Intracellular Delivery Research Articles

Tolerability of a piezoelectric microneedle electroporator in human subjects

AbstractElectroporation, or the use of electric pulses to facilitate the intracellular delivery of DNA, RNA, and other molecules, is a well‐established technique, that has been demonstrated to significantly augment the immunogenicity of DNA/mRNA vaccines and therapeutics. However, the clinical translation of traditional electroporators has been limited due to high costs, large size, complex user operation, and poor tolerability in humans due to nerve stimulation. In prior work, we introduced ePatch: an ultra‐low‐cost, handheld, battery‐free electroporator employing a piezoelectric pulser coupled with a microneedle electrode array that showed enhanced immunogenic responses to an intradermal SARS‐CoV‐2 DNA vaccine in mice. The current study shifts focus from efficacy to tolerability, hypothesizing that ePatch's microneedle array, which localizes the electric field to the superficial skin strata, will minimize nerve stimulation and improve patient comfort. We tested this hypothesis in 14 healthy adults, monitoring pain and other potential adverse effects associated with electroporation. Compared to the insertion of a traditional hypodermic needle, the ePatch was less painful. Adverse effects such as pain, tenderness, erythema and swelling at the application sites were minimal, transient, and statistically indistinguishable between the experimental and placebo ePatch application, suggesting excellent tolerability towards electroporation. In summary, ePatch has a favorable tolerability profile in humans and offers the potential for the safe use of electroporation in a variety of clinical settings, including DNA and mRNA vaccination.

Recent Advances in Synergistic Effect of Nanoparticles and Its Biomedical Application.

The synergistic impact of nanomaterials is critical for novel intracellular and/or subcellular drug delivery systems of minimal toxicity. This synergism results in a fundamental bio/nano interface interaction, which is discussed in terms of nanoparticle translocation, outer wrapping, embedding, and interior cellular attachment. The morphology, size, surface area, ligand chemistry and charge of nanoparticles all play a role in translocation. In this review, we suggest a generalized mechanism to characterize the bio/nano interface, as we discuss the synergistic interaction between nanoparticles and cells, tissues, and other biological systems. Novel perceptions are reviewed regarding the ability of nanoparticles to improve hybrid nanocarriers with homogeneous structures to enhance multifunctional biomedical applications, such as bioimaging, tissue engineering, immunotherapy, and phototherapy.

Aiming the magic bullet: targeted delivery of imaging and therapeutic agents to solid tumors by pHLIP peptides.

The family of pH (Low) Insertion Peptides (pHLIP) comprises a tumor-agnostic technology that uses the low pH (or high acidity) at the surfaces of cells within the tumor microenvironment (TME) as a targeted biomarker. pHLIPs can be used for extracellular and intracellular delivery of a variety of imaging and therapeutic payloads. Unlike therapeutic delivery targeted to specific receptors on the surfaces of particular cells, pHLIP targets cancer, stromal and some immune cells all at once. Since the TME exhibits complex cellular crosstalk interactions, simultaneous targeting and delivery to different cell types leads to a significant synergistic effect for many agents. pHLIPs can also be positioned on the surfaces of various nanoparticles (NPs) for the targeted intracellular delivery of encapsulated payloads. The pHLIP technology is currently advancing in pre-clinical and clinical applications for tumor imaging and treatment.

Universal STING mimic boosts antitumour immunity via preferential activation of tumour control signalling pathways.

The efficacy of STING (stimulator of interferon genes) agonists is due to various factors, primarily inefficient intracellular delivery, low/lack of endogenous STING expression in many tumours, and a complex balance between tumour control and progression. Here we report a universal STING mimic (uniSTING) based on a polymeric architecture. UniSTING activates STING signalling in a range of mouse and human cell types, independent of endogenous STING expression, and selectively stimulates tumour control IRF3/IFN-I pathways, but not tumour progression NF-κB pathways. Intratumoural or systemic injection of uniSTING-mRNA via lipid nanoparticles (LNPs) results in potent antitumour efficacy across established and advanced metastatic tumour models, including triple-negative breast cancer, lung cancer, melanoma and orthotopic/metastatic liver malignancies. Furthermore, uniSTING displays an effective antitumour response superior to 2'3'-cGAMP and ADU-S100. By favouring IRF3/IFN-I activity over the proinflammatory NF-κB signalling pathway, uniSTING promotes dendritic cell maturation and antigen-specific CD8+ T-cell responses. Extracellular vesicles released from uniSTING-treated tumour cells further sensitize dendritic cells via exosome-containing miRNAs that reduced the immunosuppressive Wnt2b, and a combination of LNP-uniSTING-mRNA with α-Wnt2b antibodies synergistically inhibits tumour growth and prolongs animal survival. Collectively, these results demonstrate the LNP-mediated delivery of uniSTING-mRNA as a strategy to overcome the current STING therapeutic barriers, particularly for the treatment of multiple cancer types in which STING is downregulated or absent.

Nuclear Localization Signal Enhances the Targeting and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic In Vitro and Ex Vivo Evaluation.

The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 μCi (9.25 MBq) and 500 μCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

One-dimensional diamond nanostructures: Fabrication, properties and applications

Diamonds exhibit a unique combination of numerous excellent physical and chemical properties, such as the highest hardness, high Young's modulus, wide bandgap, high carrier mobility, negative electron affinity, and high thermal conductivity. Especially, diamond has attracted significant attention in recent years for its outstanding performance in high-frequency and high-power electronic devices and thermal management. Inheriting the intrinsic physical and chemical properties of diamonds, one-dimensional (1D) diamond nanostructures exhibit many additional properties that are unavailable for bulk diamond or thin films, e.g., large specific surface area, ultrahigh elasticity, and some plasticity, tip effect as well as direct transport for photon or photo-induced carriers. These properties make 1D diamond nanostructures an ideal candidate for applications ranging from micro/nano-electromechanical systems (MEMS/NEMS) to field electron emission, optical, electrochemical, and biological areas. This review will first introduce the development of controlled synthesis methods of 1D diamond nanostructure, including bottom-up chemical vapor deposition growth and top-down selective etching of bulk diamond. Subsequently, we will summarize recent advances in the mechanical, electron field emission, optical, and biological properties of 1D diamond nanostructures and their applications in electron and photon emitter, MEMS, sensing, intracellular delivery, etc.

Mn-phenolic networks as synergistic carrier for STING agonists in tumor immunotherapy

The cGAS-STING pathway holds tremendous potential as a regulator of immune responses, offering a means to reshape the tumor microenvironment and enhance tumor immunotherapy. Despite the emergence of STING agonists, their clinical viability is hampered by stability and delivery challenges, as well as variations in STING expression within tumors. In this study, we present Mn-phenolic networks as a novel carrier for ADU-S100, a hydrophilic STING agonist, aimed at bolstering immunotherapy. These nanoparticles, termed TMA NMs, are synthesized through the coordination of tannic acid and manganese ions, with surface modification involving bovine serum albumin to enhance their colloidal stability. TMA NMs exhibit pH/GSH-responsive disintegration properties, enabling precise drug release. This effectively addresses drug stability issues and facilitates efficient intracellular drug delivery. Importantly, TMA NMs synergistically enhance the effects of ADU-S100 through the concurrent release of Mn2+, which serves as a sensitizer of the STING pathway, resulting in significant STING pathway activation. Upon systemic administration, these nanoparticles efficiently accumulate within tumors. The activation of STING pathways not only induces immunogenic cell death (ICD) in tumor cells but also orchestrates systemic remodeling of the immunosuppressive microenvironment. This includes the promotion of cytokine release, dendritic cell maturation, and T cell infiltration, leading to pronounced suppression of tumor growth. Combining with the excellent biocompatibility and biodegradability, this Mn-based nanocarrier represents a promising strategy for enhancing tumor immunotherapy through the cGAS-STING pathway.

A non-viral DNA delivery system consisting of multifunctional chimeric peptide fused with zinc-finger protein

Non-viral gene delivery systems have received sustained attention as a promising alternative to viral vectors for disease treatment and prevention in recent years. Numerous methods have been developed to enhance gene uptake and delivery in the cytoplasm; however, due to technical difficulties and delivery efficiency,these systems still face challenges in a range of biological applications, especially invivo. To alleviate this challenge, we devised a novel system for gene delivery based on a recombinant protein eTAT-ZF9-NLS, which consisted of a multifunctional chimeric peptide and a zinc-finger protein with sequence-specific DNA-binding activity. High transfection efficiency was observed in several mammalian cells after intracellular delivery of plasmid containing ZF9-binding sites mediated by eTAT-ZF9-NLS. Our new approach provides a novel transfection strategy and the transfection efficiency was confirmed both invitro and invivo, making it a preferential transfection reagent for possible gene therapy.

Tumor integrin targeted theranostic iron oxide nanoparticles for delivery of caffeic acid phenethyl ester: preparation, characterization, and anti-myeloma activities.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells preferentially in the bone marrow. Currently, emerging chemotherapy drugs with improved biosafety profiles, such as immunomodulatory agents and protease inhibitors, have been used in clinics to treat MM in both initial therapy or maintenance therapy post autologous hematopoietic stem cell transplantation (ASCT). We previously discovered that caffeic acid phenethyl ester (CAPE), a water-insoluble natural compound, inhibited the growth of MM cells by inducing oxidative stress. As part of our continuous effort to pursue a less toxic yet more effective therapeutic approach for MM, the objective of this study is to investigate the potential of CAPE for in vivo applications by using magnetic resonance imaging (MRI)-capable superparamagnetic iron oxide nanoparticles (IONP) as carriers. Cyclo (Arg-Gly-Asp-D-Phe-Cys) (RGD) is conjugated to IONP (RGD-IONP/CAPE) to target the overexpressed αvβ3 integrin on MMcells for receptor-mediated internalization and intracellular delivery of CAPE. A stable loading of CAPE on IONP can be achieved with a loading efficiency of 48.7% ± 3.3% (wt%). The drug-release studies indicate RGD-IONP/CAPE is stable at physiological (pH 7.4) and basic pH (pH 9.5) and subject to release of CAPE at acidic pH (pH 5.5) mimicking the tumor and lysosomal condition. RGD-IONP/CAPE causes cytotoxicity specific to human MM RPMI8226, U266, and NCI-H929 cells, but not to normal peripheral blood mononuclear cells (PBMCs), with IC50s of 7.97 ± 1.39, 16.75 ± 1.62, and 24.38 ± 1.71μM after 72-h treatment, respectively. Apoptosis assays indicate RGD-IONP/CAPE induces apoptosis of RPMI8226 cells through a caspase-9 mediated intrinsic pathway, the same as applying CAPE alone. The apoptogenic effect of RGD-IONP/CAPE was also confirmed on the RPMI8226 cells co-cultured with human bone marrow stromal cells HS-5 in a Transwell model to mimic the MM microenvironment in the bone marrow. In conclusion, we demonstrate that water-insoluble CAPE can be loaded to RGD-IONP to greatly improve the biocompatibility and significantly inhibit the growth of MMcells in vitro through the induction of apoptosis. This study paves the way for investigating the MRI-trackable delivery of CAPE for MM treatment in animal models in the future.

Oxidized mRNA Lipid Nanoparticles for In Situ Chimeric Antigen Receptor Monocyte Engineering

AbstractChimeric antigen receptor (CAR) monocyte and macrophage therapies are promising solid tumor immunotherapies that can overcome the challenges facing conventional CAR T cell therapy. mRNA lipid nanoparticles (mRNA‐LNPs) offer a viable platform for in situ engineering of CAR monocytes with transient and tunable CAR expression to reduce off‐tumor toxicity and streamline cell manufacturing. However, identifying LNPs with monocyte tropism and intracellular delivery potency is difficult using traditional screening techniques. Here, ionizable lipid design and high‐throughput in vivo screening are utilized to identify a new class of oxidized LNPs with innate tropism and mRNA delivery to monocytes. A library of oxidized (oLNPs) and unoxidized LNPs (uLNPs) is synthesized to evaluate mRNA delivery to immune cells. oLNPs demonstrate notable differences in morphology, ionization energy, and pKa, thereby enhancing delivery to human macrophages, but not T cells. Subsequently, in vivo library screening with DNA barcodes identifies an oLNP formulation, C14‐O2, with innate tropism to monocytes. In a proof‐of‐concept study, the C14‐O2 LNP is used to engineer functional CD19‐CAR monocytes in situ for robust B cell aplasia (45%) in healthy mice. This work highlights the utility of oxidized LNPs as a promising platform for engineering CAR macrophages/monocytes for solid tumor CAR monocyte therapy.

The application strategy of liposomes in organ targeting therapy.

Liposomes-microscopic phospholipid bubbles with bilayered membrane structure-have been a focal point in drug delivery research for the past 30 years. Current liposomes possess a blend of biocompatibility, drug loading efficiency, prolonged circulation and targeted delivery. Tailored liposomes, varying in size, charge, lipid composition, and ratio, have been developed to address diseases in specific organs, thereby enhancing drug circulation, accumulation at lesion sites, intracellular delivery, and treatment efficacy for various organ-specific diseases. For further successful development of this field, this review summarized liposomal strategies for targeting different organs in series of major human diseases, including widely studied cardiovascular diseases, liver and spleen immune diseases, chronic or acute kidney injury, neurodegenerative diseases, and organ-specific tumors. It highlights recent advances of liposome-mediated therapeutic agent delivery for disease intervention and organ rehabilitation, offering practical guidelines for designing organ-targeted liposomes. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures.

Red Blood Cell Adenylate Energetics Is Related to Endothelial and Microvascular Function in Long COVID.

Adenine nucleotides play a critical role in maintaining essential functions of red blood cells (RBCs), including energy metabolism, redox status, shape fluctuations and RBC-dependent endothelial and microvascular functions. Recently, it has been shown that infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might lead to morphological and metabolic alterations in erythrocytes in both mild and severe cases of coronavirus disease (COVID-19). However, little is known about the effects of COVID-19 on the nucleotide energetics of RBCs nor about the potential contribution of nucleotide metabolism to the long COVID syndrome. This study aimed to analyze the levels of adenine nucleotides in RBCs isolated from patients 12 weeks after mild SARS-CoV-2 infection who suffered from long COVID symptoms and to relate them with the endothelial and microvascular function parameters as well as the rate of peripheral tissue oxygen supply. Although the absolute quantities of adenine nucleotides in RBCs were rather slightly changed in long COVID individuals, many parameters related to the endothelial and microcirculatory function showed significant correlations with RBC adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentration. A particularly strong relationship was observed between ATP in RBCs and the serum ratio of arginine to asymmetric dimethylarginine-an indicator of endothelial function. Consistently, a positive correlation was also observed between the ATP/ADP ratio and diminished reactive hyperemic response in long COVID patients, assessed by the flow-mediated skin fluorescence (FMSF) technique, which reflected decreased vascular nitric oxide bioavailability. In addition, we have shown that patients after COVID-19 have significantly impaired ischemic response parameters (IR max and IR index), examined by FMSF, which revealed diminished residual bioavailability of oxygen in epidermal keratinocytes after brachial artery occlusion. These ischemic response parameters revealed a strong positive correlation with the RBC ATP/ADP ratio, confirming a key role of RBC bioenergetics in peripheral tissue oxygen supply. Taken together, the outcomes of this study indicate that dysregulation of metabolic processes in erythrocytes with the co-occurring endothelial and microvascular dysfunction is associated with diminished intracellular oxygen delivery, which may partly explain long COVID-specific symptoms such as physical impairment and fatigue.

Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.

Selective Intracellular Delivery of Antibodies in Cancer Cells with Nanocarriers Sensing Endo/Lysosomal Enzymatic Activity

AbstractThe differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody‐loaded polymeric nanocarriers having CTSB‐activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell‐specific protein delivery using stimuli‐responsive nanocarriers with controlled endosomal escape.

Selective Intracellular Delivery of Antibodies in Cancer Cells with Nanocarriers Sensing Endo/Lysosomal Enzymatic Activity.

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.

Shock wave assisted intracellular delivery of antibiotics against bone infection with Staphylococcus aureus via P2X7 receptors

BackgroundTreatment of chronic osteomyelitis (bone infection) remains a clinical challenge; in particular, it requires enhanced delivery of antibiotic drugs for the treatment of intracellular Staphylococcus aureus (S. aureus), which prevents infection recurrence and resistance. Previous studies have found that noninvasive shock waves used to treat musculoskeletal diseases can alter cell permeability, however, it is unclear whether shock waves alter cell membrane permeability in chronic osteomyelitis. Furthermore, it remains unknown whether such changes in permeability promote the entry of antibiotics into osteoblasts to exert antibacterial effects. MethodsIn our study, trypan blue staining was used to determine the shock wave parameters that had no obvious damage to the osteoblast model; the effect of shocks waves on the cell membrane permeability of osteoblast model was detected by BODIPY®FL vancomycin; high performance liquid chromatography-mass spectrometry (HLPC-MS) was used to detect the effect of shock wave on the entry of antibiotics into the osteoblast model; plate colony counting method was used to detect the clearance effect of shock wave assisted antibiotics on S. aureus in the osteoblast model. To explore the mechanism, the effect of different pulses of shock waves on S. aureus was examined by plate colony counting method, besides, P2X7 receptor in osteoblast was detected by immunofluorescence and the extracellular ATP levels was detected. Furthermore, the effect of P2X7 receptor antagonists KN-62 or A740003 on the intracellular antibacterial activity of shock-assisted antibiotics was observed. Then, we used S. aureus to establish a rat model of chronic tibial osteomyelitis and investigated the efficacy and safety of shock-wave assisted antibiotics in the treatment of chronic osteomyelitis in rats. ResultsThe viability of the osteoblast models of intracellular S. aureus infection was not significantly affected by the application of up to 400 shock wave pulses at 0.21 mJ/mm2. Surprisingly, the delivery of BODIPY®FL vancomycin to osteoblast model cells was markedly enhanced by this shock wave treatment. Furthermore, the shock wave therapy increased the delivery of hydrophilic antibiotics (vancomycin and cefuroxime sodium), but not lipophilic antibiotics (rifampicin and levofloxacin), which improved the intracellular antibacterial effect. Afterwards, we discovered that shock wave treatment increased the extracellular concentration of ATP (the P2X7 receptor activator)， while KN-62 or A740003, a P2X7 receptor inhibitor, decreased intracellular antibacterial activity. We then found that 0.1 mL of 1 × 1011 CFU/mL ATCC25923 S. aureus was suitable for modeling chronic osteomyelitis in rats. Besides, the shock wave-assisted vancomycin treatment with the strongest antibacterial and osteogenic effects among the tested treatments was confirmed in vivo by imaging examination, microbiological cultures, and histopathology, with favorable safety. ConclusionsOur results suggest that shock waves can promote the entry of antibiotics into osteoblasts for antibacteria by changing the cell membrane permeability in a P2X7 receptor-dependent manner. Besides, considering antibacterial and osteogenic efficiency and a high degree of safety in rat osteomyelitis model, shock wave-assisted vancomycin treatment may thus represent a possible adjuvant therapy for chronic osteomyelitis.

Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells

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.

The 8-hydroxyquinoline derivative, clioquinol, is an alpha-1 adrenoceptor antagonist

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.

Uptake pathways of cell-penetrating peptides in the context of drug delivery, gene therapy, and vaccine development

Cell-penetrating peptides have been extensively utilized for the purpose of facilitating the intracellular delivery of cargo that is impermeable to the cell membrane. The researchers have exhibited proficient delivery capabilities for oligonucleotides, thereby establishing cell-penetrating peptides as a potent instrument in the field of gene therapy. Furthermore, they have demonstrated a high level of efficiency in delivering several additional payloads. Cell penetrating peptides (CPPs) possess the capability to efficiently transport therapeutic molecules to specific cells, hence offering potential remedies for many illnesses. Hence, their utilization is imperative for the improvement of therapeutic vaccines. In contemporary studies, a plethora of cell-penetrating peptides have been unveiled, each characterized by its own distinct structural attributes and associated mechanisms. Although it is widely acknowledged that there are multiple pathways through which particles might be internalized, a comprehensive understanding of the specific mechanisms by which these particles enter cells has to be fully elucidated. The absorption of cell-penetrating peptides can occur through either direct translocation or endocytosis. However, it is worth noting that categories of cell-penetrating peptides are not commonly linked to specific entrance mechanisms. Furthermore, research has demonstrated that cell-penetrating peptides (CPPs) possess the capacity to enhance antigen uptake by cells and facilitate the traversal of various biological barriers. The primary objective of this work is to examine the mechanisms by which cell-penetrating peptides are internalized by cells and their significance in facilitating the administration of drugs, particularly in the context of gene therapy and vaccine development. The current study investigates the immunostimulatory properties of numerous vaccine components administered using different cell-penetrating peptides (CPPs). This study encompassed a comprehensive discussion on various topics, including the uptake pathways and mechanisms of cell-penetrating peptides (CPPs), the utilization of CPPs as innovative vectors for gene therapy, the role of CPPs in vaccine development, and the potential of CPPs for antigen delivery in the context of vaccine development.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.