The utilization of nanoparticles for the intracellular delivery of theranostic agents faces one substantial limitation. Sequestration in intracellular vesicles prevents them from reaching the desired location in the cytoplasm or nucleus to deliver their cargo. We investigated whether three different cell-penetrating peptides (CPPs), namely, octa-arginine R8, polyhistidine KH27K and histidine-rich LAH4, could promote cytosolic and/or nuclear transfer of unique model nanoparticles-pseudovirions derived from murine polyomavirus. Two types of CPP-modified pseudovirions that carry the luciferase reporter gene were created: VirPorters-IN with CPPs genetically attached to the capsid interior and VirPorters-EX with CPPs noncovalently associated with the capsid exterior. We tested their transduction ability by luciferase assay and monitored their presence in subcellular fractions. Our results confirmed the overall effect of CPPs on the intracellular destination of the particles and suggested that KH27K has the potential to improve the cytosolic release of pseudovirions. None of the VirPorters caused endomembrane damage detectable by the Galectin-3 assay. Remarkably, a noncovalent modification was required to promote high transduction of the reporter gene and cytosolic delivery of pseudovirions mediated by LAH4. Together, CPPs in different arrangements have demonstrated their potential to improve pseudovirion invasion into cells, and these findings could be useful for the development of other nanoparticle-based delivery systems.
Cell-penetrating peptides (CPPs) are a promising tool for the intracellular delivery of cargo. Due to their ability to cross membranes while also cotransporting various cargoes, they offer great potential for biomedical applications. Several CPPs have been derived from viral proteins with natural roles in the viral replication cycle that require them to breach or fuse to cellular membranes. Additionally, the ability of viruses to cross membranes makes viruses and virus-based particles a convenient model for research on nanoparticle delivery and nanoparticle-mediated gene therapy. In this chapter, we aim to characterize CPPs derived from both structural and nonstructural viral proteins. Their function as enhancers of viral infection and transduction by viral nanoparticles as well as the main features of viral CPPs employed in intracellular cargo delivery are summarized to emphasize their potential use in nanomedicine.
Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.
- MeSH
- antitumorózní látky chemie farmakokinetika farmakologie MeSH
- buněčné sféroidy účinky léků metabolismus MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- glioblastom farmakoterapie metabolismus MeSH
- hematoencefalická bariéra metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové buňky kultivované MeSH
- nádory mozku farmakoterapie metabolismus MeSH
- neuropilin-1 metabolismus MeSH
- nosiče léků chemie farmakokinetika farmakologie MeSH
- oligopeptidy chemie farmakokinetika farmakologie MeSH
- penetrační peptidy chemie farmakokinetika farmakologie MeSH
- systémy cílené aplikace léků MeSH
- tuftsin analogy a deriváty farmakokinetika farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.
- MeSH
- genetické vektory * MeSH
- HEK293 buňky MeSH
- kapsida metabolismus ultrastruktura MeSH
- lidé MeSH
- myši MeSH
- oligopeptidy chemie metabolismus MeSH
- penetrační peptidy chemie metabolismus MeSH
- Polyomavirus genetika metabolismus ultrastruktura MeSH
- transdukce genetická * MeSH
- virion genetika metabolismus ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Mitochondria-targeting peptides represent an emergent tool for cancer inhibition. Here supramolecular assemblies of novel amphiphilic cell-penetrating peptides for targeting cancer cell mitochondria are reported. The employed strategy aims at amplifying the apoptotic stimuli by weakening the mitochondrial VDAC1 (voltage-dependent anion channel-1)-hexokinase-II (HK-II) interaction. Peptide engineering is performed with the N-terminus of the HK-II protein, which binds to VDAC1. First, a designed positively charged segment (pKV) is anchored to the specific 15 amino acid sequence (MIASHLLAYFFTELN) to yield a cell-penetrating peptide (pHK-pKV). Second, a lipid chain (Pal) is conjugated to the N-terminus of pHK-pKV in order to enhance the intracellular delivery of the HK-II scaffold. The self-assembly properties of these two synthetic peptides are investigated by synchrotron small-angle X-ray scattering (BioSAXS) and cryogenic transmission electron (cryo-TEM) imaging, which evidence the formation of nanoassemblies of ellipsoid-like shapes. Circular dichroism (CD) spectroscopy demonstrates the induction of partial α-helical structures in the amphiphilic peptides. Confocal microscopy reveals the specific mitochondrial location of Pal-pHK-pKV assemblies in human non-small cell lung cancer (NSCLC) A549 cells. The cytotoxicity and apoptotic studies indicate the enhanced bioactivity of Pal-pHK-pKV self-assembled reservoirs, which cause massive A549 cell death with regard to pHK-pKV. Of significance, Pal-pHK-pKV treatment of non-cancerous NCM460 cells resulted in substantially lower cytotoxicity. The results demonstrate the potential of self-assembled lipo-peptide (HK-II-derived) conjugates as a promising strategy in cancer therapy.
- MeSH
- buněčná smrt účinky léků MeSH
- buňky A549 MeSH
- hexokinasa metabolismus MeSH
- lidé MeSH
- lipidy chemie MeSH
- lipopeptidy chemická syntéza terapeutické užití MeSH
- mitochondrie metabolismus MeSH
- nádory plic farmakoterapie patologie MeSH
- napětím ovládaný aniontový kanál 1 metabolismus MeSH
- penetrační peptidy chemická syntéza metabolismus MeSH
- povrchově aktivní látky metabolismus MeSH
- systémy cílené aplikace léků metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.
- MeSH
- arginin metabolismus fyziologie MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- fúze membrán účinky léků fyziologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membrány metabolismus MeSH
- penetrační peptidy chemie metabolismus MeSH
- peptidy chemie fyziologie MeSH
- pseudopodia metabolismus fyziologie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cell-penetrating compounds are substances that enhance the cellular uptake of various molecular cargoes that do not easily cross the cellular membrane. The majority of cell-penetrating compounds described in the literature are cell-penetrating peptides (CPPs). This review summarizes the various structural types of cell-penetrating compounds, with the main focus on CPPs. The authors present a brief overview of the history of CPPs, discuss the various types of conjugation of CPPs to biologically active cargoes intended for cell internalization, examine the cell-entry mechanisms of CPPs, and report on the applications of CPPs in research and in preclinical and clinical studies.
- MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endocytóza účinky léků fyziologie MeSH
- lidé MeSH
- penetrační peptidy aplikace a dávkování genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- systémy cílené aplikace léků metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Drug access to the CNS is hindered by the presence of the blood-brain barrier (BBB), and the intranasal route has risen as a non-invasive route to transport drugs directly from nose-to-brain avoiding the BBB. In addition, nanoparticles (NPs) have been described as efficient shuttles for direct nose-to-brain delivery of drugs. Nevertheless, there are few studies describing NP nose-to-brain transport. Thus, the aim of this work was (i) to develop, characterize and validate in vitro olfactory cell monolayers and (ii) to study the transport of polymeric- and lipid-based NPs across these monolayers in order to estimate NP access into the brain using cell penetrating peptide (CPPs) moieties: Tat and Penetratin (Pen). All tested poly(d,l-lactide-co-glycolide) (PLGA) and nanostructured lipid carrier (NLC) formulations were stable in transport buffer and biocompatible with the olfactory mucosa cells. Nevertheless, 0.7% of PLGA NPs was able to cross the olfactory cell monolayers, whereas 8% and 22% of NLC and chitosan-coated NLC (CS-NLC) were transported across them, respectively. Moreover, the incorporation of CPPs to NLC surface significantly increased their transport, reaching 46% of transported NPs. We conclude that CPP-CS-NLC represent a promising brain shuttle via nose-to-brain for drug delivery.
- MeSH
- aplikace intranazální MeSH
- biologický transport MeSH
- chemie farmaceutická metody MeSH
- chitosan chemie MeSH
- čichová sliznice cytologie metabolismus MeSH
- hematoencefalická bariéra metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina mléčná chemie MeSH
- kyselina polyglykolová chemie MeSH
- lipidy chemie MeSH
- mozek metabolismus MeSH
- nanočástice * MeSH
- nos MeSH
- nosní sliznice metabolismus MeSH
- penetrační peptidy chemie MeSH
- polymery chemie MeSH
- potkani Wistar MeSH
- systémy cílené aplikace léků * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.
- MeSH
- biologický transport MeSH
- exprese genu MeSH
- HEK293 buňky MeSH
- klonování DNA MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací MeSH
- nanočástice chemie metabolismus MeSH
- penetrační peptidy metabolismus MeSH
- plazmidy genetika MeSH
- průtoková cytometrie MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus ultrastruktura MeSH
- transfekce MeSH
- western blotting MeSH
- zelené fluorescenční proteiny genetika izolace a purifikace metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
x
- MeSH
- Escherichia coli účinky léků MeSH
- europium MeSH
- penetrační peptidy * MeSH
- Schiffovy báze * chemická syntéza MeSH
- spektrofotometrie statistika a číselné údaje MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice statistika a číselné údaje MeSH
- terbium MeSH
- Publikační typ
- práce podpořená grantem MeSH
