Search

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets. Recently, extracellular vesicles (EVs) have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility. However, fusion of protein of interest to a scaffold represents a widely used strategy for cargo enrichment in EVs, which could compromise the stability and functionality of cargo. Herein, we report intracellular delivery via EV-based approach (IDEA) that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold. As a proof-of-concept, we applied the IDEA to deliver cyclic GMP-AMP synthase (cGAS), an innate immune sensor. The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models. Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo. Mechanistically, scRNA-seq demonstrated that cGAS EVs mediated significant remodeling of intratumoral microenvironment, revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu. Collectively, IDEA, as a universal and facile strategy, can be applied to expand and advance the development of protein-based therapeutics. [ABSTRACT FROM AUTHOR]

Hydrogen‐bonded organic frameworks (HOFs) are porous nanomaterials that offer exceptional biocompatibility and versatility for integrating proteins for biomedical applications. This minireview concisely discusses recent advancements in the chemistry and functionality of protein‐HOF interfaces. It particularly focuses on strategic methodologies, such as the careful selection of building blocks and the genetic engineering of proteins, to facilitate protein‐HOF interactions. We examine the role of enzyme encapsulation within HOFs, highlighting its capability to preserve enzyme function, a crucial aspect for applications in biosensing and disease diagnosis. Moreover, we discuss the emerging utility of nanoscale HOFs for intracellular protein delivery, illustrating their applicability as nanoreactors for intracellular catalysis and neuroprotective biorthogonal catalysis within cellular compartments. We highlight the significant advancement of designing biodegradable HOFs tailored for cytosolic protein delivery, underscoring their promising application in targeted cancer therapies. Finally, we provide a perspective viewpoint on the design of biocompatible protein‐HOF assemblies, underlining their promising prospects in drug delivery, disease diagnosis, and broader biomedical applications. [ABSTRACT FROM AUTHOR]

Cellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo‐responsive phase‐separating fluorescent molecule (PPFM) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid‐liquid phase separation (LLPS), which disintegrate upon photoirradiation with a 405 nm light‐emitting diode (LED) lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo‐responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload. [ABSTRACT FROM AUTHOR]

Pulmonary delivery offers a non-invasive route for the administration of biotherapeutics. In this context, understanding and control of a transport into, and across cellular barriers is central to the design of delivery systems. Here, we report our study on receptor mediated delivery of protein cargo by a formulation comprising s ub -300 nm sized non-covalent protein complexes with biotin-conjugated PEG-poly(glutamic acid) (biotin-PEG 2k -b- GA 10) and PEG 2k -b- GA 30 copolymers blend as targeting and complexing functionalities. Designed complexes achieve intracellular delivery of the cargo in lung derived A549 epithelial cells in vitro via sodium-dependent multivitamin transporter (biotin receptor). We further show that biotin receptor driven endocytosis preferentially involves dynamin- and caveolae-dependent vesicular internalization, switching the transport pathway away from predominantly clathrin-dependent entry of free protein. Significantly for a protective intracellular delivery of biotherapeutics based on non-covalent complexation with polymeric excipients, the study provides evidence of intracellular presence of the complexing copolymer; demonstrated exploiting biotin in biotin-PEG 2k -b- GA 10 copolymer as a tag for binding with fluorescently labelled avidin. Moreover, analysis of intracellular localization of constitutive species shortly following cellular internalization suggests a co-localization of biotin-PEG 2k -b- GA 10 copolymer and protein constitutive species. The study demonstrates intracellular delivery of biotin targeted non-covalent complexes with a protein cargo, the result with important implications in a design of enabling technology platforms for protective, receptor mediated intracellular delivery of biotherapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

The treatment of malignant bone tumors by chemotherapeutics often receives poor therapeutic response due to the specific physiological bone environment, and thus calls for the development of new therapeutic options. Here, we reported a bone-targeted protein nanomedicine for this purpose. Saporin, a toxin protein, was co-assembled with a boronated polymer for intracellular protein delivery, and the formed nanoparticles were further coated with an anionic polymer poly (aspartic acid) to shield the positive charges on nanoparticles and provide the bone targeting function. The prepared ternary complex nanoparticles showed high bone accumulation both in vitro and in vivo, and could reverse the surface charge property from negative to positive after locating at tumor site triggered by tumor extracellular acidity. The boronated polymer in the de-shielded nanoparticles further promote intracellular delivery of saporin into tumor cells, exerting the anticancer activity of saporin by inactivation of ribosomes. As a result, the bone-targeted and saporin-loaded nanomedicine could kill cancer cells at a low saporin dose, and efficiently prevented the progression of osteosarcoma xenograft tumors and bone metastatic breast cancer in vivo. This study provides a facile and promising strategy to develop protein-based nanomedicines for the treatment of malignant bone tumors.

Modifying cyclic cell‐penetrating deca‐arginine (cR10) peptides with 4‐(4‐dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time‐lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL‐R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure–uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell‐uptake efficiency when conjugated to mono‐Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling. [ABSTRACT FROM AUTHOR]

Intracellular delivery of proteins has largely been relying on cationic nanoparticles to induce efficient endosome escape, which, however, poses serious concerns on the inflammatory and cytotoxic effects. Herein, a versatile noncationic nano biohybrid platform is introduced for efficient cytosolic protein delivery by utilizing a nano-confined biocatalytic reaction. This platform is constructed by co-immobilizing glucose oxidase (GOx) and the target protein into nanoscale hydrogen-bonded organic frameworks (HOFs). The biocatalytic reaction of nano-confined GOx is leveraged to induce controlled perturbation of intracellular redox homeostasis by sustained hydrogen peroxide (H 2 O 2 ) production and diminishing the flux of the pentose phosphate pathway (PPP). This in turn induces the endosome escape of nanobiohybrids. Concomitantly, GOx-mediated hypoxia leads to overexpression of azo reductase that initiated the materials' self-destruction for releasing target proteins. These biological effects collectively induce highly efficient cytosolic protein delivery. The versatility of this delivery platform is further demonstrated for various types of proteins, different protein loading approaches (in situ immobilization or post-adsorption), and in multiple cell lines. Finally, the protein delivery efficiency and biosafety are demonstrated in a tumor-bearing mouse model. This nanohybrid system opens up new avenues for intracellular protein delivery and is expected to be extensively applicable for a broad range of biomolecuels., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

The accumulation of abnormal protein deposits known as amyloid-β (Aβ) plaques contributes to the development and progression of Alzheimer's disease. Aggregated Aβ exacerbates oxidative stress by stimulating the production of reactive oxygen species (ROS) in a detrimental feedback loop. 8-Hydroxyquinoline (8-HQ) is recognized for its ability to inhibit or reverse Aβ aggregation and reduce neurotoxicity. Here, an 8-HQ-based polymer, DHQ, was developed to combat Aβ-mediated neurotoxicity by delivering an antioxidant enzyme. DHQ efficiently delivers superoxide dismutase into targeted cells, thereby downregulating the intracellular ROS level. Additionally, the polymer effectively inhibits the fibrillization of three proteins involved in fibrosis, β-lactoglobulin (BLG), insulin, and Aβ1-40, at nanomolar concentrations. Cell culture models demonstrated that DHQ reduces ROS levels induced by Aβ1-40 aggregation, rescuing cell viability and preventing apoptosis. Intracellular delivery of SOD further enhanced the ability to maintain the ROS homeostasis. This polymer offers a multifaceted approach to treating diseases associated with amyloidosis.

The delivery of functional proteins, including antibodies, into cells opens up many opportunities to regulate cellular events, with significant implications for studies in chemical biology and therapeutics. The inside of cells is isolated from the outside by the cell membrane. The hydrophilic nature of proteins prevents direct permeation of proteins through the cell membrane by passive diffusion. Therefore, delivery routes using endocytic uptake followed by endosomal escape have been explored. Alternatively, delivery concepts using transient permeabilization of cell membranes or effective promotion of endocytic uptake and endosomal escape using modified membrane-lytic peptides have been reported in recent years. Non-canonical protein delivery concepts, such as the use of liquid droplets or coacervates, have also been proposed. This review highlights some of the topics in peptide-mediated intracellular protein delivery. [ABSTRACT FROM AUTHOR]

Yu-Ling Lin,1,* Chia-Hung Chen,2,* Yen-Ku Liu,2 Tse-Hung Huang,3–6,* Nu-Man Tsai,7,8 Shey-Cherng Tzou,2,9 Kuang-Wen Liao2,9–11 1Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC; 2Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC; 3Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan, ROC; 4School of Traditional Chinese Medicine, Chang Gung University, Taoyuan, Taiwan, ROC; 5Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan, ROC; 6School of Nursing, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan, ROC; 7School of Medical Laboratory and Biotechnology, Chung Shan Medical University; 8Department of Pathology and Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC; 9Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan, ROC; 10Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC; 11Center for Intelligent Drug Systems and Smart Bio-devices, National Chiao Tung University, Hsinchu, Taiwan, ROC *These authors contributed equally to this work Background: Protein or peptide drugs are emerging therapeutics for treating human diseases. However, current protein drugs are typically limited to acting on extracellular/cell membrane components associated with the diseases, while intracellular delivery of recombinant proteins replaces or replenishes faulty/missing proteins and remains inadequate. In this study, we developed a convenient and efficient intracellular protein delivery vehicle. Materials and methods: A cationic liposomal polyethylenimine and polyethylene glycol complex (LPPC) was developed to noncovalently capture proteins for protein transfer into cells via endocytosis. β-glucuronidase (βG) was used in vitro and in vivo as a model enzyme to demonstrate the enzymatic activity of the intracellular transport of a protein. Results: The endocytosed protein/LPPC complexes escaped from lysosomes, and the bound protein dissociated from LPPC in the cytosol. The enzymatic activity of βG was well preserved after intracellular delivery in vitro and in vivo. Conclusion: Using LPPC as an intracellular protein transporter for protein therapeutics, we illustrated that LPPC may be an effective and convenient tool for studying diseases and developing therapeutics. Keywords: liposomal polyethylenimine and polyethylene glycol complex, LPPC, intracellular protein delivery, endocytosis, enhanced green fluorescent protein, EGFP, β-glucuronidase

Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH‐sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein‐modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self‐proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase‐3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild‐type caspase‐3. Only in the presence of cytosolic GFP strands 1–10 will the tagged caspase‐3 generate fluorescence to signal a non‐endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomally‐entrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp‐3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge–property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death. [ABSTRACT FROM AUTHOR]

Protein-based therapeutics have unique therapeutic potential due to their specificity, potency, and low toxicity. The vast majority of intracellular applications of proteins require access to the cytosol. Direct entry to the cytosol is challenging due to the impermeability of the cell membrane to proteins. As a result, multiple strategies have focused on endocytic uptake of proteins. Endosomally entrapped cargo, however, can have very low escape efficiency, with protein degradation occurring in acidic endolysosomal compartments. In this review, we briefly discuss endosomal escape strategies and review the strategy of cell membrane fusion, a recent strategy for direct delivery of proteins into the cell cytoplasm. Intracellular protein delivery is a powerful tool for development of protein-based therapeutics and is a challenging task due to cell membrane impermeability to large biomolecules and endosomal entrapment. Most delivery strategies rely on endosomal uptake of the carrier. Endosomally entrapped cargo generally exhibit low escape efficiency leading to eventual degradation. Numerous nanocarriers such as lipid, inorganic, and polymeric NPs have been developed to induce endosomal escape and deliver therapeutic proteins intracellularly. However, there remains considerable room for improvement in release efficiency. Development of nonendosomal uptake strategies including nanocarrier-mediated cell membrane fusion provides a promising alternative for delivering proteins directly to the cytosol. Improvements in protein delivery efficiency have enabled the translation of intracellular protein therapeutics towards the clinic. [ABSTRACT FROM AUTHOR]

The targeted delivery of therapeutic agents is a promising approach to enhance the efficacy and reduce the toxicity of cancer treatments. Understanding the intracellular endocytic mechanisms of a cell penetrating peptide (CPP) in an acidic environment is important for targeted delivery of macromolecules to tumours. In this study, we constructed a pH-sensitive CPP-based delivery system for the intracellular delivery of macromolecules. A pH-sensitive CPP, HBHAc, was fused with a model protein, enhanced green fluorescent protein (EGFP), through recombinant DNA technology. We found that is essential that negatively charged proteoglycans on the cell surface interact with HBHAc-EGFP prior to the cellular uptake of HBHAc-EGFP. The uptake was significantly restricted at 4 °C under pH conditions of both 6.5 and 7.5. The increased positive charge of HBHAc-EGFP under the acidic condition leads to a pH-dependent cellular uptake, and we observed that the internalisation of HBHAc-EGFP was significantly higher at pH 6.5 than at pH 7.5 (p <.05). Thus, with pH-sensitive activity, HBHAc is expected to improve tumour-targeted intracellular protein delivery. Moreover, our findings provide a new insight that the endocytic pathway may change under different pH conditions and suggest that this unique phenomenon benefits pH-sensitive drug delivery for tumour therapy. [ABSTRACT FROM AUTHOR]

Intracellular protein delivery systems have great potential in the fields of therapeutics development and biomedical research. However, targeted delivery, passing through the cell membrane without damaging the cells, and escaping from endosomal entrapment of endocytosed molecular cargos are major challenges of the system. Here, we present a novel intracellular protein delivery system based on modularly engineered botulinum neurotoxin type A (BoNT/A). LH N A domain, consisting of light chain and endosomal escape machinery of BoNT/A, was genetically fused with SpyCatcher (SC) and EGFR targeting affibody (EGFRAfb) to create SC-LH N A-EGFRAfb, a target-specific and protein cargo-switchable BoNT/A-based intracellular protein delivery platform. SC-LH N A-EGFRAfb was purely purified in large quantities, efficiently ligated with multiple ST-fused protein cargos individually, generating a variety of protein cargo-containing intracellular delivery complexes, and successfully delivered ligated protein cargos into the cytosol of target cells via receptor-mediated endocytosis, followed by endosomal escape and subsequent cytosolic delivery. SC-LH N A-EGFRAfb enhanced intracellular delivery efficiency of protein toxin, gelonin, by approximately 100-fold, highlighting the crucial roles of EGFRAfb and LH N A domain as a targeting ligand and an endosomal escape machinery, respectively, in the delivery process. The BoNT-based plug-and-deliverable intracellular protein delivery system has the potential to expand its applications in protein therapeutics and manipulating cellular processes. [Display omitted] • The LH N A domain of botulinum neurotoxin (BoNT/A) serves as a delivery platform. • SC-LH N A-EGFRAfb is created by combining LH N A, SpyCatcher, & EGFR binding affibody. • A variety of protein cargos are generated by genetically fusing SpyTag (ST). • ST-fused proteins are delivered into target cells by SC-LH N A-EGFRAfb on demand. • SC-LH N A-EGFRAfb is both target-specific and capable of plug-and-deliver proteins. [ABSTRACT FROM AUTHOR]

Nanocarrier-based cytoplasmic protein delivery offers opportunities to develop protein therapeutics; however, many delivery systems are positively charged, causing severe toxic effects. For enhanced therapeutics, it is also of great importance to design nanocarriers with intrinsic bioactivity that can be integrated with protein drugs due to the limited bioactivity of proteins alone for disease treatment. We report here a protein delivery system based on anionic phosphite-terminated phosphorus dendrimers with intrinsic anti-inflammatory activity. A phosphorus dendrimer termed AK-137 with optimized anti-inflammatory activity was selected to complex proteins through various physical interactions. Model proteins such as bovine serum albumin, ribonuclease A, ovalbumin, and fibronectin (FN) can be transfected into cells to exert their respective functions, including cancer cell apoptosis, dendritic cell maturation, or macrophage immunomodulation. Particularly, the constructed AK-137@FN nanocomplexes display powerful therapeutic effects in acute lung injury and acute gout arthritis models by integrating the anti-inflammatory activity of both the carrier and protein. The developed anionic phosphite-terminated phosphorus dendrimers may be employed as a universal carrier for protein delivery and particularly utilized to deliver proteins and fight different inflammatory diseases with enhanced therapeutic efficacy.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

Proteins are essential for life, as they participate in all vital processes in the body. In the past decade, delivery of active proteins to specific cells and organs has attracted increasing interest. However, most proteins cannot enter the cytoplasm due to the cell membrane acting as a natural barrier. To overcome this challenge, various proteins have been engineered to acquire cell-penetrating capacity by mimicking or modifying natural shuttling proteins. In this review, we provide an overview of the different types of engineered cell-penetrating proteins such as cell-penetrating peptides, supercharged proteins, receptor-binding proteins, and bacterial toxins. We also discuss some strategies for improving endosomal escape such as pore formation, the proton sponge effect, and hijacking intracellular trafficking pathways. Finally, we introduce some novel methods and technologies for designing and detecting engineered cell-penetrating proteins.

Abstract The design of efficient and universal carriers to transport proteins into cells is highly desirable in the development of biotherapeutics. However, intracellular protein delivery remains a major challenge owing to the multiple barriers in protein delivery process, such as protein encapsulation, endocytosis, endosomal escape and intracellular protein release. Recently, it was reported that fluorination on polymers is beneficial for their self-assembly and intracellular trafficking. We hypothesize the fluoropolymers can be used to encapsulate proteins and developed as a new class of carriers to break down the barriers during intracellular protein delivery. In this study, we successfully discovered an efficient and non-toxic polymer for protein delivery in a library of fluoropolymers. The lead material A6-2 in the library efficiently transported various proteins including bovine serum albumin, β-galactosidase, saporin and a cyclic hendecapeptide into the cytosol of living cells with minimal cytotoxicity. More importantly, activities of delivered proteins and peptides were maintained after delivery. The A6-2/saporin complex was further coated with a hyaluronic acid shell, and the yielding nanoparticle efficiently suppressed tumor growth in a breast cancer model by targeted protein delivery. This study provides a new insight into the design of efficient and biocompatible polymers for protein delivery. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Intracellular protein delivery is of significance for cellular protein analysis and therapeutic development, but remains challenging technically. Herein, we report a general and highly potent strategy for intracellular protein delivery based on commercially available cationic lipids. In this strategy, a designed double branching poly-glutamate tag is site-specifically attached onto the C-terminal of protein cargos via expressed protein ligation (EPL), which mediates the entrapment of proteins into cationic liposomes driven by electrostatic interaction. The resultant protein-lipid complexes can enter into cytosol with a high efficiency even at the low protein concentration while maintaining protein's biological activity. [ABSTRACT FROM AUTHOR]

Abstract: Efficient intracellular delivery of protein drugs and tumor‐specific activation of protein functions are critical toward anti‐cancer protein therapy. However, an omnipotent protein delivery system that can harmonize the complicated systemic barriers as well as spatiotemporally manipulate protein function is lacking. Herein, an “all‐functions‐in‐one” nanocarrier doped with photosensitizer (PS) is developed and coupled with reactive oxygen species (ROS)‐responsive, reversible protein engineering to realize cancer‐targeted protein delivery, and spatiotemporal manipulation of protein activities using long‐wavelength visible light (635 nm) at low power density (5 mW cm−2). Particularly, RNase A is caged with H2O2‐cleavable phenylboronic acid to form 4‐nitrophenyl 4‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)benzyl carbonate (NBC)‐modified RNase (RNBC), which is encapsulated in acid‐degradable, ketal‐crosslinked PEI (KPEI)‐based nanocomplexes (NCs) coated with PS‐modified hyaluronic acid (HA). Such NCs harmonize the critical processes for protein delivery, wherein HA coating renders NCs with long blood circulation and cancer cell targeting, and KPEI enables endosomal escape as well as acid‐triggered intracellular RNBC release. Tumor‐specific light irradiation generates H2O2 to kill cancer cells and restore the protein activity, thus achieving synergistic anti‐cancer efficacy. It is the first time to photomanipulate protein functions by coupling ROS‐cleavable protein caging with PS‐mediated ROS generation, and the “all‐functions‐in‐one” nanocarrier represents a promising example for the programmed anti‐cancer protein delivery. [ABSTRACT FROM AUTHOR]

Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI)-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy. [ABSTRACT FROM AUTHOR]

Intracellular delivery of protein therapeutics by cationic polymer vehicles is an emerging technique that is, however, encountering poor stability, high cytotoxicity and non-specific cell uptake. Herein, we present a facile strategy to optimize the protein-polycation complexes by encapsulating with linear-dendritic telodendrimers. The telodendrimers with well-defined structures enable the rational design and integration of multiple functionalities for efficient encapsulation of the protein-polycation complexes by multivalent and hybrid supramolecular interactions to produce sub-20 nm nanoparticles. This strategy not only reduces the polycation-associated cytotoxicity and hemolytic activity, but also eliminates the aggregation and non-specific binding of polycations to other biomacromolecules. Moreover, the telodendrimers dissociate readily from the complexes during the cellular uptake process, which restores the capability of polycations for intracellular protein delivery. This strategy overcomes the limitations of polycationic vectors for intracellular delivery of protein therapeutics. [ABSTRACT FROM AUTHOR]

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 CaF 2 :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. [ABSTRACT FROM AUTHOR]

Despite enormous efforts, achieving efficacious levels of proteins inside mammalian cells remains one of the greatest challenges in biologics-based drug discovery and development. The inability of proteins to readily cross biological membranes precludes access to the wealth of intracellular targets and applications that lie within mammalian cells. Existing methods of delivery commonly suffer from an inability to target specific cells and tissues, poor endosomal escape, and limited in vivo efficacy. The aim of the present commentary is to highlight the potential of certain classes of bacterial toxins, which naturally deliver a large protein into the cytosolic compartment of target cells after binding a host cell-surface receptor with high affinity, as robust protein delivery platforms. We review the progress made in recent years toward demonstrating the utility of these systems at delivering a wide variety of protein cargo, with special attention paid to three distinct toxin-based platforms. We contend that with recent advances in protein deimmunization strategies, bacterial toxins are poised to introduce biologics into the inner sanctum of cells and treat a wealth of heretofore untreatable diseases with a new generation of therapeutics. [ABSTRACT FROM AUTHOR]

There is ample evidence that many proteins or regions of proteins lack a well-defined folded structure under native-like conditions. These are called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). Whether this intrinsic disorder is also their main structural characteristic in living cells has been a matter of intense debate. The structural analysis of IDPs became an important challenge also because of their involvement in a plethora of human diseases, which made IDPs attractive targets for therapeutic development. Therefore, biophysical approaches are increasingly being employed to probe the structural and dynamical state of proteins, not only in isolation in a test tube, but also in a complex biological environment and even within intact cells. Here, we survey direct and indirect evidence that structural disorder is in fact the physiological state of many proteins in the proteome. The paradigmatic case of α-synuclein is used to illustrate the controversial nature of this topic. [ABSTRACT FROM AUTHOR]

Intracellular protein delivery plays a critical role in the development of biotherapeutics and biotechnologies, yet it is hampered by a number of factors including protein binding, cellular uptake, endosomal escape, and protein release. Boronate building blocks, which are frequently employed to create effective protein delivery systems, have shown significant promise in overcoming these limitations thanks to their versatile reactivities and stimuli-responsive property. Boronate ligands transport conjugated proteins into the cytosol via receptor-mediated endocytosis by forming reversible boronate disaster bonds with carbohydrates like sialic acid on the cell surface. Additionally, boronate modification gives cargo proteins extra binding sites for forming complexes with nanocarriers. After internalization, boronate-tagged proteins are released from their carriers in response to endolysosomal acidity, reactive oxygen species, and adenosine triphosphate, and sometimes transport into the nucleus via the importin α/β pathway. Besides, boronate ligands are directly decorated on nanocarriers to enhance their binding affinity to native proteins via nitrogen-boron coordination. Owing to these promising features, various supramolecular and dynamic nanoassemblies are constructed based on boronate building blocks for efficient intracellular protein delivery., (© 2022 Wiley-VCH GmbH.)

In this study, the suitability of gelatin methacryloyl (GelMA) nanogels for transdermal delivery of macromolecules was demonstrated. The synthesis of GelMA nanogels (GNs) and fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded GelMA nanogels (FGNs) were implemented when confined in water-in-oil nanoemulsion droplets via the photopolymerization of the methacryloyl substituents to create crosslinked nanogels. Both GNs and FGNs existed as fine particles in aqueous condition (pH 7.4) for 7 days. No distinct aggregation of nanogel particles were observed. In the MTT assay, high percentage of cell viability indicated that GNs did not exhibit any growth inhibitory effect or significant cytotoxicity. The skin penetration study results showed that FGNs permeated across the epidermis and into the dermis of a porcine model when compared to the FITC-BSA dissolved in PBS. Possible penetration routes of FITC-BSA through the stratum corneum (SC) were illustrated by visualizing the SC structure with fluorescent signals of FITC-BSA. The penetration mechanism of FGNs across the SC layer was successfully demonstrated by explaining three penetration routes (intercellular, follicular, and transcellular route). The results suggest that GNs have a potential as a transdermal delivery carrier for hydrophilic macromolecules. [ABSTRACT FROM AUTHOR]

To date, plant medicine research has focused mainly on the chemical compositions of plant extracts and their medicinal effects. However, the therapeutic or toxic effects of nanoparticles in plant extracts remain unclear. In this study, large numbers of spherical nanoparticles were discovered in some plant extracts. Nanoparticles in Turkish galls extracts were used as an example to examine their pH responsiveness, free radical scavenging, and antibacterial capabilities. By utilizing the underlying formation mechanism of these nanoparticles, a general platform to produce spherical nanoparticles via direct self-assembly of Turkish gall extracts and various functional proteins was developed. The results showed that the nanoparticles retained both the antibacterial ability and intracellular carrier ability of the original protein or catechol. This work introduces a new member of the plant-derived edible nanoparticle (PDEN) family, establishes a simple and versatile platform for mass production nanoparticles, and provides new insight into the formation mechanism of nanoparticles during plant extraction.

The intracellular delivery of proteins is of great significance. For diseases such as cancer, heart disease and neurodegenerative diseases, many important pharmacological targets are located inside cells. For genetic engineering and cell engineering, various functional proteins need to be delivered into cells for gene editing or cell state regulation. However, most existing protein delivery strategies involve endosomal escape (endocytosis-dependent), resulting in inefficient delivery due to endosome trapping. In contrast, endocytosis-independent intracellular delivery, which refers to the directly delivery of proteins across the cell membrane to the cytoplasm, will bypass the low efficiency of early endosomal escape, avoid protein inactivation caused by late endosome/lysosome, fundamentally improve the intracellular delivery efficiency, and open up a new way for intracellular protein delivery. In this review, the latest advances in direct intracellular delivery of proteins through membrane perforation, membrane translocation, and membrane fusion were summarized. The mechanisms, related materials and potential therapeutic in living cells/in vivo for each approach were discussed in detail, and the future development in this promising field was briefly presented. [ABSTRACT FROM AUTHOR]

In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery. [ABSTRACT FROM AUTHOR]

Abstract: Intracellular delivery of functionally active proteins into cells is emerging as a novel strategy for research and therapeutic applications. Here, we present the properties of a self-assembling pyridylthiourea-modified polyethylenimine (πPEI), which interacts with proteins and promotes their delivery into the cytosol of mammalian cells. In aqueous medium at pH7.4, self-association of πPEI in the presence of green fluorescent proteins (GFP) leads to supramolecular protein-entrapped assemblies. These assemblies protect GFP from losing its fluorescence upon pH variation and assist delivery/translocation into the cytosol of mammalian cells via the endocytic pathway. The scope of application of this delivery system was extended to antibodies against intracellular targets as illustrated using a monoclonal antibody directed against the HPV-16 viral E6 oncoprotein and an antibody directed against the threonine-927 phosporylation site of the EG5 kinesin spindle protein. The πPEI-mediated delivery of native anti-E6 antibodies or anti-E6 antibodies equipped with a nuclear localization signal (NLS), led to regeneration of the p53 tumor suppression protein in E6-transformed CaSki cells. Delivery of functionally active anti-EG5 antibodies, with the same polymer, reduced HeLa cell viability and appeared to perturb, as expected, chromosome segregation during mitosis. Altogether, these results provide an easy to use delivery system for extending the scope of application of antibodies for epitope recognition within living cells and may provide novel opportunities for selective interference of cell function by a steric hindrance modality. [Copyright &y& Elsevier]

Intracellular protein delivery has attracted increasing attentions in biomedical applications. However, current delivery systems usually have poor serum stability due to the competitive binding of serum proteins to the polymers during delivery. Here, we report a reversible cross-linking strategy to improve the serum stability of polymers for robust intracellular protein delivery. In the proposed delivery system, nanoparticles are assembled by cargo proteins and cationic polymers and further stabilized by a glutathione-cleavable and traceless cross-linker. The cross-linked nanoparticles show high stability and efficient cell internalization in serum containing medium and can release the cargo proteins in response to intracellular glutathione and acidic pH in a traceless manner. The generality and versatility of the proposed strategy were demonstrated on different types of cationic polymers, cargo proteins, as well as cell lines. The study provides a facile and efficient method for improving the serum tolerance of cationic polymers in intracellular protein delivery.

[Display omitted] We have developed a series of attenuated cationic amphiphilic lytic (ACAL) peptides that can efficiently bring immunoglobulin G (IgG) and other functional proteins into cells. Delivery is generally achieved through the coadministration of ACAL peptides with cargo proteins. However, conjugation of ACAL peptides with cargos may be a promising approach for in vivo application to link in vivo outcomes of ACAL peptides and cargos. This study describes the creation of a new cell-permeable ACAL peptide, L17ER4. L17E is an optimized prototype of ACAL peptides previously developed in our laboratory for efficient delivery of IgGs into cells. Delivery was improved by functionalizing L17E with a tetra-arginine (R4) tag. Compared to the use of R8, a representative cell-penetrating peptide with high intracellular delivery efficacy, conjugation with L17ER4 afforded approximately four-fold higher cellular uptake of model small-molecule cargos (fluorescein isothiocyanate and HiBiT peptide). L17ER4 was also able to deliver proteins to cells. Fused with L17ER4, Cre recombinase was delivered into cells. Intracerebroventricular injection of Cre-L17ER4 into green red reporter mice, R26GRR, led to significant in vivo gene recombination in ependymal cells, suggesting that L17ER4 may be used as a cell-penetrating peptide for delivering protein therapeutics into cells in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reversal of the polymeric carrier from negative to positive. The concomitant endosomal buffering effect and increased polymer–endosomal membrane interactions are considered to lead to increased protein delivery into the cytosol. Besides destabilization of the polymer–protein nanoparticles by the charge reversal effect, intracellular cleavage of disulfide linkages in the polymer ensure further unpacking of the protein in the cytosol. Cellinternalization and cytotoxicity experiments with primary human umbilical vein endothelial cells (HUVEC) showed that the SS-PAA-based nanocomplexes were essentially non-toxic, and that lysozyme is successfully internalized into HUVEC. The results indicate that these charge reversal SS-PAAs have excellent properties as non-toxic intracellular delivery systems for cationic proteins. [Copyright &y& Elsevier]

Abstract: An effective intracellular protein delivery system was developed based on linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Two differently functionalized PAAs were synthesized by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA) and to bisacryloylpiperazine (BAP), yielding p(CBA-ABOL) and p(BAP-ABOL), respectively. These water-soluble PAAs efficiently condense human serum albumin (HSA) by self-assembly into stable nanoscaled and positively-charged complexes. The disulfide-containing p(CBA-ABOL)/HSA nanocomplexes exhibited high mucoadhesive properties and, while stable under neutral (extracellular) conditions, rapidly destabilized in a reductive (intracellular) environment due to the cleavage of the repetitive disulfide linkages in the CBA units of the polymer. Human-derived intestinal Caco-2/TC7 cells and HT29-MTX mucus secreting cells were exposed to these PAAs/HSA nanoparticles and the extent of their uptake and the localization within endosomal compartments were examined. The higher uptake of p(CBA-ABOL)/HSA than that of p(BAP-ABOL)/HSA suggests that the mucoadhesive properties of the p(CBA-ABOL) are beneficial to the uptake process. The transported HSA was located within early endosomes, lysosomes and the cytosol. The enhanced uptake of the p(CBA-ABOL)/HSA nanoparticles, observed in the presence of Cyclosporin A, a non-specific Multi Drug Resistance (MDR) blocker, indicates the possible efflux of these nanoparticles through MDR transporters. The results show that bioreducible PAAs have excellent properties for intracellular protein delivery, and should be applicative in oral protein delivery. [Copyright &y& Elsevier]

Abstract: An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense β-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. β-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing β-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery. [Copyright &y& Elsevier]

Improving the efficacy of drug delivery via nanocarriers has been a major issue in the field of intravenous delivery. In this study, a polymeric nanogel was developed to enhance the stability, redox responsiveness, and the efficacy for intracellular protein delivery. The thiolated heparin-Pluronic conjugate was self-assembled and oxidized to form a disulfide-crosslinked nanogel network under a diluted aqueous condition. The disulfide-crosslinked heparin-Pluronic (DHP) nanogels with encapsulated RNase A were characterized by in vitro release and cytotoxicity tests depending on the existence of glutathione (GSH). The DHP nanogels exhibited reduced hydrodynamic size, higher encapsulation degree, and augmentable release responding to the GSH concentration. The ctotoxicity data confirmed that DHP nanogels were more effective for the intracellular delivery of RNase A compared to non-crosslinked nanogel. [ABSTRACT FROM AUTHOR]

Abstract: To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells. [Copyright &y& Elsevier]

Abstract: Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid Saint-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with Saint-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using β-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy. [Copyright &y& Elsevier]

Cell penetrating peptides consist of short sequences of amino acids containing a large net positive charge that are able to penetrate almost any cell, carrying with them relatively large cargoes such as proteins, oligonucleotides, and drugs. During the 10 years since their discovery, the question of how they manage to translocate across the membrane has remained unanswered. The main discussion has been centered on whether they follow an energy-independent or an energy-dependent pathway. Recently, we have discovered the possibility of an energy-independent pathway that challenges fundamental concepts associated with protein-membrane interactions (Herce and Garcia, PNAS, 104: 20805 (2007) []). It involves the translocation of charged residues across the hydrophobic core of the membrane and the passive diffusion of these highly charged peptides across the membrane through the formation of aqueous toroidal pores. The aim of this review is to discuss the details of the mechanism and interpret some experimental results consistent with this view. [ABSTRACT FROM AUTHOR]

Abstract: To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(l-lysine)–poly(ethylene glycol)–folate (PLL–PEG–FOL), was synthesized. The PLL–PEG–FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL–PEG–FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL–PEG–FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL–PEG–FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface. [Copyright &y& Elsevier]

Abstract: Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides. [Copyright &y& Elsevier]