 4 ºC, a condition that endocytosis was inhibited.62 The guanidinium groups on CPPs could form bidentate hydrogen bonds and electrostatic interactions with cell-surface anion groups, such as sulfate, phosphate and carboxylate moieties, which are usually present as part of proteoglycans, phospholipids or sialic acids. These interactions could promote cell-surface accumulation and subsequent internalization. Several translocation mechanisms have been proposed to account for CPP direct penetration, for example, inverted micelle formation, pore formation, the carpet-like model and the membrane-thinning model.57 Once the CPPs are translocated intracellularly, they could be detached from the membrane by another counterion exchange with intracellular anions. The Cardoso group has provided experimental evidence that deprotonated fatty acids could interact with guanidinium groups and facilitate transportation of arginine-rich peptides. The cell-membrane pH gradient was shown as a universal driving force that could stimulate translocation.61 The internalization mechanism depends on various factors such as peptide concentration and sequence, as well as lipid composition. If the translocation is too slow, the competing endocytosis occurs and CPPs end up getting trapped in endosomes. Generally, high CPP concentrations would promote direct penetration. The possibility of translocation could also be enhanced in the presence of hydrophobic counteranions in the membranes through ion-pair stabilizaotn.63,64 Screening of several hydrophobic counteranions identified



pyrenebutyrate as an efficient delivery additive.65,66 Fluorescently labelled R8 peptide could be directly translocated into the cytosol in the presence of pyrenebutyrate. Efficient cytosolic delivery of the peptide-fused enhanced green fluorescent protein (R8-EGFP) into rat primary neurons occurred within a few minutes.65 However this approach was not applicable in the presence of serum. Cyclic TAT Peptides. Several studies showed that, when CPPs were fused with large cargoes such as proteins, direct transduction through plasma membrane could be involved in the uptake of such protein complexes.56 It is noteworthy that this direct translocation is not a general phenomenon but may occur in some circumstances, and both the ttansduction mode and efficiency could greatly depend on the cargo size. This has limited the RRP-based non-endocytic delivery methods to mostly small cargos, such as small-molecule fluorophores, peptides and smallsized proteins.67 A recent study with HIV-Tat peptide showed that higher peptide structural rigidity could significantly enhance the transduction efficiency of arginine-rich peptides.68 Cyclic TAT showed better cellular uptake kinetics relative to their linear and more ﬂexible counterparts. In order to investigate whether a cyclic CPP could also enable cellular uptake of full-length proteins in a non-endocytic manner, Hackenberger et al. used alkyne-modified GFPs and azide-functionalized CPPs to form different cyclic and linear CPP-GFP conjugates.69 It was observed that cyclic CPP-GFP conjugates were internalized into live cells with immediate bioavailability in both the cytosol and the nucleus, whereas its linear counterpart did not confer GFP transduction. Based on these encouraging results, the approach was transferred to the design of antibody delivery (Figure 4).15 The authors presented a modular strategy to create functional cell-permeable nanobodies with efficient non-endocytic cellular uptake. Nanobodies were used instead of conventional full-length antibodies because a large cargo size would likely prohibit CPP-assisted membrane transduction. By making use of expressed protein ligation (EPL), the authors produced GFP-binding proteins (GFP nanobodies) site-specifically attached to a cyclic CPP peptide (cR10 in Figure 4A). It was shown that these cellpermeable nanobodies were capable of entering fibroblast cells and binding to intracellularly expressed GFP (Figure 4B). These CPP-nanobody conjugates could label and manipulate antigens and their interacting partners. This was demonstrated by the delivery of two GFP fusion nanobodies, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumor suppressor p53 to the nucleolus. Furthermore, cR10–nanobody constructs allowed transport of their respective antigens and antigen-coupled proteins, for example, GFPfused Mecp2, into the cells. As cR10 led to a nucleolar redistribution of the antigen, a cleavable version of the cR10– nanobody chimeras was prepared by using an intracellularly cleavable disulfide bond (Figure 4A). These conjugates were efficiently taken up by cells and shown to bind to their target GFP–PCNA fusion protein upon localization in the nucleus (Figure 4C).

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A

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cytosol

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Figure 4. Cell-penetrating peptides (CPPs) for cytosolic delivery of proteins. (A) The full-length nanobody was expressed as an intein–CBD fusion and subsequently reacted with a Cys-containing CPP through expressed protein ligation (EPL). (B) Schematic of cR10-mediated cellular uptake of a nanobody that binds intracellularly expressed GFP. (C) The cleavable cR10 moiety facilitates efficient cellular uptake and GFP binding but without nucleolar relocalization. Reproduced from reference 15 with permission from Springer Nature.

6. CELL-PENETRATING POLY(DISULFIDE)S The field of cell-penetrating poly(disulfide)s (CPDs) is rapidly emerging in recent years, making them highly promising candidates to complement other existing cellpenetrating molecules. As synthetic mimics of poly-arginine CPPs, in which the polypeptide backbone is replaced with poly(disulfide)s, CPDs undergo rapid degradation intracellularly upon cell uptake, a process catalyzed by endogenous glutathione (GSH), leading to spontaneous cargo release. Cytosolic degradation of CPDs effectively lowers their toxicity, which is a main problem of most CPPs. Perhaps one of the biggest advantages of CPDs is their endocytosis-independent cellular uptake mechanisms, which we will discuss in the following sections. Despite growing interests in the use of CPDs, their synthesis remains largely similar to what was originally reported, from monomers that already contain a disulfide.70 From the initial application in noncovalent polyplexes used for gene transfection, to the more recent expansion into the intracellular delivery of proteins and materials, the application of CPDs has been fueled by the seminal work of Matile et al., who first introduced the concept of substrateinitiated synthesis (siCPDs),71 followed by detailed studies of the relationship between the structure and cell-penetrating properties of CPDs,72 as well as their unique modeof-action in crossing the plasma membrane.73 The Yao group expanded the potential of CPD-assisted cytosolic delivery of proteins to achieve immediate bioavailability.17 This was made possible with the successful combination of


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polymer chemistry, bioorthogonal chemistry and protein chemistry. Substrate-initiated cell-penetrating poly(disulfide)s. In 2013, Matile’s group introduced substrate-initiated polymerization of cell-penetrating poly(disulfide)s (siCPDs).71 It was the first report of growing CPDs directly on substrates of free choice in situ under mild conditions. The inspiration for obtaining siCPDs stemmed from the finding that poly(disulfide)s can be grown directly on solid substrates by surface-initiated ring-opening disulfide-exchange polymerization. The synthesis of siCPDs (henceforth named CPDs) requires three components (Figure 5A): an initiator (I) (in this case the substrate), a monomer (M) and a terminator (T). The monomer contains a strained disulfide for ring-opening disulfide-exchange polymerization and a guanidinium cation to assure cellpenetrating activity. The formation of CPDs occurred rapidly in neutral pH at room temperature. By using CPDs made from a fluorophore-containing initiator, it was discovered some of the simplest yet most active CPDs, upon incubation with HeLa cells, could reach the cytosol in 5 min and underwent rapid depolymerization to release the native substrate.72 CPD depolymerization prevented continuing membrane-perturbing activities and eliminated toxicity at all tested concentrations (up to 10 μM). The authors next confirmed the cell uptake of CPDs was insensitive to common endocytosis inhibitors, but dependent weakly on temperature and strongly on a thiol blocker. Therefore, CPDs overcame two main limitations of most delivery approaches: endosomal trapping and cytotoxicity.73 It was subsequently discovered that CPDs achieved effective cell uptake through dynamic covalent disulfide exchange with thiols on cell surfaces and reach the cytosol by bypassing the endocytosis pathways (a more detailed discussion of CPD cell uptake mechanism was reviewed by Gasparini et al.74). Further studies showed that even the monomer itself, or its strain-promoted analogs such as diselenolanes and epidithiodiketopiperazines, could effectively enter cells as well.75-77 The unique cell-entry mechanism is thus in direct contrast with CPP-assisted protein delivery as earlier discussed; with increases in cargo sizes, most CPP-based methods tend to enter cells through endocytosis, eventually ending up in endolysosomal vesicles destined for degradation. Leveraging on the potential presented by CPDs, this platform was utilized to achieve intracellular delivery of proteins and their therapeutic applications.16-18,78,79 Based on biotin-streptavidin biotechnology, Matile’s group demonstrated non-covalent protein delivery strategy as a proof-of-concept;16 upon mixing with a CPD made from an initiator containing a biotin and fluorescein, streptavidin was shown to be efficiently delivered into HeLa cells, eventually reaching the cell nucleoli. CLSM images showed identical CPD localization with and without streptavidin, thereby suggesting that the unique mode-of-action of CPDs was not perturbed by the presence of large biomolecules. The same system was further modified for cytosolic delivery of quantum dots (QDs) with surface-functionalized streptavidin.78 Successful delivery of these QDs was observed (about 70 per cell on average); most of the CPD-

functionalized QDs were verified to reach the cytosol without being trapped in endosomal compartments or at the plasma by various independent methods. Upon further functionalization with biotinylated GFP or anti-GFP nanobodies, these QDs were shown to retain their fluorescence or binding properties, respectively, thus demonstrating the potential to deliver proteins in their native, functional state. Protein transduction with CPDs. In above examples, only biotinylated initiators were incorporated into CPD synthesis, and as a result cargo proteins needed to be conjugated to the CPD through a streptavidin adaptor, which is not desirable. During CPD synthesis, the need for millimolar concentrations of initiator/monomer/terminator, as well as the use of organic co-solvents, rendered it extremely difficult if not impossible for a protein cargo to be directly used (e.g. as an initiator or terminator). In order for the CPD strategy to be used as a general approach for effective cellular delivery of different functional proteins, it would be more practical to eliminate proteins altogether during the CPD polymerization process and instead conjugate CPDs with proteins through bioorthogonal reactions and other well-established protein chemistry. With this hypothesis in mind, two-step CPD-assisted approaches for protein delivery were developed (Figure 5A).17,79 A series of novel CPDs bearing different bioorthogonal/affinity tags (BiotinCPD, Ni-NTACPD, TzCPD and AOCPD) were designed and synthesized by using the corresponding initiators (Figure 5A). These resulting CPDs could readily bind to available protein cargos (including recombinant proteins and suitably modified antibodies) through bioorthogonal “conjugation”, either covalently or non-covalently, in a simple “mixand-go” protocol under mild conditions and used immediately for subsequent cell delivery. Protein delivery was observed after 1-h incubation with as low as 50 nM protein loading. Functional caspase-3 was successfully transduced into HeLa cells and efficiently induced apoptosis, indicating this strategy was compatible with delicate proteins. The successful intracellular delivery of functional enzymes, coupled with its ability to deliver therapeutic antibodies, suggested that this method could be universally applicable in the future for effective cytosolic delivery of many different protein biologics.17 In above studies, both non-covalent and covalent conjugations of CPDs were dependent on the presence of a suitable “tag” in the protein cargo (i.e., histidine or transcyclooctene (TCO) tags), which needed to be genetically or chemically introduced.17 Upon CPD depolymerization, these tags were still attached to the successfully delivered cargo, and such “artificial” modifications might affect protein activities and conformations. Therefore in a follow-up study, new CPDs were designed, and two complementary approaches were developed to conjugate CPDs with native proteins, transduce them across the cell plasma membrane and subsequently release them in their “native” functional



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(A)

(B)

Conjugation Chemistry

Covalent

Non-covalent • Avidin/Biotin

• ThioLinker • Chemoenzymatic

Interaction • (His)6-tag/Ni-NTA Interaction

• Traceless tagging • Oxime conjugation

(C) 1 & others

Tz CPD

I

II

Azo Monomer (1)

III

Released Protein

hypoxia

Figure 5. Overview of CPD-facilitated intracellular delivery of proteins. (A) Summary of newly developed initiators, monomer (M), terminator (T), the polymerization/depolymerization process of CPDs, and the various two-step approaches for “conjugation” of protein cargos. (B) Chemistry of traceless tagging and its spontaneous cleavage of the self-immolative linker. (C) Scheme showing the nanocapsule approach of CPD-facilitated protein delivery, including the preparation of CPDprotein@nanocapsule and its endocytosis-independent cell uptake (step I), endogenous GSH-assisted CPD depolymerization (step II), and hypoxia-triggered intracellular protein release (step III). (Boxed) nanocapsule degradation mechanism. Reproduced from references 17, 18 and 79, with permission from American Chemical Society and Wiley.

forms with immediate bioavailability (Figure 5A-B).79 The approaches, termed PTM-tagging and traceless-tagging, take advantage of naturally occurring “glycan” tags present in many eukaryotic proteins or the surface-abundant lysine residues present in all proteins, respectively, to label native proteins with CPDs. The traceless tagging in particular, was able to label both glycosylated and non-glycosylated proteins alike, and moreover, the resulting norbornene biorthogonal tag introduced on the labeled protein by the reagent NBL underwent self-immolating cleavage following intracellular CPD-assisted protein delivery, leading to spontaneous release of the functional, native protein (Figure 5B). NBL is a disulfide-containing bifunctional linker containing a primary amine-reactive p-nitrophenylcarbonate moiety and norbornene capable of bioorthogonal ligation to TzCPD. Similarly, the PTM-tagged glycoprotein, following intracellular delivery and CPD depolymerization, was essentially “native” with minimal changes to its modified glycan as well as the protein activity.

In contrast with direct CPD-conjugation of proteins described above, another recently developed method involves the encapsulation of proteins in a biodegradable organosilica shell (Figure 5C).18 The resulting nanocapsule was then surface-modified with CPDs which enabled efficient cell uptake followed by cytosolic nanocapsule disintegration to release the encapsulated protein in its native form. With this nanocapsule delivery system, it was shown that native proteins of different sizes, including therapeutic antibody Cetuximab™ (an FDA-approved drug) could be effectively delivered into mammalian cells in endocytosis-independent manner and released on-demand under hypoxic conditions. The hypoxia-responsive property of the nanocapsule was constructed by the addition of a bisorthosilicate-containing azo monomer (1 in Figure 5C) during the nanocapsule-forming step. Inside normal cells, the nanocapsule was completely stable, thus effectively shielding its encapsulated protein from the cytosolic milieu and minimizing degradation. By doping the silica coating of the nanocapsule with a fluorescent dye and a quencher, the protein nanocarrier was further endowed


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with a hypoxia-responsive fluorescence turn-on property. This protein delivery system thus provides a platform capable of stable encapsulation, efficient cellular uptake, ondemand cytosolic protein release, and endogenous imaging of cargo release/cell state. It is noteworthy that during the nanocapsule-forming process, the protein was exposed to relatively harsh conditions. While most antibodies were expected to remain stable under such conditions, it is likely that this method is not suitable for more delicate proteins. Given the prolonged time needed for the nanocapsule to degrade intracellularly (24 h), this strategy does not provide immediate bioavailability of the delivered protein. Instead, it focuses on targeted and on-demand cargo release. In addition to above examples of CPD-assisted protein delivery, the utility of CPDs in the delivery of other cargoes, for example, small-molecule drugs,17 antisense oligonucleotides80,81 and microRNAs (miRNAs),82 for detection and therapeutic purposes, has been successfully explored. More recently, a nanosensor made of MSNs coated with an anti-GSH monoclonal antibody and CPDs were shown to be able to detect intracellular protein glutathionylation in live mammalian cells.83 In all cases, the cargos were transduced into cytoplasm with high efficiency and minimal endosomal trapping. The major issue commonly associated with CPPs and other means of intracellular delivery has therefore been greatly alleviated.

7. CONCLUSION AND OUTLOOK

Protein therapy, when compared to other means of therapies, has provided the obvious advantages of high specificity and potency, as well as low oncogenic risks. Despite such benefits, there remain major challenges in efficient cytosolic delivery of proteins especially therapeutic antibodies. The most difficult hurdle has been the inability to release sufficient amount of functional cargos from endocytic pathways to their site-of-action. Therefore in this perspective, we have summarized recent advances in various types of protein delivery methods (Table 1), while specifically focusing on approaches that allow direct cytosolic protein delivery while bypassing endocytosis. All of the strategies described in this perspective have shown successful applications in cell culture context, but their utility in translational research has yet to be demonstrated. Each of these approaches currently still has limitations and challenges that will need to be overcome (Table 1). Moreover, in vivo delivery faces far more challenges than cellular uptake, such as biodistribution, pharmacokinetics, immune response and many others.8 It should be noted that the endocytosis-independent uptake mechanism for nearly all cytosolic delivery strategies summarizied in this perspective was established by using live-cell experiments including bioimaging, co-localization and endocytosis inhibition.15-1828,32,36,39,47,69,72-74 In recent years, most quantitative assays that enable direct measurements of cytosolic protein delivery have emerged.84-86 It’s our view that many of these novel protein

Table 1 Summary of different protein delivery methods described in this perspective. Delivery Methods Mechanism Physical Method Microinjection Membrane Puncture Electroporation

Protein Cargo TRIM21, antibody

Nanocarriers

EGFP, Dendra2, and R-phycoerythrin

Fusogenic liposomes

Membrane Fusion/Endocytosis

Cytochrome-C (encapsulated in MSN)

Exosomes Nanomotor

Membrane mCherry, Bax, super-repressor IkB Fusion/Endocytosis protein and Cre enzyme Membrane Penetration Tumor-necrosis factor-alpha (TNFa) Caspase-3 Cas9-sgRNA complex

Small Molecule Tag

Nanoparticlestabilized nanocapsules (NPSCs)

Membrane Fusion

Esterification

Diffusion

Cell-Penetrating Cyclic TAT Peptides (CPPs)

Cell-Penetrating Poly(disulfide)s (CPDs)

GFP and Caspase-3 dsRed and β-galactosidase GFP, Cre Recombinase, Granzyme A, Histone 2A, Prothymosin-α Cas9-sgRNA GFP

Membrane TransductionGFP Nanobody

SubstrateThiol-mediated initiated cellTranslocation penetrating poly(disulfide)s (siCPDs)

Streptavidin

Limitation Ref Low-throughput 21 Disruptive Specialized Instrumentation Limited utility for in vivo applications Positively charged proteins cannot 27 be delivered Restricted to proteins small enough 28 to be encapsulated in the pores of MSNs Complex and laborious procedure 32 required for carrier preparation Short actuation time 34 Possible toxicity to organisms or to 36 the environment 37 In vivo efficiency to be demonstrated Restricted to delivering recombinant proteins with E-tag Yet to be tested in functional therapeutic proteins or proteins with higher molecular weights Large cargo size would likely prohibit CPP-assisted membrane transduction; endosomal capture and toxicity In vivo efficiency to be demonstrated

39 41 42 43 47

69 15

16

GFP or biotinylated anti-GFP nanobodies, quantum dots (QDs) Avidin, BSA, BRD-4, Caspase-3

78

BSA, IgG, HRP, RNase A

79

BSA, RNase A, Cetuximab

18


17


delivery methods, especially the CPD-based strategies,16-18 could gain significant benefits from much closer scrutiny with such assays. Although there is no single solution to all systems, we have taken a much closer look at recent developments in CPD-assisted protein delivery methods. With their unique mechanisms and highly efficient cellular uptake on different types of protein cargos ranging from highmolecular-weight therapeutic monoclonal antibodies to delicate functional enzymes, we believe the CPD technologies hold great potentials in routine laboratory and future clinical applications. In the latter, there may be concerns of haptenization of cellular proteins and unwanted immune responses to neoantigen as a result of disulfide shuffling. Nevertheless looking ahead, we foresee this exciting field will continue to evolve at a rapid pace and the following are some of the most pressing challenges, in our own perspective, that must be met. First, new conjugation chemistries need to be developed for convenient and efficient attachment of CPDs to different therapeutic proteins. To date, both covalent and non-covalent chemistries are available for “conjugation” of native and recombinant proteins alike. For covalent conjugation, lysines, disulfides and glycans present in a native protein have been used successfully.17,79 Other site- and residue-specific protein modification chemistries could be explored to further expand such chemistry toolboxes.87-89 Similarly, other types of post-translational modifications,90 in addition to the “glycan” used in the PTM-tagging, may be investigated. All these new developments will allow multiple-site labeling, as well as attachment of other appendants besides CPDs, within the same protein cargo. For non-covalent CPD attachment, the current strategy of using either an streptavidin or (His)6-tag is not ideal, as it necessitates the use of recombinant proteins with genetic fusions. With our ongoing interest in intracellular delivery of therapeutic antibodies,18 we envisage a convenient yet potentially effective non-covalent approach to “conjugate” CPD to a native monoclonal antibody might be the use of well-known antibody-binding adaptors (i.e. protein A/G/L) pre-modified with CPDs. Such CPD-containing adaptors would be rendered cell-permeable, which upon mixing with a native antibody, may effectively facilitate cytosolic delivery of the antibody via formation of a binary complex. Second, CPD-facilitated organelle-specific protein delivery methods need to be established. With current methods of CPD delivery,17,18,79 the protein cargo stayed mostly in the cytoplasm and was unable to enter sub-cellular organelles such as mitochondria or nucleus, where many novel therapeutic targets, including those normally considered undruggable (e.g. transcription factors), reside. Organelle-specific targeting by small-molecule drugs are well-known to show improved therapeutic efficacies.91 We expect organelle-specific delivery of therapeutic antibodies (and other proteins) will hold great potential in further expanding the popularity of antibody-based drugs. This conceivably could be accomplished by clever implementation of different conjugation chemistries as mentioned above, to attach CPDs and organelle-targeting modalities at different sites within the same cargo.

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Finally, new cell type-specific, on-demand protein delivery methods need to be discovered. Our already-established biodegradable nanocapsules have provided the first glimpse of such systems.18 But the slow release of the encapsulated cargo calls for urgent development of fast-release novel nanocarriers that can respond to different disease and cellular signals. A variety of chemistries are already available for small-molecule prodrugs and drug-carrying responsive polymers,92,93 and we anticipate future adaptation of some of them will fuel the development of different types of protein-encapsulated nanocapsules capable of on-demand cytosolic protein delivery under different endogenous and exogenous stimuli. With the continuous evolution of different disciplines in chemistry, biology and medicine, we foresee proteinbased therapy will be propelled to even greater heights in the coming years.

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

ACKNOWLEDGMENT This work was financially supported by the Synthetic Biology Research & Development Programme (SBP) of National Research Foundation (NRF), the GSK-EDB Trust Fund (R-143000-688-592) and MOE-T1 (R-143-000-694-114) of Singapore, the National Natural Science Foundation of China (81672508, 61505076) and Jiangsu Provincial Foundation for Distinguished Young Scholars (BK20170041).

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TOC:

Physical methods

Nanocarriers

Cell-penetrating peptides Cell-penetrating poly(disulfide)s

Non-endocytic Pathway

Cytoplasm nucleus

13 ACS Paragon Plus Environment


Figure 2 830x581mm (192 x 192 DPI)


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Figure 3 516x293mm (192 x 192 DPI)



Figure 4 773x602mm (192 x 192 DPI)


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Figure 5 972x675mm (192 x 192 DPI)



Graphical TOC 944x369mm (192 x 192 DPI)


Page 18 of 19

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Table 1 985x705mm (192 x 192 DPI)


Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins

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