Intracellular Delivery Of Biomolecules Research Articles

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

Characterization and Evaluation of Cell-Penetrating Activity of Brevinin-2R: An Amphibian Skin Antimicrobial Peptide

Natural peptides have been the source of some important tools to address challenges in protein therapy of diseases. Bypassing cell plasma membrane has been a bottleneck in the intracellular delivery of biomolecules. Among others, cell-penetrating peptides (CPPs) provide an efficient strategy for intracellular delivery of various cargos. Brevinin-2R peptide is an antimicrobial peptide isolated from the skin secretions of marsh frog, Rana ridibunda with semi-selective anticancer properties. Here, we investigated cell-penetrating properties of Brevinin-2R peptide and its ability to deliver functional protein cargos. Bioinformatics studies showed that Brevinin-2R is a cationic peptide with a net charge of + 5 with an alpha-helix structure and a heptameric ring at the carboxylic terminal due to disulfide bond between C19 and C25 amino acids and a hinge region at A10. To evaluate the ability of this peptide as a CPP, β-galactosidase protein and GFP were transfected into HeLa cells. The entry pathway of the peptide/protein complex into the cell was investigated by inhibiting endocytic pathways at 4°C. It was observed that Brevinin-2R can efficiently transfer β-galactosidase and GFP with 21% and 90% efficacy, respectively. Brevinin-2R opts for endocytosis pathways to enter cells. The cytotoxicity of this peptide against HeLa cells was studied using MTT assay. The results showed that at the concentration of 131.5μg/ml of Brevinin-2R peptide, the proliferation of 50% of HeLa cells was inhibited. The results of this study suggest that Brevinin-2R peptide can act as a CPP of natural origin and low cytotoxicity.

Development of multifunctional nanopipettes for controlled intracellular delivery and single-entity detection.

The intracellular delivery of biomolecules and nanoscale materials to individual cells has gained remarkable attention in recent years owing to its wide applications in drug delivery, clinical diagnostics, bio-imaging and single-cell analysis. It remains a challenge to control and measure the delivered amount in one cell. In this work, we developed a multifunctional nanopipette - containing both a nanopore and nanoelectrode (pyrolytic carbon) at the apex - as a facile, minimally invasive and effective platform for both controllable single-cell intracellular delivery and single-entity counting. While controlled by a micromanipulator, the baseline changes of the nanopore ionic current (I) and nanoelectrode open circuit potential (V) help to guide the nanopipette tip insertion and positioning processes. The delivery from the nanopore barrel can be facilely controlled by the applied nanopore bias. To optimize the intracellular single-entity detection during delivery, we studied the effects of the nanopipette tip geometry and solution salt concentration in controlled experiments. We have successfully delivered gold nanoparticles and biomolecules into the cell, as confirmed by the increased scattering and fluorescence signals, respectively. The delivered entities have also been detected at the single-entity level using either one or both transient I and V signals. We found that the sensitivity of the single-entity electrochemical measurement was greatly affected by the local environment of the cell and varied between cell lines.

A Universal Platform for High‐Efficiency “Engineering” Living Cells: Integration of Cell Capture, Intracellular Delivery of Biomolecules, and Cell Harvesting Functions

AbstractA universal platform for the efficient intracellular delivery of biomacromolecules with minimal trauma to the cells is highly desirable for biological research and clinical applications. Moreover, such a platform should include the ability to harvest the “engineered” cells, for particular in vitro or ex vivo conditions. Herein, a broadly applicable platform is presented with integrated multifunctions based on silicon nanowire arrays (SiNWAs) modified with a sugar‐responsive polymer containing phenylboronic acid (PBA) groups. Due to the synergistic effects of the specific recognition of PBA groups by sialic acid and “nanoenhancement” by the SiNWAs, this system shows a high capture capacity for both surface adherent and suspension cells overexpressing sialic acid on the membrane. Under appropriate near‐infrared irradiation, the photothermal properties of SiNWAs endow this system with high efficiency to deliver biomacromolecules into the captured cells by a membrane disruption mechanism. The cells thus “engineered” can be harvested simply by treatment with a nontoxic sugar solution, thereby maintaining good viability for subsequent applications. This method appears to have strong potential for the intracellular delivery of diverse biomacromolecules into both surface adherent and suspension cells, including hard‐to‐transfect suspension T cells, and may open up new pathways for engineering living cells.

Functionalized Spiky Particles for Intracellular Biomolecular Delivery.

The intracellular delivery of biomolecules is of significant importance yet challenging. In addition to the conventional delivery of nanomaterials that rely on biochemical pathways, vertical nanowires have been recently proposed to physically penetrate the cell membrane, thus enabling the direct release of biomolecules into the cytoplasm circumventing endosomal routes. However, due to the inherent attachment of the nanowires to a planar 2D substrate, nanowire cell penetrations are restricted to in vitro applications, and they are incapable of providing solution-based delivery. To overcome this structural limitation, we created polyethylenimine-functionalized microparticles covered with nanospikes, namely, “spiky particles”, to deliver biomolecules by utilizing the nanospikes to penetrate the cell membrane. The nanospikes might penetrate the cell membrane during particle engulfment, and this enables the bound biomolecules to be released directly into the cytosol. TiO2 spiky particles were fabricated through hydrothermal routes, and they were demonstrated to be biocompatible with HeLa cells, macrophage-like RAW cells, and fibroblast-like 3T3-L1 cells. The polyethylenimine-functionalized spiky particles provided direct delivery of fluorescent siRNA into cell cytosol and functional siRNA for gene knockdown as well as successful DNA plasmid transfection which were difficult to achieve by using microparticles without nanospikes. The spiky particles presented a unique direct cell membrane penetrant vehicle to introduce biomolecules into cell cytosol, where the biomolecules might bypass conventional endocytic degradation routes.

A Delivery Method for Poly‐histidine Tagged Proteins and Peptides for Transient Protein Expression with Light Control via Hollow Gold Nanoshells: Successful delivery of CRISPR Cas9 and Apoptotic Peptide NuBCP with NIR light control.

Intracellular delivery of biomolecules is hindered by the size and charge of the biomolecule. While a number of delivery methods are available for genetic material, few are available for larger biomolecules like peptides and proteins. The delivery of these biomolecules are often limited to viral transfections of the genetic material which can lead to off target gene editing due to prolonged expression of a protein within a cell. We have developed a delivery method for poly‐histidine tagged proteins and peptides of variable sizes and charges for transient protein expression with light control via hollow gold nanoshells (HGNs). The construct of our delivery system involves a thiolated DNA strand containing an NTA modification on the 3′ end attached to the gold surface. A his‐tagged protein or peptide of interest is attached to the HGN via copper‐NTA affinity. Endosomal uptake of these particles is mediated through an orthogonal strand containing a cell penetrating peptide (CPP). Protein release and endosomal disruption is achieved after irradiation with a focused femtosecond pulsed‐ laser of biologically benign near‐infrared light. With this technology we have successfully delivered multiple proteins of interest including the gene editing protein Cas9 for efficient knockout of GFP with light control as well as the Nur77 derived apoptotic peptide NuBCP which allowed for an increase in efficiency with our delivery method.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

単一細胞への生体分子デリバリーを実現するMEMSナノニードル

単一細胞への生体分子デリバリーを実現するMEMSナノニードル

Ex vivo cytosolic delivery of functional macromolecules to immune cells.

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach’s potential to engineer cell function is demonstrated in HIV infection studies.

MATra - Magnet Assisted Transfection: Combining Nanotechnology and Magnetic Forces to Improve Intracellular Delivery of Nucleic Acids

Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules.