Intracellular Cargo Delivery Research Articles

Using viruses as nanomedicines

The field of nanomedicine involves the design and fabrication of novel nanocarriers for the intracellular delivery of therapeutic cargo or for use in molecular diagnostics. Although traditionally recognized for their ability to invade and infect host cells, viruses and bacteriophages have been engineered over the past decade as highly promising molecular platforms for the targeted delivery and treatment of many human diseases. Inherently biodegradable, the outer capsids of viruses are composed entirely of protein building blocks, which can be genetically or chemically engineered with molecular imaging reagents, targeting ligands and therapeutic molecules. While there are several examples of viruses as in vitro molecular cargo carriers, their potential for applications in nanomedicine has only recently emerged. Here we highlight recent developments towards the design and engineering of viruses for the treatment of cancer, bacterial infections and immune system-related diseases.

Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery.

Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors

Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.

LAH4 enhances CD8+ T cell immunity of protein/peptide-based vaccines

It is now clear that CD8+ T cells are crucial for therapeutic immunity against chronic viral infections and/or tumors. We reason that a strategy capable of improving CD8+ T cell activation would improve the efficacy of protein-based vaccines, which predominantly generate CD4+ T cell-mediated responses. Herein, we explore the ability of a novel cell-penetrating peptide (CPP), LAH4, to facilitate intracellular delivery of protein-based vaccines adjuvanted with Toll-like receptor 9 agonist CpG oligonucleotide (CpG) to generate enhanced CD8+ T cell immune responses and antitumor effects. LAH4 was found to mediate the intracellular delivery of both protein and nucleotide cargo and facilitate protein internalization using mechanisms involving endosomal acidification and processing through the proteasome pathway, leading to enhanced cross presentation of protein antigen by dendritic cells to CD8+ T cells. LAH4 also improved the internalization of CpG, resulting in NFkB activation, thus potentiating the adjuvant effect of CpG. We found that protein-based vaccine comprised of LAH4 mixed with model antigen and CpG generated significantly improved antigen-specific CD8+ T cell immune responses and/or antitumor effects. Furthermore, we found that LAH4 was able to enhance the ability of a tyrosinase-related protein 2 (TRP-2) peptide-based vaccine to generate TRP2-specific CD8+ T cells and antitumor effects against TRP2-expressing tumors. Thus, our results suggest that CPP technology using LAH4 is able to enhance both protein-based and peptide-based vaccine potency to generate antigen-specific CD8+ T cells and antitumor effects. Our findings serve as an important foundation for future clinical applications of CPP technology to improve protein/peptide-based vaccine potency.

Abstract 2479: Detecting Abelson tyrosine kinase activation by ATM and DNA-PK after exposure to ionizing radiation using a peptide biosensor

Abstract Abelson (Abl) kinase is a non-receptor tyrosine kinase that is involved in many cellular processes, including survival, differentiation and apoptosis. Abl is activated in the response to DNA damage, and is involved in the decision between DNA repair versus apoptosis. It is widely accepted that Abl is regulated in response to DNA double-strand breaks (DSBs) by ataxia telangiectasia mutated protein (ATM) via phosphorylation of Abl at S465. However, other aspects of this complex signaling response have been unclear, including the role of other kinases such as DNA-dependent protein kinase (DNA-PK), because of the difficulties of detecting subtle changes in Abl activation through endogenous substrates. Here, we show Abl is activated in a mutant cell line that cannot be phosphorylated by ATM at S465 (Abl-S465A-EGFP) after exposure to ionizing radiation (IR) using a peptide biosensor we developed for Abl kinase. This biosensor contains the following functional modules: a highly specific Abl kinase substrate, an Abl SH3 binding ligand, a photocleavable linker, a biotin tag and a cell penetrating peptide derived from Hiv-TAT to aid intracellular delivery of cargo across the plasma membrane. Using a combination of chemical biology approaches, Western blot for the phosphorylation of the biosensor peptide and high-resolution MS-based phosphoproteomics, we identified a potential role for DNA-PK in activating Abl kinase after IR, independent of ATM and S465. This information about the importance of DNA-PK in this signaling pathway may lead to a better understanding of c-Abl's function in the response to IR, and could be useful to develop strategies to combat radioresistance or improve radiosensitivity in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2011-2479

Construction of Molecular Shuttles Based on Kinesin Motor Proteins and Microtubules

ABSTRACTThe intracellular cargo delivery performed by kinesin motor proteins can be biomimetically employed to engineer tailor-made artificial nanotransport systems. Kinesin (expressed on an Escherichia coli system) and microtubules (obtained from the polymerization of tubulin proteins) were prepared and characterized. We report recent results and explore the aim of the construction of biomotor-based NanoElectroMechanical Systems (NEMS) and their potential applications, e.g. as drug delivery systems.

Reducible Poly(oligo-D-arginine) for Enhanced Gene Expression in Mouse Lung by Intratracheal Injection

Nonarginine (D-R9) has been reported to be one of the most efficacious protein transduction domains (PTDs) for the intracellular cargo delivery such as DNA, RNA, proteins, and particles. Although oligoarginines are capable of forming polyplex with DNA by electrostatic interaction, the length of oligoarginine can affect the toxicity and gene expression. The reducible poly(oligo-D-arginine) (rPOA) composed of the Cys-(D-R9)-Cys repeating unit forming disulfide bonds between terminal cysteinyl-thiol groups of short peptides was hypothesized to show efficient gene transfection without toxicity. The reducible high molecular weight poly(oligo-D-arginine) may fragment into the Cys-(D-R9)-Cys in cellular environments such as cytosol, cell surface, endosomes, and lysosomes, and enhance DNA transfection efficiency. In the present study, in vitro stability, cytotoxicity, and transfection efficiency of DNA/poly(oligo-D-arginine) polyplex were evaluated. In addition, in vivo delivery of DNA into the lung was performed by intratracheal injection of DNA/poly(oligo-D-arginine) polyplex. The in vivo study with rPOA showed higher level of gene expression than PEI, sustaining for 1 week without toxicity. Reducible high molecular weight poly(oligo-D-arginine) based on R9 PTD is a very promising nonviral gene carrier for lung diseases by efficiently condensing, stabilizing, and transfecting DNA.

Uptake and trafficking of liposomes to the endoplasmic reticulum

Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low-density lipoprotein receptors for endocytosis and enter cells through a caveolin- and microtubule-dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22-25 min, which was confirmed by fluorescence-dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER-assembling entities, such as the ER-budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV-1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.

New mechanisms for non-porative ultrasound stimulation of cargo delivery to cell cytosolwith targeted perfluorocarbon nanoparticles

The cell membrane constitutes a major barrier for non-endocytotic intracellular delivery oftherapeutic molecules from drug delivery vehicles. Existing approaches to breaching the cellmembrane include cavitational ultrasound (with microbubbles), electroporationand cell-penetrating peptides. We report the use of diagnostic ultrasound forintracellular delivery of therapeutic bulky cargo with the use of molecularly targetedliquid perfluorocarbon (PFC) nanoparticles. To demonstrate the concept, we useda lipid with a surrogate polar head group, nanogold-DPPE, incorporated intothe nanoparticle lipid monolayer. Melanoma cells were incubated with nanogoldparticles and this was followed by insonication with continuous wave ultrasound(2.25 MHz, 5 min, 0.6 MPa). Cells not exposed to ultrasound showed gold particlespartitioned only in the outer bilayer of the cell membrane with no evidence of theintracellular transit of nanogold. However, the cells exposed to ultrasound exhibitednumerous nanogold-DPPE components inside the cell that appeared polarizedinside intracellular vesicles demonstrating cellular uptake and trafficking. Further,ultrasound-exposed cells manifested no incorporation of calcein or the release of lactatedehydrogenase. These observations are consistent with a mechanism that suggests thatultrasound is capable of stimulating the intracellular delivery of therapeutic moleculesvia non-porative mechanisms. Therefore, non-cavitational adjunctive ultrasoundoffers a novel paradigm in intracellular cargo delivery from PFC nanoparticles.

Intracellular Cargo Delivery by an Octaarginine Transporter Adapted to Target Prostate Cancer Cells through Cell Surface Protease Activation

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

1111. TAT-Streptavidin: A Novel Drug Delivery Vector for the Intracellular Uptake of Macromolecular Cargo

Top of pageAbstract Summary |[ndash]| The TAT protein transduction domain has been used extensively for directing the intracellular delivery of proteins to which it is conjugated, complexed, or fused 1,2,3,4. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-Streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. We demonstrate that TAT-SA can be used to direct intracellular delivery of various bioactive cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired. Results |[ndash]| FACS analysis of TAT-SA-treated Jurkat cells confirmed nearly 100% uptake as compared with WT-SA-treated cells after 3 h. Trypsinization and treatment with an impermeable fluorescence quenching antibody seperately confirmed that the majority of cell-associated fluorescence was from internalized protein and not due to an extracellular membrane-bound fraction. Confocal microscopy of live WT-SA-treated Jurkat cells 3 h after treatment showed little to no fluorescence, consistent with low-level, non-specific uptake, while cells treated with TAT-SA displayed punctate patches of intracellular fluorescence, indicative of an endosomal entry pathway. The ability of a pH-responsive polymer poly(propylacrylic acid) (PPAA) to enhance cytoplasmic delivery of TAT-SA was investigated 5,6. Images of live Jurkat cells 4 h after incubation with TAT-SA:PPAA complexes were characterized by a diffuse cytoplasmic distribution, in contrast to the compartmentalization seen in the absence of an endosomal disrupting polymer. Subcellular fractionation was performed to quantify the uptake of TAT-SA and the effect of PPAA on the intracellular distribution within various subcellular compartments. The ability of TAT-SA:PPAA complexes to deliver a bioactive cargo protein to the cytoplasm was investigated with the nonphosphorylatable, nondegradable superrepressor I|[kappa]|B|[alpha]| (srI|[kappa]|B|[alpha]|) mutant which has been demonstrated as a potent inhibitor of NF-|[kappa]|B activity when expressed in cells 7. Here, purifed, biotinylated srI|[kappa]|B|[alpha]| was delivered to a 57A HeLa cell line which had been genetically engineered with a Luc reporter gene downstream of NF-|[kappa]|B promoter elements 8. TAT-SA:PPAA:srI|[kappa]|B|[alpha]| complexes inhibited TNF-|[alpha]|-induced NF-|[kappa]|B activation by nearly 50% (p<0.001) in treated cells after 4 h, demonstrating the potential of the TAT-SA:PPAA complex as an effective intracellular delivery vector. This work thus demonstrates that TAT-SA and PPAA can be used jointly as a biomolecular vector for delivering heterogeneous cargo proteins of large size in an apparent step-wise uptake and endosomal release mechanism.

Intracellular Cargo Delivery Using Tat Peptide and Derivatives

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Intracellular cargo delivery using tat peptide and derivatives

Efficient delivery of therapeutic and diagnostic agents across the plasma membrane is crucial in developing novel therapies. Membrane permeating peptides, in particular HIV-1 tat peptide and its derivatives, have enabled the intracellular delivery of cargos of various sizes and physicochemical properties. This review summarizes the current knowledge of tat-derived cell permeating peptides in the transduction of exogenous molecules/complexes.

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7.

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.