Intracellular Delivery Of Functional Proteins Research Articles

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.

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.

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.

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.

"Clickable" ZIF-8 for Cell-Type-Specific Delivery of Functional Proteins.

Protein therapy provides a powerful alternative to small-molecule-based therapy, especially on cellular targets that are normally considered to be less druggable. Intracellular protein delivery, in particular, in a cell-type-specific manner, is still highly challenging. At present, few general strategies are available for the robust and selective intracellular delivery of proteins. In this Letter, by using zeolitic imidazolate framework-8 (ZIF-8) as protein-encapsulated nanoparticles and simultaneous doping with norbornene-modified imidazole (MIM-Nor), followed by surface attachment of the resulting nanoparticles with cetuximab (Cet) through click chemistry, we successfully synthesized Cet@protein@ZIF-8N, which was subsequently used for the selective intracellular delivery of functional proteins to epidermal-growth-factor-receptor (EGFR)-overexpressed cells. Both in-cell and in vivo experiments proved that Cet@RNase A@ZIF-8N can effectively deliver RNase A with the retention of selective inhibition. Furthermore, the same strategy was successfully applied to cell-type-specific gene editing through the delivery of a Cas9/sgRNA complex to knockdown the endogenous expression of glutathione peroxidase (GPX4), a key protein in ferroptosis. Our new system thus has potential implications in future cancer treatment and the development of precision medicine.

Magnetically Navigated Protein Transduction In Vivo using Iron Oxide-Nanogel Chaperone Hybrid.

Systems for "protein transduction," intracellular delivery of functional proteins, are needed to address deliverability challenges of protein therapeutics. However, in vivo protein transduction remains challenging because of instability in serum, extracellular protease digestion and rapid excretion from the bloodstream. Here, a magnetically guided in vivo protein transduction using magnetic nanogel chaperone (MC) composed of iron oxide nanoparticles and a polysaccharide nanogel, a protein carrier inspired by "catch and release" mechanisms of molecular chaperones is demonstrated. The MC system enables efficient delivery of anti-cancer proteins, saporin and RNaseA, into cultured tumor lines and inhibits cell proliferation, mainly via apoptosis. Magnetic in vivo protein transduction via intravenous whole body administration is demonstrated in a fibrosarcoma model. By in vivo optical imaging, MC accumulated in tumor tissues under magnetic field three times more than without irradiation. With subcutaneous injection, saporin is delivered by MC to the cytoplasm in magnetically targeted tissues. In an oral cancer model, MC-delivered magnetically targeted saporin decreased tumor volume without significant body weight changes and no regrowth of tumor at 3 months after complete regression. Protein transduction with MC shows promise for cancer therapeutics and, potentially, for regenerative medicine and other biomedical applications.

Engineered Interactions with Mesoporous Silica Facilitate Intracellular Delivery of Proteins and Gene Editing.

Intracellular delivery of functional proteins is a promising, but challenging, strategy for many therapeutic applications. Here, we report a new methodology that overcomes drawbacks of traditional mesoporous silica (MSi) particles for protein delivery. We hypothesize that engineering enhancement in interactions between proteins and delivery vehicles can facilitate efficient encapsulation and intracellular delivery. In this strategy, surface lysines in proteins were modified with a self-immolative linker containing a terminal boronic acid for stimulus-induced reversibility in functionalization. The boronic acid moiety serves to efficiently interact with amine-functionalized MSi through dative and electrostatic interactions. We show that proteins of different sizes and isoelectric points can be quantitatively encapsulated into MSi, even at low protein concentrations. We also show that the proteins can be efficiently delivered into cells with retention of activity. Utility of this approach is further demonstrated with gene editing in cells, through the delivery of a CRISPR/Cas9 complex.

High-efficiency protein delivery into transfection-recalcitrant cell types.

Intracellular delivery of functional proteins is of great interest forbasic biological research as well as for clinical applications. Transfection is the most commonly used method, however, itis not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

AbstractDirect and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound‐powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA‐loaded nanomotors to directly penetrate through the plasma membrane of GFP‐expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA‐loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor‐based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.

Silica nanospheres entrapped with ultra-small luminescent crystals for protein delivery

Constructing smart nano-systems for intracellular delivery of functional proteins has been endeavored for diverse biomedical applications, but suffered daunting challenges. Herein silica nanospheres entrapped with photoluminescent CaF2:Tm,Yb nanocrystals were synthesized and decorated with amino molecules for protein delivery. Amino-modified nanospheres presented high protein loading capacity and sustained release phenomenon. The photoluminescence of particles highly corresponded to protein release progress. The preliminary in-vitro study confirmed markedly enhanced cell up-taking efficiency of protein molecules with the nanocomposite developed.

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications. Statement of SignificanceRegenerative medicine aims to reform functional biological tissues by controlling cell behaviour. Growth factors (GFs) are soluble cues presented to cells in spatio-temporal gradients and play important roles programming cell fate and gene expression. The efficient transduction of cells by GET (Glycosaminoglycan-enhanced transducing)-tagged transcription factors (TFs) can be used to by-pass GF-stimulation and directly program cells. For the first time we demonstrate diffusion of GET proteins generate stable protein transduction gradients. We demonstrated the feasibility of creating spatio-temporal gradients of GET-MyoD and show differential programing of myogenic differentiation. We believe that GET could provide a powerful tool to program cell behaviour using gradients of recombinant proteins that allow tissue generation directly by programming gene expression with TFs.

Intracellular Delivery of Functional Proteins and Native Drugs by Cell-Penetrating Poly(disulfide)s.

The efficient delivery of bioactive compounds into cells is a major challenge in drug discovery. We report herein the development of novel methods for intracellular delivery of functional proteins (including antibodies) and native small-molecule drugs by making use of cell-penetrating poly(disulfide)s (CPDs). CPDs were recently shown to be rapidly taken up by mammalian cells in endocytosis-independent pathways, but their applications for delivery of proteins and native small-molecule drugs have not been demonstrated. With our newly developed, CPD-assisted approaches, rapid and "bioorthogonal" loading of cargos was carried out with pre-synthesized CPDs, in two steps and in a matter of minutes under aqueous conditions. The resulting CPD-cargo conjugates were used immediately for subsequent cell delivery studies. With the versatility and flexibility of these methods, we further showed that they could be used for immediate delivery of a variety of functional cargos with minimum chemical and genetic manipulations. The minimal cell cytotoxicity of these CPDs and their cargo-loaded conjugates further highlights the unique advantage of this new cell-transduction method over other existing strategies and ensures that our entire delivery protocol is compatible with subsequent live-cell experiments and biological studies.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins.

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Engineering the nanoparticle-protein interface for cancer therapeutics.

Intracellular delivery of functional proteins using nanoparticles can be a game-changing approach for cancer therapy. However, cytosolic release of functional protein is still a major challenge. In addition, formation of protein corona on the surface of the nanoparticles can also alter the behavior of the nanoparticles. Here, we will review recent strategies for protein delivery into the cell. Finally we will discuss the issue of protein corona formation in light of nanoparticle-protein interactions.

PH-sensitive carbonate apatite as an intracellular protein transporter

The transfer of specific proteins into living cells to enable the regulation of cell function or the tracking of the intracellular distribution of proteins is a desirable objective for offering a potential alternative to gene therapy. Here, protein/carbonate apatite complexes were successfully fabricated for intracellular delivery of functional proteins since the carbonate apatite being highly water solubility under an acidic condition could easily be dissolved in endosomes following endocytosis, thus releasing the electrostatically associated proteins in cytoplasm. In this study, we characterized protein/carbonate apatite complexes as an intracellular protein delivery system and we checked intracellular delivery of proteins by carbonate apatite nanoparticles in vitro. Fluorescently-labeled bovine serum albumin as a model protein was effectively delivered into nearly 100% of HeLa cells by the simple addition of protein/carbonate apatite complexes to the cells. Confocal microscopic imaging suggested the endosomal release of protein delivered with carbonate apatite. And intracellularly delivered ß-galactosidase did not lose its enzymatic activity. These results suggested that intracellular delivery system of protein using pH-sensitive carbonate apatite carrier with a very simple procedure will be a highly effective method to the biological and clinical researches.

Efficient intracellular delivery of functional proteins using cationic polymer core/shell nanoparticles

Cationic core/shell nanoparticles self-assembled from biodegradable, cationic and amphiphilic copolymer poly{ N-methyldietheneamine sebacate)- co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate}, P(MDS- co-CES), were fabricated and employed to deliver lectin A-chain, an anticancer glycoprotein. Lectin A-chain was efficiently bound onto the surfaces of the nanoparticles at high mass ratios of nanoparticles to lectin A-chain. The nanoparticle/lectin A-chain complexes had an average size of approximately 150 nm with zeta potential of about +30 mV at the mass ratio of 50 or above while the BioPorter/lectin A-chain complexes had a larger particle size and relatively lower zeta potential (150 nm vs. 455 nm; +30 mV vs. +20 mV). Therefore, the cellular uptake of nanoparticle/lectin A-chain complexes was much greater than that of BioPorter/lectin A-chain complexes. The results obtained from cytotoxicity tests show that lectin A-chain delivered by the nanoparticles was significantly more toxic against MDA-MB-231, HeLa, HepG2 and 4T1 cell lines when compared to BioPorter, and IC50 of lectin A-chain delivered by the nanoparticles was 0.2, 0.5, 10 and 50 mg/l, respectively, while that of lectin A-chain delivered by BioPorter was higher than 100 mg/l in all cell lines tested. These nano-sized particles may provide an efficient approach for intracellular delivery of biologically active proteins.