Expression and purification of cell-penetrating Cas9 and Cas12a enzymes for peptide-assisted genome editing.

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells.

Categorizing interaction modes of antimicrobial peptides with extracellular vesicles: Disruption, membrane trespassing, and clearance of the protein corona.

Host antimicrobial peptides (AMPs) and extracellular vesicles (EVs) are known to play important roles as part of the immune system, from antimicrobial actions to immune regulation. Recent results also demonstrate that EVs could serve as carriers for AMPs. Related, it was shown that some AMPs can remove the protein corona (PC), the externally adsorbed layer of proteins, from EVs which can be exploited for subtractive proteomics strategies. The interaction of these compounds is thus interesting for multiple reasons from better insight to natural processes to direct applications in EV-based bioengineering. However, we have only limited information on the various ways these species may interact with each other. To reach a broader overview, here we selected twenty-six AMPs, including cell-penetrating peptides (CPPs), and investigated their interactions with red blood cell-derived vesicles (REVs). For this, we employed a complex lipid biophysics including linearly polarized light spectroscopy, flow cytometry, nanoparticle tracking analysis, electron microscopy and also zeta-potential measurements. This enabled the categorization of these peptides into distinct groups. At specific low concentrations, peptides such as LL-37 and lasioglossin-III were effective in PC elimination with minimal disruption of the membrane. In contrast, AMPs like KLA, bradykinin, histatin-5, and most of the tested CPPs (e.g. octa-arginine, penetratin, and buforin II), demonstrate cell-penetrating mechanisms as they could sustain large peptide concentrations with minimal membrane damage. The systematic overview presented here shows the potential mechanism of how AMPs and EVs could interact in vivo, and also how certain peptides may be employed to manipulate EVs for specific applications.

Exploring the Utility of Cell-Penetrating Peptides as Vehicles for the Delivery of Distinct Antimalarial Drug Cargoes.

The devastating impact of malaria includes significant mortality and illness worldwide. Increasing resistance of the causative parasite, Plasmodium, to existing antimalarial drugs underscores a need for additional compounds with distinct modes of action in the therapeutic development pipeline. Here we showcase peptide-drug conjugates (PDCs) as an attractive compound class, in which therapeutic or lead antimalarials are chemically conjugated to cell-penetrating peptides. This approach aims to enhance selective uptake into Plasmodium-infected red blood cells and impart additional cytotoxic actions on the intraerythrocytic parasite, thereby enabling targeted drug delivery and dual modes of action. We describe the development of PDCs featuring four compounds with antimalarial activity - primaquine, artesunate, tafenoquine and methotrexate - conjugated to three cell-penetrating peptide scaffolds with varied antiplasmodial activity, including active and inactive analogs of platelet factor 4 derived internalization peptide (PDIP), and a cyclic polyarginine peptide. Development of this diverse set of PDCs featured distinct and adaptable conjugation strategies, to produce conjugates with in vitro antiplasmodial activities ranging from low nanomolar to low micromolar potencies according to the drug cargo and bioactivity of the partner peptide. Overall, this study establishes a strategic and methodological framework for the further development of dual mode of action peptide-drug antimalarial therapeutics.

Visualization of the Plasmid DNA Delivery System by Complementary Fluorescence Labeling of Arginine-Rich Peptides.

Cell-penetrating peptides, such as arginine-rich peptides, encapsulate nucleic acid drugs and deliver them to intracellular compartments. Comprehensive tracking of drug delivery systems (DDSs) provides information about the behavior of the drug as well as the fate of the drug carrier after drug release, which is crucial for minimizing side effects. In this study, we labeled peptides designed to carry plasmid DNA with two types of dyes, traditional dye fluorescein and aggregation-induced emission (AIE) dye tetraphenylethylene, and subsequently tracked the DDS through the complementary ON and OFF fluorescence behaviors of the dyes. Traditional fluorescent dyes are susceptible to aggregation-caused quenching during bioimaging, a problem that is mitigated by using AIE dyes. However, by using both of these contrasting fluorescent labels, we were able to clearly visualize the DDS at different stages of its deployment, from drug transport and delivery to carrier dissociation and migration, demonstrating the feasibility of accurate DDS visualization by complementary fluorescence labeling.

Lysosome-Targeted Bifunctional Therapeutics Induce Autodynamic Cancer Therapy.

Autodynamic cancer therapy possesses tremendous potential for enhancing therapeutic efficacy by initiating the treatment process autonomously within targeted cells. However, challenges related to biocompatibility and targeted delivery have hindered its clinical translation owing to the induction of adverse effects and cytotoxicity in healthy cells. In this study, a novel approach for auto-initiated dynamic therapy by conjugating zwitterionic near-infrared fluorophores to a cell-penetrating peptide is proposed. This enables efficient cellular uptake and specific targeting of therapy to desired cells while avoiding off-target uptake. The zwitterionic bioconjugate causes cancer-specific toxicity following its internalization into the targeted cells, triggered by specific intracellular conditions in lysosomes. This innovative approach enables selective targeting of lysosomes in malignant cells while minimizing cytotoxic effects on normal cells. By targeting lysosomes, the method overcomes inherent risks and side effects associated with conventional cancer treatments, offering a selective and effective approach to cancer therapy.

The Roadmap of Plant Antimicrobial Peptides Under Environmental Stress: From Farm to Bedside.

Antimicrobial peptides (AMPs) are the most favorable alternatives in overcoming multidrug resistance, alone or synergistically with conventional antibiotics. Plant-derived AMPs, as cysteine-rich peptides, widely compensate the pharmacokinetic drawbacks of peptide therapeutics. Compared to the putative genes encrypted in the genome, AMPs that are produced under stress are active forms with the ability to combat resistant microbial species. Within this study, plant-derived AMPs, namely, defensins, nodule-specific cysteine-rich peptides, snakins, lipid transfer proteins, hevein-like proteins, α-hairpinins, and aracins, expressed under biotic and abiotic stresses, are classified. We could observe that while α-hairpinins and snakins display a helix-turn-helix structure, conserved motif patterns such as ß1αß2ß3 and ß1ß2ß3 exist in plant defensins and hevein-like proteins, respectively. According to the co-expression data, several plant AMPs are expressed together to trigger synergistic effects with membrane disruption mechanisms such as toroidal pore, barrel-stave, and carpet models. The application of AMPs as an eco-friendly strategy in maintaining agricultural productivity through the development of transgenes and bio-pesticides is discussed. These AMPs can be consumed in packaging material, wound-dressing products, coating catheters, implants, and allergology. AMPs with cell-penetrating properties are verified for the clearance of intracellular pathogens. Finally, the dominant pharmacological activities of bioactive peptides derived from the gastrointestinal digestion of plant AMPs, namely, inhibitors of renin and angiotensin-converting enzymes, dipeptidyl peptidase IV and α-glucosidase inhibitors, antioxidants, anti-inflammatory, immunomodulating, and hypolipidemic peptides, are analyzed. Conclusively, as phytopathogens and human pathogens can be affected by plant-derived AMPs, they provide a bright perspective in agriculture, breeding, food, cosmetics, and pharmaceutical industries, translated as farm to bedside.

Spotlight on HIV-derived TAT peptide as a molecular shuttle in drug delivery.

HIV-derived TAT peptide, with a high penetration rate into cells and its nonimmunogenic and minimally toxic nature, is an attractive tool for enhancing the biodistribution of drugs and their systemic administration. Despite the presence of numerous promising preclinical investigations illustrating its capability to specifically target distinct tissues and deliver a diverse range of pharmacological agents, the efficacy of various clinical trials incorporating TAT has been impeded by several considerable obstacles. Hence, there is much need for an in-depth investigation concerning the application of TAT in drug delivery mechanisms. In this review, we have elucidated the structure of TAT and its utility in the proficient delivery of various types of bioactive molecules.

siRNA Nanoparticle Dry Powder Formulation with High Transfection Efficiency and Pulmonary Deposition for Acute Lung Injury Treatment.

Acute lung injury (ALI) is a severe inflammatory syndrome, which was caused by diverse factors. The COVID-19 pandemic has resulted in a higher mortality rate of these conditions. Currently, effective treatments are lacking. Although siRNA nucleotide-based drugs are promising therapeutic approaches, their poor stability and inability to efficiently reach target cells limit their clinical translation. This study identified a peptide from known cell-penetrating peptides that can form an efficient anti-inflammatory complex with TNF-α siRNA, termed SAR6EW/TNF-α siRNA. This complex can effectively transport TNF-α siRNA into the cytoplasm and achieve potent gene silencing in vitro as well as in vivo. By using lactose and triarginine as coexcipients and optimizing the spray-drying process, a powder was produced with micrometer-scale spherical and porous structures, enhancing aerosol release and lung delivery efficiency. The dry powder formulation and process preserve the stability and integrity of the siRNA. When administered intratracheally to ALI model mice, the complex powder demonstrated specific pulmonary gene silencing activity and significantly reduced inflammation symptoms caused by ALI, suggesting a potential strategy for the clinical therapeutic approach of respiratory diseases.

Enhancing peptide and PMO delivery to mouse airway epithelia by chemical conjugation with the amphiphilic peptide S10.

Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.

Investigation of Dual-Loaded Doxorubicin and Sorafenib Liposomes Co-Modified with Glycyrrhetinic Acid and Cell-Penetrating Peptide TAT.

BACKGROUND: Combining Doxorubicin (DOX) with sorafenib (SF) is a promising strategy for treating Hepatocellular Carcinoma (HCC). However, strict dosage control is required for both drugs, and there is a lack of target selectivity. OBJECTIVE: This study aims to develop a novel nano-drug delivery system for the combined use of DOX and SF, aiming to reduce their respective dosages, enhance therapeutic efficacy, and improve target selectivity. METHODS: DOX/SF co-loaded liposomes (LPs) were prepared using the thin-film hydration method. The liposomes were modified with 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE)- polyethylene glycol (PEG2000), DSPE-PEG1000-cell penetrating peptide TAT, and Glycyrrhetinic Acid (GA). The basic properties of the liposomes were characterized. CCK-8 cell viability assays were conducted using HepG2, MHCC97-H, and PLC cell models, and apoptosis experiments were performed using HepG2 cells to determine if this delivery system could reduce the respective dosages of DOX and SF and enhance HCC cytotoxicity. Liposome uptake experiments were performed using HepG2 cells to validate the target selectivity of this delivery system. RESULTS: A GA/TAT-DOX/SF-LP liposomal nano drug delivery system was successfully constructed, with a particle size of 150 nm, a zeta potential of -7.9 mV, a DOX encapsulation efficiency of 92%, and an SF encapsulation efficiency of 88.7%. Cellular experiments demonstrated that this delivery system reduced the required dosages of DOX and SF, exhibited stronger cytotoxicity against liver cancer cells, and showed better target selectivity. CONCLUSION: A simple and referenceable liposomal nano drug delivery system has been developed for the combined application of DOX and SF in hepatocellular carcinoma treatment.

Mucosal Penetrative Polymeric Micelle Formulations for Insulin Delivery to the Respiratory Tract.

Purpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers. Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract. Results: The nano-micelles, with a particle size of <100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (Cmax) and AUC0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h. Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract.

IMT-P8 potentiates Gram-positive specific antibiotics in intrinsically resistant Gram-negative bacteria.

Gram-negative bacteria (GNB) pose a major global public health challenge as they exhibit a remarkable level of resistance to antibiotics. One of the factors responsible for promoting resistance against a wide range of antibiotics is the outer membrane (OM) of Gram-negative bacteria. The OM acts as a barrier that prevents the entry of numerous antibiotics by reducing their influx (due to membrane impermeability) and enhancing their efflux (with the help of efflux pumps). Our study focuses on analyzing the effect of IMT-P8, a cell-penetrating peptide (CPP), to enhance the influx of various Gram-positive specific antibiotics in multi-drug resistant Gram-negative pathogens. In the mechanistic experiments, IMT-P8 permeabilizes the OM at the same concentrations at which it enhances the activity of various antibiotics against GNB. Cytoplasmic membrane permeabilization was also observed at these concentrations, indicating that IMT-P8 acts on both the outer and cytoplasmic membranes. IMT-P8 interferes with the intrinsic resistance mechanism of GNB and has the potential to make Gram-positive specific antibiotics effective against GNB. IMT-P8 extends the post-antibiotic effect and in combination with antibiotics shows anti-persister activity. The IMT-P8/fusidic acid combination is effective in eliminating intracellular pathogens. IMT-P8 with negligible toxicity displayed good efficacy in murine lung and thigh infection models. Based on these findings, IMT-P8 is a potential antibiotic adjuvant to treat Gram-negative bacterial infections that pose a health hazard.

Towards Cell-Permeable Hepatitis B Virus Core Protein Variants as Potential Antiviral Agents.

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile.

Crotamine derived from Crotalus durissus terrificus venom combined with drugs increases in vitro antibacterial and antifungal activities.

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

Configuration of two cysteine residues in a ring within a stapled Bim peptide affects the secondary structure and apoptotic activity.

Many reports have shown that stabilization of secondary structure by stapling functional peptides enhances the intracellular bioactivity. However, no report has discussed the correlation between stabilization and biological activity based on the configuration of amino acid residues used as anchors for stapling. To clarify this, we investigated the helix content and apoptotic efficiency of an apoptosis-inducing peptide, Bim, and four stapled Bim peptides containing stapling-related Cys residues introduced with different configurations within the sequence. The results demonstrated that the configuration of Cys residues in stapled Bim peptides affected the secondary structure and intracellular activity of the peptides, and furthermore, there was a correlation between these latter two variables.

Development and evaluation of a radiolabelling agent for white blood cell scans.

Radiolabelled autologous leukocytes have been used for the clinical diagnosis of inflammation and infection. To develop a stable and efficient radiopharmaceutical for labelling leukocytes, we prepared a novel radioiodinated cell-penetrating peptide, 125I-TAT, using a bi-functional linker. 125I-TAT was stable for two days under three different temperature conditions of -20 °C, 4 °C, and 40 °C, with its radiochemical purity remaining over 99%. Iodinated TAT was non-toxic to leukocytes with an IC50 value of over 100 µM. The labelling efficiency of 125I-TAT using 1ｘ107 cells ranged from 27% to 53% when the three leukocyte cell lines were pre-treated with DMSO. This is comparable to the labelling efficiency recommended by the guideline for conventional labelling agents using 2ｘ108 cells. Radioiodinated cell-penetrating peptide may be an improved radiopharmaceutical for white blood cell scans by further optimization.

Design, Synthesis, and Evaluation of Oleyl-WRH Peptides for siRNA Delivery.

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1-4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 µM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of <100 nm, formed nano-complexes with siRNA and exhibited a stable range of zeta potential values (13-18 mV at N/P = 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of a successful cellular internalization of siRNA. The peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells.

Inhibition of enhanced green fluorescent protein cytosolic delivery mediated by modified cell-penetrating peptide due to high overexpression of Caveolin-1.

The modified cell-penetrating peptide Pas2r12 can deliver antibodies (IgG, 150 kDa) and enhanced green fluorescent protein (EGFP1, 27 kDa) into the cytosol through caveolae-dependent endocytosis. In this study, we determined the effect of Caveolin-1 overexpression on the cytosolic delivery of EGFP by Pas2r12. Three types of Caveolin-1 overexpressing strains were isolated, including Cav1L (low), Cav1M (medium), and Cav1H (high), using HEK293 as the parent cell line. We found that the number of caveolae on the surface of the Caveolin-1-overexpressing strains was similar to that of HEK293. We examined the cytosolic delivery rate of EGFP by Pas2r12. In the Cav1L and Cav1M cells, there was little change compared with HEK293; however, in Cav1H, the rate was significantly decreased. Moreover, the amount of EGFP uptake into the cells (total intracellular EGFP) showed an increasing trend in Cav1H compared with HEK293. These results indicate that in Cav1H, the amount of EGFP uptake into the cells increases, whereas the cytosolic delivery rate of EGFP decreases. This suggests that high overexpression of Caveolin-1 inhibits the transition of EGFP from endosomes to the cytosol.

Octa-Arginine-Conjugated Liposomal Nimodipine Incorporated in a Temperature-Responsive Gel for Nasoencephalic Delivery.

Nimodipine is the primary clinical drug used to treat cerebral vasospasm following subarachnoid hemorrhage. Currently, tablets have low bioavailability when taken orally, and injections contain ethanol. Therefore, we investigated a new method of nimodipine administration, namely, nasoencephalic administration. Nasal administration of nimodipine was carried out by attaching the cell-penetrating peptide octa-arginine (R8) to liposomes of nimodipine and incorporating it into a temperature-sensitive in situ gel. The prepared liposomes and gels underwent separate evaluations for in vitro characterization. In vitro release exhibited a significant slow-release effect. In vitro toad maxillary cilia model, RPMI 2650 cytotoxicity, and in vivo SD rat pathological histotoxicity experiments showed that all the dosage from the groups had no significant toxicity to toad maxillary cilia, RPMI 2650 cells, and SD rat tissues and organs, and the cilia continued to oscillate up to 694 ± 10.15 min, with the survival rate of the cells being above 85%. A transwell nasal mucosa cell model and an isolated porcine nasal mucosa model were established, and the results showed that the osmolality of the R8-modified nimodipine liposomal gel to nasal mucosal cells and isolated porcine nasal mucosa was 30.41 ± 2.14 and 65.9 ± 7.34 µg/mL, respectively, which was significantly higher than that of the NM-Solution and PEGylated nimodipine liposome gel groups. Animal fluorescence imaging studies revealed that the R8-modified nimodipine liposomal gel displayed increased brain fluorescence intensity compared to the normal liposomal gel. Pharmacokinetic results showed that after transnasal administration, the AUC(0-∞) of the R8-modified nimodipine liposomal gel was 11.662 ± 1.97 µg·mL-1, which was significantly higher than that of the plain nimodipine liposomal gel (5.499 ± 2.89 µg·mL-1). Brain-targeting experiments showed that the brain-targeting efficiencies of the PEGylated nimodipine liposome gel and R8-modified PEGylated nimodipine liposome gels were 20.44 and 33.45, respectively, suggesting that R8/PEG/Lip-NM-TSG significantly increased the brain-targeting of the drug.

Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 µg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 µg/mL and 465.2 µg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 µg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.