RESUMEN

Telomere length plays a crucial role in cellular aging and the risk of diseases. Unlike normal cells, cancer cells can extend their own survival by maintaining telomere stability through telomere maintenance mechanism. Therefore, regulating the lengths of telomeres have emerged as a promising approach for anti-cancer treatment. In this study, we introduce a nanoscale octopus-like structure designed to induce physical entangling of telomere, thereby efficiently triggering telomere dysfunction. The nanoscale octopus, composed of eight-armed PEG (8-arm-PEG), are functionalized with cell penetrating peptide (TAT) to facilitate nuclear entry and are covalently bound to N-Methyl Mesoporphyrin IX (NMM) to target G-quadruplexes (G4s) present in telomeres. The multi-armed configuration of the nanoscale octopus enables targeted binding to multiple G4s, physically disrupting and entangling numerous telomeres, thereby triggering telomere dysfunction. Both in vitro and in vivo experiments indicate that the nanoscale octopus significantly inhibits cancer cell proliferation, induces apoptosis through telomere entanglement, and ultimately suppresses tumor growth. This research offers a novel perspective for the development of innovative anti-cancer interventions and provides potential therapeutic options for targeting telomeres.

Asunto(s)

Apoptosis , Telómero , Telómero/metabolismo , Apoptosis/efectos de los fármacos , Humanos , Animales , Línea Celular Tumoral , Ratones , G-Cuádruplex/efectos de los fármacos , Ratones Desnudos , Polietilenglicoles/química , Proliferación Celular/efectos de los fármacos , Ratones Endogámicos BALB C , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Femenino , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Nanoestructuras/química

RESUMEN

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.

Asunto(s)

Péptidos de Penetración Celular , Insulina , Micelas , Polietilenglicoles , Insulina/administración & dosificación , Insulina/farmacocinética , Insulina/química , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Animales , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Humanos , Tamaño de la Partícula , Administración por Inhalación , Masculino , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Ratas Sprague-Dawley , Moco/química , Moco/metabolismo , Moco/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Glucemia/efectos de los fármacos , Glucemia/análisis

RESUMEN

Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.

Asunto(s)

Membrana Celular , Péptidos de Penetración Celular , Citoplasma , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Células HeLa , Animales

RESUMEN

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Asunto(s)

Membrana Celular , Histona Desacetilasas , Proteína 2 de Unión a Metil-CpG , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/química , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Histona Desacetilasas/metabolismo , Histona Desacetilasas/química , Histona Desacetilasas/genética , Células HEK293 , Transporte de Proteínas , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética

RESUMEN

One of the bottlenecks to bringing new therapies to the clinic has been a lack of vectors for delivering novel therapeutics in a targeted manner. Cell penetrating peptides (CPPs) have received a lot of attention and have been the subject of numerous developments since their identification nearly three decades ago. Known for their transduction abilities, they have generally been considered inert vectors. In this review, we present a schema for their classification, highlight what is known about their mechanism of transduction, and outline the existing literature as well as our own experience, vis a vis the intrinsic anti-inflammatory properties that certain CPPs exhibit. Given the inflammatory responses associated with viral vectors, CPPs represent a viable alternative to such vectors; furthermore, the anti-inflammatory properties of CPPs, mostly through inhibition of the NF-κB pathway, are encouraging. Much more work in relevant animal models, toxicity studies in large animal models, and ultimately human trials are needed before their potential is fully realized.

Asunto(s)

Antiinflamatorios , Péptidos de Penetración Celular , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Humanos , Antiinflamatorios/farmacología , Antiinflamatorios/química , Animales , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

Asunto(s)

Membrana Celular , Péptidos de Penetración Celular , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Humanos , Membrana Celular/metabolismo , Células Jurkat , Transporte de Proteínas

RESUMEN

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.

Asunto(s)

Radioisótopos de Yodo , Leucocitos , Radiofármacos , Humanos , Radiofármacos/farmacocinética , Péptidos de Penetración Celular/química , Marcaje Isotópico/métodos

RESUMEN

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.

Asunto(s)

Antibacterianos , Péptidos de Penetración Celular , Gonorrea , Neisseria gonorrhoeae , Neisseria gonorrhoeae/efectos de los fármacos , Humanos , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/química , Antibacterianos/farmacología , Antibacterianos/química , Animales , Ratones , Femenino , Biopelículas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Ciclización , Lipopolisacáridos/metabolismo , Arginina/farmacología , Arginina/química

RESUMEN

The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.

Asunto(s)

Mucosa Nasal , Polifenoles , Taninos , Taninos/química , Polifenoles/química , Polifenoles/administración & dosificación , Mucosa Nasal/metabolismo , Mucosa Nasal/inmunología , Animales , Nanopartículas/química , Humanos , Péptidos de Penetración Celular/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Adhesivos/química , Depuración Mucociliar/efectos de los fármacos , Inmunoglobulina A , Ratones

RESUMEN

Targeted protein degradation through the lysosomal pathway has attracted increasing attention and expanded the scope of degradable proteins. However, the endogenous lysosomal degradation strategies are mainly based on antibodies or nanobodies. Effective small molecule lysosomal degraders are still rather rare. Herein, a new lysosomal degradation approach, termed peptide-mediated small molecule lysosome-targeting chimeras (PSMLTACs), was developed by the incorporation of small molecule ligands with a lysosome-sorting NPGY motif containing the cell-penetrating peptide. PSMLTACs were successfully applied to degrade both membrane and intracellular targets. In particular, the PSMLTAC strategy demonstrated higher degradation efficiency on membrane target PD-L1 and intracellular target PDEδ than corresponding PROTAC degraders. Taken together, this proof-of-concept provides a convenient and effective strategy for targeted protein degradation.

Asunto(s)

Lisosomas , Proteolisis , Lisosomas/metabolismo , Humanos , Proteolisis/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo

RESUMEN

Despite ongoing research on antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), their precise translocation mechanism remains elusive. This includes Buforin 2 (BF2), a well-known AMP, for which spontaneous translocation across the membrane has been proposed but a high barrier has been calculated. Here, we used computer simulations to investigate the effect of a nonequilibrium situation where the peptides are adsorbed on one side of the lipid bilayer, mimicking experimental conditions. We demonstrated that the asymmetric membrane adsorption of BF2 enhances its translocation across the lipid bilayer by lowering the energy barrier by tens of kJ mol-1. We showed that asymmetric membrane adsorption also reduced the free energy barrier of lipid flip-flop but remained unlikely even at BF2 surface saturation. These results provide insight into the driving forces behind membrane translocation of cell-penetrating peptides in nonequilibrium conditions, mimicking experiments.

Asunto(s)

Membrana Dobles de Lípidos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Adsorción , Terpenos/química , Terpenos/farmacología , Simulación de Dinámica Molecular , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Termodinámica , Membrana Celular/metabolismo , Membrana Celular/química , Proteínas

RESUMEN

Proteolysis targeting chimeras (PROTAC) are bifunctional chimeric molecules capable of directly degrading binding proteins through the ubiquitin-proteasome pathway. PROTACs have demonstrated significant potential in overcoming drug resistance and targeting previously untreatable targets. However, several limitations still need to be addressed, including their high molecular weight resulting in poor membrane permeability and bioavailability. In this study, we proposed that cancer-targeted penetrating peptides could enhance the cell permeability of PROTACs. We developed 26 novel targeted penetrating peptides for leukemia and lymphoma cells, among which C9C-f(3Bta) and Cyclo-C9C-R exhibited superior membrane permeability, targetability, and stability. By combining C9C-f(3Bta) and Cyclo-C9C-R with IMA-PROTAC, we effectively enhanced the anti-proliferative activity of IMA-PROTAC, facilitated degradation of Bcr-Abl protein in K562 cells, and reduced downstream STAT5 phosphorylation. Furthermore, the combined application promoted cell apoptosis while blocking G1 phase progression. HPLC-MRM-MS revealed that the combination of C9C-f(3Bta) or Cyclo-C9C-R with IMA-PROTAC significantly enhanced intracellular IMA-PROTAC content. In summary, our proof-of-concept study validated the hypothesis that combining PROTACs with targeted penetrating peptides can improve protein degradation efficiency as well as anti-proliferative capabilities.

Asunto(s)

Antineoplásicos , Apoptosis , Proliferación Celular , Leucemia , Linfoma , Proteolisis , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Leucemia/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proteolisis/efectos de los fármacos , Linfoma/tratamiento farmacológico , Linfoma/patología , Linfoma/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Relación Estructura-Actividad , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/síntesis química , Estructura Molecular , Células K562 , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos

RESUMEN

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.

Asunto(s)

Péptidos de Penetración Celular , Neurotoxinas , Venenos de Araña , Venenos de Araña/química , Venenos de Araña/genética , Venenos de Araña/toxicidad , Animales , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/química , Neurotoxinas/toxicidad , Neurotoxinas/farmacología , Neurotoxinas/genética , Lectinas de Plantas/farmacología , Lectinas de Plantas/genética , Lectinas de Plantas/química , Insecticidas/farmacología , Insecticidas/toxicidad

RESUMEN

Cell-penetrating peptides (CPPs) are crucial for delivering macromolecules such as nucleic acids into cells. This study investigates the effectiveness of dual-modified penetratin peptides, focusing on the impact of stapling structures and an endosomal escape domain (EED) on enhancing intracellular uptake. Some CPPs were synthesized with an EED at either the N- or C-terminus and stapling structures, and then complexed with plasmid DNA (pDNA) to evaluate their cellular uptake. Results revealed that the combination of stapling and an EED significantly improved delivery efficiency, primarily via macropinocytosis and clathrin-mediated endocytosis. These findings underscore the importance of optimizing CPP sequences for effective nucleic acid delivery systems.

Asunto(s)

Péptidos de Penetración Celular , Endosomas , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/farmacología , Humanos , Endosomas/metabolismo , ADN/química , Plásmidos , Células HeLa

RESUMEN

Advancements in medicine and pharmacology have led to the development of systems that deliver biologically active molecules inside cells, increasing drug concentrations at target sites. This improves effectiveness and duration of action and reduces side effects on healthy tissues. Cell-penetrating peptides (CPPs) show promise in this area. While traditional medicinal chemistry methods have been used to develop CPPs, machine learning techniques can speed up and reduce costs in the search for new peptides. A predictive algorithm based on machine learning models was created to identify novel CPP sequences using molecular descriptors using a combination of algorithms like k-nearest neighbors, gradient boosting, and random forest. Some potential CPPs were found and tested for cytotoxicity and penetrating ability. A new low-toxicity CPP was discovered from the Rhopilema esculentum venom proteome through this study.

Asunto(s)

Algoritmos , Péptidos de Penetración Celular , Aprendizaje Automático , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Humanos , Animales , Secuencia de Aminoácidos , Venenos de Avispas/química , Proteoma

RESUMEN

Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001's interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.

Asunto(s)

Péptidos de Penetración Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Peptidilprolil Isomerasa de Interacción con NIMA , Osteogénesis , Factor de Transcripción Sp7 , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Osteogénesis/efectos de los fármacos , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Ratones , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/química , Factor de Transcripción Sp7/metabolismo , Factor de Transcripción Sp7/genética , Humanos , Femenino , Estabilidad Proteica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/citología

RESUMEN

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL25-35) and ε-poly-l-ß-lysine consisting of l-ß-lysine residues (ε-PßL4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL5-14-FAM, with HeLa cells. Unlike ε-PαL25-35-FAM, ε-PαL5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 µM) was required for the internalization events. ε-PαL5-14 has a chain length almost equal to that of the membrane permeable ε-PßL4-13, which can enter cells at low concentrations. Considering that the basicity of the ß-amino group is higher than that of α-amino acid at physiological pH, ε-PßL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PßL would be more advantageous for cellular internalization of cargo proteins than ε-PαL25-35.

Asunto(s)

Péptidos de Penetración Celular , Endocitosis , Polilisina , Humanos , Células HeLa , Polilisina/química , Polilisina/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Polielectrolitos/química , Química Clic

RESUMEN

The messenger RNA (mRNA) SARS-CoV-2 vaccines have demonstrated the therapeutic potential of this novel drug modality. Protein expression is the consequence of a multistep delivery process that relies on proper packaging into nanoparticle carriers to protect the mRNA against degradation enabling effective cellular uptake and endosomal release, and liberating the mRNA in the cytosol. Bottlenecks along this route remain challenging to pinpoint. Although methods to assess endosomal escape of carriers have been developed, versatile strategies to identify bottlenecks along the delivery trajectory are missing. Here, it is shown that co-incubating an inefficient nanoparticle formulation with an efficient one solves this problem. Cells were co-incubated with mRNA nanoparticles formed with either the efficient cell-penetrating peptide (CPP) PepFect14 or the inefficient CPP nona-arginine (R9). Co-transfection enhanced cellular uptake and endosomal escape of R9-formulated mRNA, resulting in protein expression, demonstrating that both vectors enter cells along the same route. In addition, cells were transfected with a galectin-9-mCherry fusion protein to detect endosomal rupture. Remarkably, despite endosomal release, mRNA remained confined to punctate structures, identifying mRNA liberation as a further bottleneck. In summary, co-transfection offers a rapid means to identify bottlenecks in cytosolic mRNA delivery, supporting the rational design and optimization of intracellular mRNA delivery systems.

Asunto(s)

Péptidos de Penetración Celular , Endosomas , Nanopartículas , ARN Mensajero , Transfección , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Humanos , Péptidos de Penetración Celular/química , Endosomas/metabolismo , Nanopartículas/química , Transfección/métodos , SARS-CoV-2/genética , SARS-CoV-2/efectos de los fármacos , Vacunas contra la COVID-19/administración & dosificación , Portadores de Fármacos/química , COVID-19 , Técnicas de Transferencia de Gen , Sistemas de Liberación de Medicamentos/métodos

RESUMEN

Glioblastoma is a common brain tumor that poses considerable challenges in drug delivery. In this study, we investigated the potential of cell-based nanoparticles for targeted drug delivery to the glioblastoma sites. The anticancer drug of temozolomide (TMZ)-loaded T7-cholesterol nanoparticle micelles efficiently delivered nanoparticles to neutrophils and, subsequently, to the tumors. T7 is a cell-penetrating peptide that enhances the delivery of T7/TMZ to the target cells. T7 also serves as a transferrin target peptide, enabling targeted delivery to tumors. T7-conjugated cholesterol can self-assemble into micelles in aqueous solution and attach to the membrane of neutrophils. We confirmed that T7/TMZ nanoparticle micelles were efficiently located inside the neutrophils. Thereafter, T7/TMZ-conveyed neutrophils were administered to a glioblastoma mouse model, enabling neutrophils to penetrate the blood-brain barrier and deliver drugs directly to the tumor site. We evaluated the drug delivery efficiency and therapeutic effects of intravenous injection of T7/TMZ-conveyed neutrophils to a glioblastoma mouse model. These results demonstrate the promising role of neutrophil-based nanoparticle delivery systems in the targeted therapy of glioblastoma.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Neutrófilos , Temozolomida , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Animales , Ratones , Temozolomida/química , Temozolomida/farmacología , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Humanos , Nanopartículas/química , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Micelas , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Colesterol/química , Portadores de Fármacos/química , Péptidos de Penetración Celular/química , Fragmentos de Péptidos , Colágeno Tipo IV

RESUMEN

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Asunto(s)

Péptidos de Penetración Celular , Cisteína Endopeptidasas , Inmunoglobulina G , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/química , Humanos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Cationes/química , Péptidos/química , Péptidos/farmacología , Células HeLa , Sistemas de Liberación de Medicamentos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química