Delivery Of Functional Proteins Research Articles

Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

Non-covalent delivery of native proteins and peptides by phenylboronic cell-penetrating poly(disulfide)s

Poly(disulfide)s have been proposed as delivery carriers, yet their design for native protein delivery without covalent conjugation remain elusive and challenging. Here, we present a type of poly(disulfide)s randomly copolymerized from cell-penetrating cyclic five-membered disulfide (CFMD) monomer (M1) and phenylboronic CFMD monomer (M2) by ring-opening polymerization. The resulted poly(disulfide)s can directly complex a broad range of native, unmodified proteins and peptides via multiple non-covalent forces, regardless of their chemical structure, molecular weight and isoelectric point. The complexation between poly(disulfide)s and proteins can be predominantly internalized by cells via strain-promoted, thiol-mediated translocation, bypassing the classical endocytic pathway. The degradation of the poly(disulfide) is induced by rich intracellular glutathione, thereby timely releasing protein or peptide cargoes in their active form and minimize the cytotoxicity of the carrier. Of note, the surface coating of poly(disulfide) complexes by hyaluronic acid enables the systemic delivery of functional proteins, demonstrating their therapeutic potentials in vivo.

Boronic Acid-Linked Cell-Penetrating Peptide for Protein Delivery.

Studying functional protein delivery into live cells is important, ranging from fundamental research to therapeutics. Cell-penetrating peptides (CPPs) are known to deliver proteins with applauded efficacy and have gained importance for applications in protein therapeutics and exploration of versatile cellular mechanisms. The primary aim of the work is to design a CPP as a tool and delivery vehicle for macromolecules, including proteins. In this work, boronic acid-linked cyclic deca arginine (cR10) is reported as an efficient CPP that exhibited 3-fold higher delivery of chemically synthesized ubiquitin (Ub) than pristine cR10-linked Ub, examined with live U2OS cells. As a futuristic plan, an artificial intelligence machine learning-based rationale has been designed and proposed.

An optogenetic method for the controlled release of single molecules

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 μM) underlines potential interest of this nanoplatform for biomedical applications.

Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins.

The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure-activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

Guanidyl-Rich Poly(β Amino Ester)s for Universal Functional Cytosolic Protein Delivery and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 Ribonucleoprotein Based Gene Editing.

Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(β amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.

Functional small peptides for enhanced protein delivery, solubility, and secretion in microbial biotechnology.

In microbial biotechnology, there is a constant demand for functional peptides to give new functionality to engineered proteins to address problems such as direct delivery of functional proteins into bacterial cells, enhanced protein solubility during the expression of recombinant proteins, and efficient protein secretion from bacteria. To tackle these critical issues, we selected three types of functional small peptides: cell-penetrating peptides (CPPs) enable the delivery of diverse cargoes into bacterial cytoplasm for a variety of purposes, protein-solubilizing peptide tags demonstrate remarkable efficiency in solubilizing recombinant proteins without folding interference, and signal peptides play a key role in enabling the secretion of recombinant proteins from bacterial cells. In this review, we introduced these three functional small peptides that offer effective solutions to address emerging problems in microbial biotechnology. Additionally, we summarized various engineering efforts aimed at enhancing the activity and performance of these peptides, thereby providing valuable insights into their potential for further applications.

Inside Cover: Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins (Angew. Chem. Int. Ed. 38/2023)

Inside Cover: Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins (Angew. Chem. Int. Ed. 38/2023)

Innentitelbild: Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins (Angew. Chem. 38/2023)

Innentitelbild: Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins (Angew. Chem. 38/2023)

Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins

AbstractCurrently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome‐to‐cytosol delivery based on an extension of the “histidine switch” principle. By substituting the Arg/Lys residues in cationic cell‐penetrating peptides (CPPs) with histidine, we obtained peptides with pH‐dependent membrane‐perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16‐residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody‐targeted delivery of diverse protein cargoes—including the pro‐apoptotic protein BID (BH3‐interacting domain death agonist) and Cre recombinase—into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab‐hsLMWP‐BID fusion conferred strong anti‐tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins.

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Two birds one stone: β-fluoropyrrolyl-cysteine SNAr chemistry enabling functional porphyrin bioconjugation.

Bioconjugation, a synthetic tool that endows small molecules with biocompatibility and target specificity through covalent attachment of a biomolecule, holds promise for next-generation diagnosis or therapy. Besides the establishment of chemical bonding, such chemical modification concurrently allows alteration of the physicochemical properties of small molecules, but this has been paid less attention in designing novel bioconjugates. Here, we report a "two birds one stone" methodology for irreversible porphyrin bioconjugation based on β-fluoropyrrolyl-cysteine SNAr chemistry, in which the β-fluorine of porphyrin is selectively replaced by a cysteine in either peptides or proteins to generate novel β-peptidyl/proteic porphyrins. Notably, due to the distinct electronic nature between fluorine and sulfur, such replacement makes the Q band red-shift to the near-infrared region (NIR, >700 nm). This facilitates intersystem crossing (ISC) to enhance the triplet population and thus singlet oxygen production. This new methodology features water tolerance, a fast reaction time (15 min), good chemo-selectivity, and broad substrate scope, including various peptides and proteins under mild conditions. To demonstrate its potential, we applied porphyrin β-bioconjugates in several scenarios, including (1) cytosolic delivery of functional proteins, (2) metabolic glycan labeling, (3) caspase-3 detection, and (4) tumor-targeting phototheranostics.

Biomimetic Mineralization of Iron-Fumarate Nanoparticles for Protective Encapsulation and Intracellular Delivery of Proteins.

Biomimetic mineralization of proteins and nucleic acids into hybrid metal−organic nanoparticles allows for protection and cellular delivery of these sensitive and generally membrane-impermeable biomolecules. Although the concept is not necessarily restricted to zeolitic imidazolate frameworks (ZIFs), so far reports about intracellular delivery of functional proteins have focused on ZIF structures. Here, we present a green room-temperature synthesis of amorphous iron-fumarate nanoparticles under mildly acidic conditions in water to encapsulate bovine serum albumin (BSA), horseradish peroxidase (HRP), green fluorescent protein (GFP), and Cas9/sgRNA ribonucleoproteins (RNPs). The synthesis conditions preserve the activity of enzymatic model proteins and the resulting nanoparticles deliver functional HRP and Cas9 RNPs into cells. Incorporation into the iron-fumarate nanoparticles preserves and protects the activity of RNPs composed of the acid-sensitive Cas9 protein and hydrolytically labile RNA even during exposure to pH 3.5 and storage for 2 months at 4 °C, which are conditions that strongly impair the functionality of unprotected RNPs. Thus, the biomimetic mineralization into iron-fumarate nanoparticles presents a versatile platform for the delivery of biomolecules and protects them from degradation during storage under challenging conditions.

Efficient transdermal delivery of functional protein cargoes by a hydrophobic peptide MTD 1067

The skin has a protective barrier against the external environment, making the transdermal delivery of active macromolecules very difficult. Cell-penetrating peptides (CPPs) have been accepted as useful delivery tools owing to their high transduction efficiency and low cytotoxicity. In this study, we evaluated the hydrophobic peptide, macromolecule transduction domain 1067 (MTD 1067) as a CPP for the transdermal delivery of protein cargoes of various sizes, including growth hormone-releasing hexapeptide-6 (GHRP-6), a truncated form of insulin-like growth factor-I (des(1-3)IGF-I), and platelet-derived growth factor BB (PDGF-BB). The MTD 1067-conjugated GHRP-6 (MTD-GHRP-6) was chemically synthesized, whereas the MTD 1067-conjugated des(1-3)IGF-I and PDGF-BB proteins (MTD-des(1-3)IGF-I and MTD-PDGF-BB) were generated as recombinant proteins. All the MTD 1067-conjugated cargoes exhibited biological activities identical or improved when compared to those of the original cargoes. The analysis of confocal microscopy images showed that MTD-GHRP-6, MTD-des(1-3)IGF-I, and MTD-PDGF-BB were detected at 4.4-, 18.8-, and 32.9-times higher levels in the dermis, respectively, compared to the control group without MTD. Furthermore, the MTD 1067-conjugated cargoes did not show cytotoxicity. Altogether, our data demonstrate the potential of MTD 1067 conjugation in developing functional macromolecules for cosmetics and drugs with enhanced transdermal permeability.

Specific Activation of Photosensitizer with Extrinsic Enzyme for Precisive Photodynamic Therapy.

Delivery of functional proteins into the intracellular space has been a challenging task that could lead to a myriad of therapeutic applications. We report herein a novel bioconjugation strategy for enzyme modification and selective delivery into cancer cells for lock-and-key-type activation of photosensitizers. Using a bifunctional linker containing a bis(bromomethyl)phenyl group and an o-phthalaldehyde moiety, it could induce cyclization of the peptide sequence Ac-NH-CRGDfC-CONH2 through site-specific dibenzylation with the two cysteine residues and further coupling with β-galactosidase via the phthalaldehyde-amine capture reaction. This facile two-step one-pot procedure enabled the preparation of cyclic RGD-modified β-galactosidase readily, which could be internalized selectively into αvβ3 integrin-overexpressed cancer cells. Upon encountering an intrinsically quenched distyryl boron dipyrromethene-based photosensitizer conjugated with a galactose moiety through a self-immolative linker inside the cells, the extrinsic enzyme induced specific cleavage of the β-galactosidic bond followed by self-immolation to release an activated derivative, thereby restoring the photodynamic activities and causing cell death effectively. The high specificity of this extrinsic enzyme-activated photosensitizing system was also demonstrated in vivo using nude mice bearing an αvβ3 integrin-positive U87-MG tumor. The specific activation at the tumor site resulted in lighting up and complete eradication of the tumor upon laser irradiation, while by using the native β-galactosidase, the effects were largely reduced. In contrast to the conventional activation using intrinsic enzymes, this extrinsic enzyme activatable approach can further minimize the nonspecific activation toward precisive photodynamic therapy.

Covalent Labeling-Mass Spectrometry Provides a Molecular Understanding of Noncovalent Polymer-Protein Complexation.

The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.

Engineering of Extracellular Vesicles for Small Molecule-Regulated Cargo Loading and Cytoplasmic Delivery of Bioactive Proteins.

Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain to sort bioactive proteins into EVs using the FRB-FKBP system. When CD81, a typical EV marker protein, and POI were fused with FKBP and FRB, respectively, rapamycin induced the binding of these proteins through the FKBP-FRB interaction and recruited the POIs into EVs. The released EVs, displaying the virus-derived membrane fusion protein, delivered the POI cargo into recipient cells and their functionality in the recipient cells was confirmed. Furthermore, we demonstrated that CD81 could be replaced with other EV-enriched proteins, such as CD63 or HIV Gag. Thus, the FRB-FKBP system enables the delivery of functional proteins and paves the way for EV-based protein delivery platforms.

Intracellular Delivery of Functional Proteins

Leveraging proteins as therapeutics has revolutionized the way we treat diseases. Recent advances in protein biologics and antibody engineering represent promising new treatment strategies for addressing complex etiologies. Thus far, these advances have been restricted to only accessible extracellular proteins, leaving a significant portion of the proteome undruggable. Efficient intracellular delivery of proteins would reveal uncharted therapeutic avenues and cell surface engineering opportunities. This presentation describes combinatorial strategies towards delivering proteins to the cytosol. By modifying a protein of interest through surface esterification or functionalization with a cell penetrating peptide we have observed robust intracellular delivery. In this presentation, I will first describe the development of a functional assay using a model protein to assess the efficacy and localization of delivery agents. Applying these findings to HaloTag has demonstrated the generality of our approach. I will show that our delivery strategy has enabled stable cell surface engineering. This study highlights that multiple intracellular delivery strategies can be used synergistically to enable cytosolic access with functionally active protein/biologic cargo.

Carbamoylated Guanidine-Containing Polymers for Non-Covalent Functional Protein Delivery in Serum-Containing Media.

Despite the high potential of controlling cellular processes and treating various diseases by intracellularly delivered proteins, current delivery systems exhibit poor efficiency due to poor serum stability, cellular entry, and cytosolic availability of proteins. Here, we report a novel functional group, phenyl carbamoylated guanidine (Ph-CG), that greatly enhances the delivery efficiency to various types of cells. Owing to the substantially lowered pKa , the hydrophobic Ph-CG offers optimized inter-macromolecular interactions via enhanced hydrogen-bonding and hydrophobic interactions. The coplanarity of Ph-CG also leads to the better intracellular entry of protein complexes. Intracellularly delivered apoptosis-inducing enzymes and antibodies significantly induce cell viability inhibitions in a serum-containing medium. The newly developed Ph-CG can be introduced to various existing carriers, leading to the realization of future therapeutic protein delivery.