Efficient Cytosolic Delivery Research Articles

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo.

AbstractThere is enormous interest in strategies to efficiently traffic biologics–proteins, nucleic acids, and complexes thereof–into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic knowledge regarding the bioinorganic chemistry of zinc(II) coordination in small proteins, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity upon arrival in the cytosol. Delivery of the pH-sensitive protein dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins.Abstract Figure

Abstract A027: Enhancing pancreatic cancer chemotherapy through photochemical internalisation

Abstract Introduction Pancreatic cancer is the deadliest common cancer. Its poor prognosis is associated with its complex biology and lack of early detection strategies. Although much progress has been made in finding more effective therapeutic strategies, pancreatic tumours remain difficult to treat. A major reason why chemotherapy is relatively ineffective against this type of cancer is inefficient delivery of drugs to their target sites combined with multidrug resistance. Our aim is to use the technique called Photochemical Internalisation (PCI), a light-triggered intracellular drug delivery method which combines low dose Photodynamic Therapy (PDT) with chemotherapy, to induce efficient cytosolic delivery of therapeutic compounds to their specific subcellular targets. Methodology Treatment outcomes using saporin (a type I ribosome-inactivating protein) and gemcitabine as monotherapies, or in combination with a light-activated photosensitizer (the porphyrin TPPS2a), were thoroughly analysed in both established pancreatic cancer cell lines and patient-derived xenograft (PDX) models. The efficacy of these treatments was assessed in both 2D and 3D tumour models through various cell viability assays, including MTT, crystal violet staining, and neutral red staining. Additionally, molecular techniques such as western blotting, protein arrays, and flow cytometry were employed to determine the effects on cell viability and to elucidate changes in signalling pathways leading to the therapeutic response. Results In this study, we observed minimal cytotoxicity induced by saporin, gemcitabine or TPPS2a + light monotherapies. However, PCI synergistically enhanced saporin and gemcitabine cytotoxicity (p<0.001) using very low concentrations in all tumour models. Moreover, we determined the mechanism of cell death triggered by these protocols. PCI induced endosomal disruption following light excitation and apoptosis leading to caspases 3/7 activation, in a time dependent manner. Conclusions Overall, our findings demonstrate the potential of PCI to enhance the efficacy of cancer chemotherapy for pancreatic cancer using lower doses than in monotherapy and open a new window for future translational studies. By using low doses of light therapy, we have found a promising avenue for future studies that could eventually lead to better treatments for this challenging disease. Citation Format: Maria Rosado, Ismahan Mahamed, Andres Garcia-Sampedro, Sandy MacRoberts, Pål Selbo, Stephen Pereira, Pilar Acedo. Enhancing pancreatic cancer chemotherapy through photochemical internalisation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A027.

Efficient cytosolic delivery of proteins enabled by modular fusion protein

The development of protein-based biotechnology and drugs holds tremendous potential for various applications. However, the effective intracellular delivery of natural proteins remains a challenge due to their inability to spontaneously penetrate the cell membrane. In this study, we have developed a modular fusion protein sequence that overcomes this limitation and enables efficient cytoplasmic protein delivery . This modular fusion protein we designed has demonstrated efficient lysosomal escape ability, allowing for successful delivery of various protein cargoes into the cytoplasm of different cell types. Importantly, the fusion with the mCherry tag does not affect the biological activity of the protein. The fusion protein can still exert its catalytic activity when delivered into cells. We also designed a fluorescent protein probe capable of precisely targeting lysosomes, providing powerful fluorescent probe tools for intracellular localization and tracking in biomedical research. This approach contributes to the advancement of protein drug development and opens up new possibilities for therapeutic interventions.

An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

Rational design of diblock copolymer enables efficient cytosolic protein delivery

Polymer-mediated cytosolic protein delivery demonstrates a promising strategy for the development of protein therapeutics. Here, we propose a new designed diblock copolymer which realizes efficient cytosolic protein delivery both in vitro and in vivo. The polymer contains one protein-binding block composed of phenylboronic acid (PBA) and N-(3-dimethylaminopropyl) (DMAP) pendant units for protein binding and endosomal escape, respectively, followed by the response to ATP enriched in the cytosol which triggers the protein release. The other block is PEG designed to improve particle size control and circulation in vivo. By optimizing the block composition, sequence and length of the copolymer, the optimal one (BP20) was identified with the binding block containing 20 units of both PBA and DMAP, randomly distributed along the chain. When mixed with proteins, the BP20 forms stable nanoparticles and mediates efficient cytosolic delivery of a wide range of proteins including enzymes, toxic proteins and CRISPR/Cas9 ribonucleoproteins (RNP), to various cell lines. The PEG block, especially when further modified with folic acid (FA), enables tumor-targeted delivery of Saporin in vivo, which significantly suppresses the tumor growth. Our results shall inspire the design of novel polymeric vehicles with robust capability for cytosolic protein delivery, which holds great potential for both biological research and therapeutic applications.

A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.

The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.

Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye.

The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.

Liquid crystalline inverted lipid phases encapsulating siRNA enhance lipid nanoparticle mediated transfection

Efficient cytosolic delivery of RNA molecules remains a formidable barrier for RNA therapeutic strategies. Lipid nanoparticles (LNPs) serve as state-of-the-art carriers that can deliver RNA molecules intracellularly, as exemplified by the recent implementation of several vaccines against SARS-CoV-2. Using a bottom-up rational design approach, we assemble LNPs that contain programmable lipid phases encapsulating small interfering RNA (siRNA). A combination of cryogenic transmission electron microscopy, cryogenic electron tomography and small-angle X-ray scattering reveals that we can form inverse hexagonal structures, which are present in a liquid crystalline nature within the LNP core. Comparison with lamellar LNPs reveals that the presence of inverse hexagonal phases enhances the intracellular silencing efficiency over lamellar structures. We then demonstrate that lamellar LNPs exhibit an in situ transition from a lamellar to inverse hexagonal phase upon interaction with anionic membranes, whereas LNPs containing pre-programmed liquid crystalline hexagonal phases bypass this transition for a more efficient one-step delivery mechanism, explaining the increased silencing effect. This rational design of LNPs with defined lipid structures aids in the understanding of the nano-bio interface and adds substantial value for LNP design, optimization and use.

Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy

We report a family of luminescent lanthanide bioprobes featuring a two-photon absorbing antenna and a dimer of the TAT cell penetrating peptides. They penetrate live cells efficiently and allow two-photon microscopy with commercial microscopes.

Fusogenic Cell-Derived nanocarriers for cytosolic delivery of cargo inside living cells

A surface-engineered cell-derived nanocarrier was developed for efficient cytosolic delivery of encapsulated biologically active molecules inside living cells. Thus, a combination of aromatic-labeled and cationic lipids, instrumental in providing fusogenic properties, was intercalated into the biomimetic shell of self-assembled nanocarriers formed from cell membrane extracts. The nanocarriers were loaded, as a proof of concept, with either bisbenzimide molecules, a fluorescently labeled dextran polymer, the bicyclic heptapeptide phalloidin, fluorescently labeled polystyrene nanoparticles or a ribonucleoprotein complex (Cas9/sgRNA). The demonstrated nanocarrieŕs fusogenic behavior relies on the fusogen-like properties imparted by the intercalated exogenous lipids, which allows for circumventing lysosomal storage, thereby leading to efficient delivery into the cytosolic milieu where cargo regains function.

Dual-responsive bioconjugates bearing a bifunctional adaptor for robust cytosolic peptide delivery.

Peptide drugs have been successfully used for the treatment of various diseases. However, it is still challenging to develop therapeutic peptides working on intracellular targets due to their poor membrane permeability. Here, we proposed a type of dual-responsive bioconjugates bearing a heterobifunctional adaptor containing both aldehyde and catechol moieties for efficient cytosolic peptide delivery. Hydrazine-terminated cargo peptides were tagged to a boronated dendrimer with the help of the adaptor via dynamic acylhydrazone and catechol‑boronate linkages. The bioconjugates efficiently delivered peptides with distinct physicochemical properties into various cells, and could release the cargo peptides triggered by intracellular reactive oxygen species and endolysosomal acidity, restoring the biofunctions of delivered peptides. In addition, the designed complexes efficiently delivered a pro-apoptotic peptide into osteosarcoma cancer cells and successfully inhibited the tumor growth both in vitro and in vivo. This study provides a universal and efficient platform for cytosolic therapeutic peptide delivery to intracellular targets for treating various diseases.

Efficient intracellular and in vivo delivery of toxin proteins by a ROS-responsive polymer for cancer therapy.

Rational design of efficient cytosolic protein delivery carriers holds enormous promise for biotherapeutics development. Several delivery systems have been developed during the past decades, while tailoring the balance between extracellular protein binding and intracellular cargo release is still challenging. In this study, we synthesized a series of oxygen-sensitive reactive polymers, rich in boron, by radical polymerization and post-modification for cytosolic protein delivery in vitro and in vivo. The introduction of boronate building blocks into the polymer scaffold significantly enhanced its protein binding affinity, and the polymer/protein complexes with high stability were obtained by tailoring the molecular ratios between the boronate ligands and the amine groups. The lead material screened from the polymer library exhibited efficient protein delivery efficacy that can release cargo proteins in cytosol in a reactive oxygen species responsive manner, which enables intracellular delivery of proteins with maintained bioactivity. In addition, the polymer-based nanoformulations efficiently delivered saporin, a toxin protein, into osteosarcoma cells and tumor tissues, and exhibited high therapeutic efficacy in an osteosarcoma mouse model. The synthesized polymer in this study can be developed as a promising nanocarrier for cytosolic delivery of protein therapeutics to treat a variety of diseases.

Direct Cytosolic Delivery of Citraconylated Proteins.

Current intracellular protein delivery strategies face the challenge of endosomal entrapment and consequent degradation of protein cargo. Methods to efficiently deliver proteins directly to the cytosol have the potential to overcome this hurdle. Here, we report the use of a straightforward approach of protein modification using citraconic anhydride to impart an overall negative charge on the proteins, enabling them to assemble with positively charged nano vectors. This strategy uses anhydride-modified proteins to electrostatically form polymer-protein nanocomposites with a cationic guanidinium-functionalized polymer. These supramolecular self-assemblies demonstrated the efficient cytosolic delivery of modified proteins through a membrane fusion-like mechanism. This approach was validated on five cell lines and seven proteins as cargo. Retention of protein function was confirmed through efficient cell killing via the intracellular enzymatic activity of RNase A. This platform provides a versatile, straightforward, and single-step method of protein modification and efficient direct cytosolic protein delivery.

Histidine-based coordinative polymers for efficient intracellular protein delivery via enhanced protein binding, cellular uptake, and endosomal escape.

Polymers are one of the most promising protein delivery carriers; however, their applications are hindered by low delivery efficacy owing to their undesirable performance in protein binding, cellular uptake and endosomal escape. Here, we designed a series of histidine-based coordinative polymers for efficient intracellular protein delivery. Coordination of metal ions such as Ni2+, Zn2+, and Cu2+ with histidine residues on a polymer greatly improved its performance in protein binding, complex stability, cellular uptake and endosomal escape, therefore achieving highly improved protein delivery efficacy. Among the coordinative polymers, the Zn2+-coordinated one exhibited the highest cellular uptake, while the Cu2+-coordinated one exhibited the highest endosomal escape. The Ni2+-coordinated polymer formed large-sized aggregates with cargo proteins and showed insufficient protein release after endocytosis. The results obtained in this study provided new insight into the development of coordinative polymer-based protein delivery systems.

Improved intracellular delivery of exosomes by surface modification with fluorinated peptide dendrimers for promoting angiogenesis and migration of HUVECs.

Exosomes exhibit great potential as novel therapeutics for tissue regeneration, including cell migration and angiogenesis. However, the limited intracellular delivery efficiency of exosomes might reduce their biological effects. Here, exosomes secreted by adipose-derived mesenchymal stem cells were recombined with fluorinated peptide dendrimers (FPG3) to form the fluorine-engineered exosomes (exo@FPG3), which was intended to promote the cytosolic release and the biological function of exosomes. The mass ratio of FPG3 to exosomes at 5 was used to investigate its cellular uptake efficiency and bioactivity in HUVECs, as the charge of exo@FPG3 tended to be stable even more FPG3 was applied. It was found that exo@FPG3 could enter HUVECs through a variety of pathways, in which the clathrin-mediated endocytosis played an important role. Compared with exosomes modified with peptide dendrimers (exo@PG3) and exosomes alone, the cellular uptake efficiency of exo@FPG3 was significantly increased. Moreover, exo@FPG3 significantly enhanced the angiogenesis and migration of HUVECs in vitro as compared to exo@PG3 and exosomes. It is concluded that surface fluorine modification of exosomes with FPG3 is conducive to the cellular uptake and bioactivity of the exosome, which provides a novel strategy for engineered exosomes to enhance the biological effects of exosome-based drug delivery.

One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.

Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.

Benzaldehyde-tethered fluorous tags for cytosolic delivery of bioactive peptides.

Development of intracellular delivery systems for bioactive peptides remains challenging. Herein, we report a facile strategy to address this issue by conjugating peptides with benzaldehyde-tethered fluorous tags to generate dynamic peptide amphiphiles via a hydrazone bond for efficient cytosolic delivery. Those dynamic peptide fluoroamphiphiles could self-assemble into nanoparticles that readily cross the cell membrane. Using this strategy, several bioactive peptides were efficiently internalized by cancer cells and released into the cytosol to exert their biological functions, which showed much higher efficacies than non-fluorous lipids and cell penetrating peptide decorated peptides. Moreover, the fluorous tagged proapoptotic peptide was able to efficiently inhibit tumor growth in vivo. This report provides a new family of fluorous tags based on benzaldehyde for efficient cytosolic peptide delivery.

Combined Self-Assembled iRGD Polymersomes for Effective Targeted siRNA Anti-Tumor Therapy.

iRGD is usually used as a motif to modify siRNA-nanodelivery vectors to improve tumor-targeting and penetration. However, most of the modifications are realized by covalent conjugation, which normally requires complex preparation processes possibly with low conjugation efficiency and yield, and might lower its bioactivity. To avoid this, here, we presented an alternative physical method to decorate iRGD on nanopolymersomes via facile self-assembly in water. siVEGF was chosen as a siRNA model, and lipopolysaccharide-amine nanopolymersomes (NPs), an efficient cytosolic delivery vector developed by our group, was used as an original vector. By successively incubating siVEGF with NPs, followed by adding iRGD, a siVEGF-loaded NPs functionalized with iRGD (siRNA/iRGD-NPs) was obtained. The properties of iRGD-NPs or siRNA/iRGD-NPs were evaluated in vitro and in vivo. iRGD is efficiently introduced onto NPs with different amounts, which can be precisely controlled by the feeding ratio. The introduced iRGD keeps tumor-targeting and -penetrating bioactivity, which endows iRGD-NPs with ~100% of tumor-cell uptake and excellent tumor spheroid-penetration, and thus iRGD-NPs can efficiently deliver siVEGF to significantly inhibit angiogenesis in zebrafish and tumor growth in nude mice bearing breast cancer without obvious toxicity. This study provides a facile physical method to decorate nanodelivery vectors with iRGD for effective targeted siRNA anti-tumor therapy.

Reactive oxygen species-responsive branched poly (β-amino ester) with robust efficiency for cytosolic protein delivery

Protein therapy targeting the intracellular machinery holds great potentials for disease treatment, and therefore, effective cytosolic protein delivery technologies are highly demanded. Herein, we developed reactive oxygen species (ROS)-degradable, branched poly(β-amino ester) (PBAE) with built-in phenylboronic acid (PBA) in the backbone and terminal-pendent arginine for the efficient cytosolic protein delivery. The PBAE could form stable and cell-ingestible nanocomplexes (NCs) with proteins via electrostatic interaction, nitrogen-boronate (N-B) coordination, and hydrogen bonding, while it can be degraded into small segments by the over-produced H2O2 in tumor cells to enable cytoplasmic protein release. As thus, PBAE exhibited high efficiency in delivering varieties of proteins with distinct molecular weights (12.4–430 kDa) and isoelectric points (4.7-10.5) into tumor cells, including enzymes, toxins, and antibodies. Moreover, PBAE mediated efficient delivery of saporin into tumor cells in vivo, provoking pronounced anti-tumor outcomes. This study provides a robust and versatile platform for cytosolic protein delivery, and the elaborately tailored PBAE may find promising applications for protein-based biological research and disease management. Statement of significanceCytosolic delivery of native proteins holds great therapeutic potentials, which however, is limited by the lack of robust delivery carriers that can simultaneously feature strong protein encapsulation yet effective intracellular protein release. Herein, ROS-degradable, branched poly(β-amino ester) (PBAE) with backbone-embedded phenylboronic acid (PBA) and terminal-pendent arginine was developed to synchronize these two processes. PBA and arginine moieties allowed PBAE to encapsulate proteins via N-B coordination, electrostatic interaction, hydrogen bonding, and salt bridging, while PBA could be oxidized by over-produced H2O2 inside cancer cells to trigger PBAE degradation and intracellular protein release. As thus, the top-performing PBAE mediated efficient cytosolic delivery of various proteins including enzymes, toxins, and antibodies. This study provides a powerful platform for cytosolic protein delivery, and may find promising utilities toward intracellular protein therapy against cancer and other diseases such as inflammation.