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Abstract The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol. [ABSTRACT FROM AUTHOR]

Abstract Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide’s ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Abstract Background RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results: BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion: The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form. [ABSTRACT FROM AUTHOR]

The lack of effective treatments for neurodegenerative diseases (NDs) is an important current concern. Lipid nanoparticles can deliver innovative combinations of active molecules to target the various mechanisms of neurodegeneration. A significant challenge in delivering drugs to the brain for ND treatment is associated with the blood–brain barrier, which limits the effectiveness of conventional drug administration. Current strategies utilizing lipid nanoparticles and cell-penetrating peptides, characterized by various uptake mechanisms, have the potential to extend the residence time and bioavailability of encapsulated drugs. Additionally, bioactive molecules with neurotropic or neuroprotective properties can be delivered to potentially mediate the ND targeting pathways, e.g., neurotrophin deficiency, impaired lipid metabolism, mitochondrial dysfunction, endoplasmic reticulum stress, accumulation of misfolded proteins or peptide fragments, toxic protein aggregates, oxidative stress damage, and neuroinflammation. This review discusses recent advancements in lipid nanoparticles and CPPs in view of the integration of these two approaches into nanomedicine development and dual-targeted nanoparticulate systems for brain delivery in neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications. [ABSTRACT FROM AUTHOR]

Exosomes (diameter 30–200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Simple Summary: This study focuses on the desert locust, a critical insect pest species for agriculture. We aimed at investigating how to induce RNA interference by feeding in this species. RNA interference is a gene-silencing mechanism that promises to contribute to pest control strategies. We studied cell-penetrating peptides (CPPs) as potential dsRNA/siRNA delivery systems. We found CPPs that can complex with dsRNA and siRNAs, as well as protect them from the degradation of midgut enzymes. In addition, we report that intra-hemocoelic injection of naked siRNAs does not trigger a gene-silencing response in the desert locust but, for siRNAs complexed with one of the investigated CPPs, it does. Although we could not find a suitable CPP to induce RNAi by feeding in the locust, our results stimulate future research on this topic. In addition, our findings contribute to the understanding of the RNA interference response and its complexity in insects as well as emphasizing the importance of research in living insects when it comes to dsRNA/siRNA oral delivery systems. RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems. [ABSTRACT FROM AUTHOR]

The lack of effective treatments for neurodegenerative diseases (NDs) is an important current concern. Lipid nanoparticles can deliver innovative combinations of active molecules to target the various mechanisms of neurodegeneration. A significant challenge in delivering drugs to the brain for ND treatment is associated with the blood–brain barrier, which limits the effectiveness of conventional drug administration. Current strategies utilizing lipid nanoparticles and cell-penetrating peptides, characterized by various uptake mechanisms, have the potential to extend the residence time and bioavailability of encapsulated drugs. Additionally, bioactive molecules with neurotropic or neuroprotective properties can be delivered to potentially mediate the ND targeting pathways, e.g., neurotrophin deficiency, impaired lipid metabolism, mitochondrial dysfunction, endoplasmic reticulum stress, accumulation of misfolded proteins or peptide fragments, toxic protein aggregates, oxidative stress damage, and neuroinflammation. This review discusses recent advancements in lipid nanoparticles and CPPs in view of the integration of these two approaches into nanomedicine development and dual-targeted nanoparticulate systems for brain delivery in neurodegenerative disorders.

The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP]3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery. [ABSTRACT FROM AUTHOR]

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes., Competing Interests: The authors declare no conflict of interest., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect. [ABSTRACT FROM AUTHOR]

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP] 3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery.