Tool For Intracellular Delivery Research Articles

Analysis of In Situ Electroporation Utilizing Induced Electric Field at a Wireless Janus Microelectrode.

In situ electroporation, a non-invasive technique for enhancing the permeability of cell membranes, has emerged as a powerful tool for intracellular delivery and manipulation. This method allows for the precise introduction of therapeutic agents, such as nucleic acids, drugs, and proteins, directly into target cells within their native tissue environment. Herein, we introduce an innovative electroporation strategy that employs a Janus particle (JP)-based microelectrode to generate a localized and controllable electric field within a microfluidic chip. The microfluidic device is engineered with an indium tin oxide (ITO)-sandwiched microchannel, where the electric field is applied, and suspended JP microelectrodes that induce a stronger localized electric field. The corresponding simulation model is developed to better understand the dynamic electroporation process. Numerical simulations for both single-cell and chain-assembled cell electroporation have been successfully conducted. The effects of various parameters, including pulse voltage, duration medium conductivity, and radius of Janus microelectrode, on cell membrane permeabilization are systematically investigated. Our findings indicate that the enhanced electric intensity near the poles of the JP microelectrode significantly contributes to the electroporation process. In addition, the distribution for both transmembrane voltage and the resultant nanopores can be altered by conveniently adjusting the relative position of the JP microelectrode, demonstrating a selective and in situ electroporation technique for spatial control over the delivery area. Moreover, the obtained differences in the distribution of electroporation between chain cells can offer insightful directives for the electroporation of tissues or cell populations, enabling the precise and targeted modulation of specific cell populations. As a proof of concept, this work can provide a robust alternative technique for the study of complex and personalized cellular processes.

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.

Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

Engineering of Multivalent Membrane-Anchored DNA Frameworks for Precise Profiling of Variable Membrane Permeability During Reversible Electroporation.

Electroporation techniques have emerged as attractive tools for intracellular delivery, rendering promising prospects towards clinical therapies. Transient disruption of membrane permeability is the critical process for efficient electroporation-based cargo delivery. However, smart nanotools for precise characterization of transient membrane changes induced by strong electric pulses are extremely limited. Herein, multivalent membrane-anchored fluorescent nanoprobes (MMFNPs) that take advantages of flexible functionalization and spatial arrangement of DNA frameworks are developed for in situ evaluation of electric field-induced membrane permeability during reversible electroporation . Single-molecule fluorescence imaging techniques are adopted to precisely verify the excellent analytical performance of the engineered MMFNPs. Benefited from tight membrane anchoring and sensitive adenosine triphosphate (ATP)profiling, varying degrees of membrane disturbances are visually exhibited under different intensities of the microsecond pulse electric field (µsPEF). Significantly, the dynamic process of membrane repair during reversible electroporation is well demonstrated via ATP fluctuations monitored by the designed MMFNPs. Furthermore, molecular dynamics (MD) simulations are performed for accurate verification of electroporation-driven dynamic cargo entry via membrane nanopores. This work provides an avenue for effectively capturing transient fluctuations of membrane permeability under external stimuli, offering valuable guidance for developing efficient and safe electroporation-driven delivery strategies for clinical diagnosis and therapeutics.

Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds

Virus-like particles (VLPs) are nanostructures assemble from viral proteins. Besides widely used for vaccine development, VLPs have also been explored as nanocarriers for cargo delivery as they combine the key advantages of viral and non-viral vectors. While it protects cargo molecules from degradation, the VLP has good cell penetrating property to mediate cargo passing the cell membrane and released into cells, making the VLP an ideal tool for intracellular delivery of biomolecules and drugs. Great progresses have been achieved and multiple challenges are still on the way for broad applications of VLP as delivery vectors. Here we summarize current advances and applications in VLP as a delivery vector. Progresses on delivery of different types of biomolecules as well as drugs by VLPs are introduced, and the strategies for cargo packaging are highlighted which is one of the key steps for VLP mediated intracellular delivery. Production and applications of VLPs are also briefly reviewed, with a discussion on future challenges in this rapidly developing field.

FRET-Based In-Cell Detection of Highly Selective Supramolecular Complexes of meso-Tetraarylporphyrin with Peptide/BODIPY-Modified Per-O-Methyl-β-Cyclodextrins.

Artificial supramolecular systems capable of self-assembly and that precisely function in biological media are in high demand. Herein, we demonstrate a highly specific host-guest-pair system that functions in living cells. A per-O-methyl-β-cyclodextrin derivative (R8-B-CDMe ) bearing both an octaarginine peptide chain and a BODIPY dye was synthesized as a fluorescent intracellular delivery tool. R8-B-CDMe was efficiently taken up by HeLa cells through both endocytosis and direct transmembrane pathways. R8-B-CDMe formed a 2 : 1 inclusion complex with tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a guest molecule in water, from which fluorescence resonance energy transfer (FRET) from R8-B-CDMe to TPPS was observed. The FRET phenomenon was clearly detected in living cells using confocal microscopy techniques, which revealed that the formed supramolecular R8-B-CDMe /TPPS complex was maintained within the cells. The R8-B-CDMe cytotoxicity assay revealed that the addition of TPPS counteracts the strong cytotoxicity (IC50 =16 μM) of the CD cavity due to complexation within the cells. A series of experiments demonstrated the bio-orthogonality of the supramolecular per-O-methyl-β-CD/tetraarylporphyrin host-guest pair in living cells.

Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

Synthesis of six-membered carbocyclic ring α,α-disubstituted amino acids and arginine-rich peptides to investigate the effect of ring size on the properties of the peptide

Cell-penetrating peptides (CPPs) have been attracting attention as tools for intracellular delivery of membrane-impermeant functional molecules. Among the variety of CPPs that have been developed, many are composed of both natural and unnatural amino acids. We previously synthesized α,α-disubstituted α-amino acids (dAAs) containing a five-membered carbocyclic ring in its side chain and revealed the utility of dAAs for the development of novel CPPs. In the present study, we designed a six-membered carbocyclic ring dAA with an amino group on the ring and introduced it into arginine (Arg)-rich peptides to further investigate the value of dAAs for developing CPPs. We also assessed the effects of the size of the dAA carbocyclic ring on cellular uptake of dAA-containing peptides. The stability of the peptide’s secondary structure and its membrane permeability were both greater in dAA-containing peptides than in an Arg nonapeptide. However, the number of carbon atoms in the dAA side chain ring had little effect. Nevertheless, these results show the utility of cyclic dAAs in the design of novel CPPs containing unnatural amino acids.

Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel.

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a microfluidic system for precise and rapid single-cell electroporation and simultaneous impedance monitoring in a constriction microchannel. When single cells (A549) were continuously passing through the constriction microchannel, a localized high electric field was applied on the cell membrane, which resulted in highly efficient (up to 96.6%) electroporation. During a single cell entering the constriction channel, an abrupt impedance drop was noticed and demonstrated to be correlated with the occurrence of electroporation. Besides, while the cell was moving in the constriction channel, the stabilized impedance showed the capability to quantify the electroporation extent. The correspondence of the impedance variation and electroporation was validated by the intracellular delivery of the fluorescence indicator (propidium iodide). Based on the obtained results, this system is capable of precise control of electroporation and real-time, label-free impedance assessment, providing a potential tool for intracellular delivery and other biomedical applications.

Control of Mitochondrial Localization Using Thermoresponsive Sulfobetaine Polymer.

Fast intracellular migration and controlled localization of molecules represent significant challenges for future applications of drug discovery and related fields. In this study, thermoresponsive sulfobetaine polymers with pyridinium cations are evaluated as biocompatible and mitochondria-localizing agents. Among the polymers, poly(3-(4-(2-methacrylamido)ethyl pyridinio-1-yl)propane-1-sulfonate), P(E-PySMAAm)14k (Mn = 14 000g mol-1 ) exhibit thermoresponsiveness with an upper critical solution temperature like behavior in cell culture medium containing serum with minimal cytotoxicity. Upon the addition of P(E-PySMAAm)14k to HeLa cells at temperatures above the clearing point at 37 °C, effective localization is observed in mitochondria. However, increased intensity but nonspecific localization is observed below the clearing point at 4 °C. Doxorubicin is conjugated to the P(E-PySMAAm) and achieves effective mitochondrial delivery while maintaining drug efficacy. Such sulfobetaine polymers represent promising tools for intracellular delivery of molecules.

Targeting extracellular vesicles-mediated hepatic inflammation as a therapeutic strategy in liver diseases.

Extracellular Vesicles (EVs) are nano- to micro-sized membranous vesicles that can be produced by normal and diseased cells. As carriers of biologically active molecules including proteins, lipids and nucleic acids, EVs mediate cell-to-cell communication and execute diverse functions by delivering their cargoes to specific cell types. Hepatic inflammation caused by virus infection, autoimmunity and malignancy is a common driver of progressive liver fibrosis and permanent liver damage. Emerging evidence has shown that EVs-mediated inflammation as critical player in the progression of liver diseases since they shuttle within the liver as well as between other tissues with inflammatory signals. Therefore, targeting inflammatory EVs could represent a potential therapeutic strategy in liver diseases. Moreover, EVs are emerging as a promising tool for intracellular delivery of therapeutic nucleic acid. In this review, we will discuss not only recent advances on the role of EVs in mediating hepatic inflammation and present strategies for targeted therapy on the context of liver diseases but also the challenging questions that need to be answered in the field.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α-Amino Acids with Basic Functional Groups.

In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,α-disubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool.

Enhanced intracellular delivery of macromolecules by melittin derivatives mediated cellular uptake

Various cell-penetrating peptides (CPPs) have been utilized as a promising tool for intracellular delivery of therapeutic macromolecules. In this study, hemolytic membrane-active peptide (Melittin) has been utilized for intracellular uptake of macromolecules by its sequential fragmentation. Among its fragmented derivatives, melittin(1-14) showed highly efficient delivery of proteins into HeLa cells, while maintaining their biological activities. The highest level of intracellular delivery was observed when ß-galactosidase and Cytochrome C were co-delivered with melittin(1-14). These data suggested that melittin(1-14) could be a useful adjuvant for the enhanced intracellular delivery of therapeutic macromolecules.

Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.

Development of helix-stabilized cell-penetrating peptides containing cationic α,α-disubstituted amino acids as helical promoters

Cell-penetrating peptides (CPP) have attracted many scientists’ attention as intracellular delivery tools due to their high cargo molecule transportation efficiency and low cytotoxicity. Therefore, in many research fields CPP, such as HIV-Tat and oligoarginine (Rn), are used to deliver hydrophilic drugs and biomolecules, including proteins, DNA, and RNA. We designed four types of CPP that contained cationic α,α-disubstituted amino acids (ApiC2Gu and ApiC4Gu) as helical promoters; i.e., 1–4 [FAM-β-Ala-(l-Arg-l-Arg-Xaa)3-(Gly)3-NH2 (1: Xaa=ApiC2Gu, 2: Xaa=ApiC4Gu), 3: FAM-β-Ala-(l-Arg)8-ApiC2Gu-(Gly)3-NH2, and 4: FAM-β-Ala-(l-Arg)5-ApiC2Gu-(l-Arg)2-ApiC2Gu-(Gly)3-NH2], and investigated their preferred secondary structures and cell membrane-penetrating ability. As a result, we found that the permeation efficiency of the CPP was affected by the number of helical promoters in their sequences. Specially, peptide 1, which contained three ApiC2Gu residues, formed a stable helical structure and passed through the cell membrane more efficiently than the other peptides. Moreover, it was demonstrated that the spatial arrangement of the peptides’ side chains also influenced their permeability and the helical stabilization of their main chains.

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R8 ) and CHK6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment.

Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier.

Cover Picture: A Helix‐Stabilized Cell‐Penetrating Peptide as an Intracellular Delivery Tool (ChemBioChem 2/2016)

Cover Picture: A Helix‐Stabilized Cell‐Penetrating Peptide as an Intracellular Delivery Tool (ChemBioChem 2/2016)