Investigating the Cell Entry Mechanism, Disassembly, and Toxicity of the Nanocage PCC-1: Insights into Its Potential as a Drug Delivery Vehicle.

The porous coordination cage PCC-1 represents a new platform potentially useful for the cellular delivery of drugs with poor cell permeability and solubility. PCC-1 is a metal-organic polyhedron constructed from zinc metal ions and organic ligands through coordination bonds. PCC-1 possesses an internal cavity that is suitable for drug encapsulation.

Deciphering variations in the endocytic uptake of a cell-penetrating peptide: the crucial role of cell culture protocols.

Unlabelled: Delivery tools, including cell-penetrating peptides (CPPs), are often inefficient due to a combination of poor endocytosis and endosomal escape. Aspects that impact the delivery of CPPs are typically characterized using tissue culture models. One problem of using cell culture is that cell culture protocols have the potential to contribute to endosomal uptake and endosomal release of CPPs.

Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems.

Cell-penetrating peptides (CPPs) are promising tools for the intracellular delivery of various biological payloads. However, the impact of payload conjugation on the cell-penetrating activity of CPPs is poorly understood. This study focused on dfTAT, a modified version of the HIV-TAT peptide with enhanced endosomal escape activity, to explore how different payloads affect its cell-penetrating activity.

Lipids uniquely alter rates of insulin aggregation and lower toxicity of amyloid aggregates.

Amyloid formation is a hallmark of many medical diseases including diabetes type 2, Alzheimer's and Parkinson diseases. Under these pathological conditions, misfolded proteins self-assemble forming oligomers and fibrils, structurally heterogeneous aggregates that exhibit a large variety of shapes and forms. A growing body of evidence points to drastic changes in the lipid profile in organs affected by amyloidogenic diseases.

In vivo peptide-based delivery of a gene-modifying enzyme into cells of the central nervous system.

We report on the successful delivery of the Cre recombinase enzyme in the neural cells of mice in vivo by simple coinjection with peptides derived from HIV-TAT. Cre delivery activates the expression of a reporter gene in both neurons and astrocytes of the cortex without tissue damage and with a transduction efficiency that parallels or exceeds that of a commonly used adeno-associated virus. Our data indicate that the delivery peptides mediate efficient endosomal leakage and cytosolic escape in cells that have endocytosed Cre.

Hydrophobicity is a key determinant in the activity of arginine-rich cell penetrating peptides.

To deliver useful biological payloads into the cytosolic space of cells, cell-penetrating peptides have to cross biological membranes. The molecular features that control or enhance this activity remain unclear. Herein, a dimeric template of the arginine-rich HIV TAT CPP was used to establish the effect of incorporating groups and residues of various chemical structures and properties.

Superparamagnetic iron oxide-gold nanoparticles conjugated with porous coordination cages: Towards controlled drug release for non-invasive neuroregeneration.

This paper reports a smart intracellular nanocarrier for sustainable and controlled drug release in non-invasive neuroregeneration. The nanocarrier is composed by superparamagnetic iron oxide-gold (SPIO-Au) core-shell nanoparticles (NPs) conjugated with porous coordination cages (PCCs) through the thiol-containing molecules as bridges. The negatively charged PCC-2 and positively charged PCC-3 are compared for intracellular targeting.

Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway.

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies.

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT Peptide and Bis(monoacylglycero)phosphate.

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Here, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes.

Expressed Protein Ligation: General Experimental Protocols.

Expressed protein ligation allows for the attachment of a chemically labeled peptide to the N- or C-terminus of a recombinant protein. In this book chapter, the practical considerations involved in using this protein engineering technology are described. In particular, approaches used to design optimal ligation sites are discussed.

Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol.

Heavy Pnictogenium Cations as Transmembrane Anion Transporters in Vesicles and Erythrocytes.

Our work on the complexation of fluoride anions using group 15 Lewis acids has led us to investigate the use of these main group compounds as anion transporters. In this paper, we report on the anion transport properties of tetraarylstibonium and tetraarylbismuthonium cations of the general formula [PhPnAr] with Pn = Sb or Bi and with Ar = phenyl, naphthyl, anthryl, or pyrenyl. Using EYPC-based large unilamellar vesicles, we show that these main group cations transport hydroxide, fluoride and chloride anions across phospholipid bilayers.

Efficient and Innocuous Live-Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry: How Better Cellular Delivery Tools Can Contribute to Precise and Quantitative Cell Biology Assays.

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry.

Endosomal Escape and Cytosolic Penetration of Macromolecules Mediated by Synthetic Delivery Agents.

Cell delivery reagents often exploit the endocytic pathway as a route of cell entry. Once endocytosed, these reagents must overcome endosomal entrapment to ensure the release of their macromolecular cargo into the cytosol of cells. In this review, we describe several examples of prototypical synthetic reagents that are capable of endosomal escape and examine their mechanisms of action, their efficiencies, and their effects on cells.

Investigating Subcellular Compartment Targeting Effect of Porous Coordination Cages for Enhancing Cancer Nanotherapy.

Understanding the key factors for successful subcellular compartment targeting for cargo delivery systems is of great interest in a variety of fields such as bionanotechnology, cell biology, and nanotherapies. However, the fundamental basis for intracellular transportation with these systems has thus far rarely been discussed. As a cargo vector, porous coordination cages (PCCs) have great potential for use in cancer nanotherapy and to elucidate fundamental insight regarding subcellular compartment targeting.

Efficient Delivery of Macromolecules into Human Cells by Improving the Endosomal Escape Activity of Cell-Penetrating Peptides: Lessons Learned from dfTAT and its Analogs.

Cell-penetrating peptides (CPPs) are typically prone to endocytic uptake into human cells. However, they are often inefficient at escaping from endosomes, which limits their ability to deliver cargos into cells. This review highlights the efforts that our laboratory has devoted toward developing CPPs that can mediate the leakage of endosomal membranes, and consequently gain better access to the intracellular milieu.

Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides.

Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density.

Enzyme-MOF Nanoreactor Activates Nontoxic Paracetamol for Cancer Therapy.

Prodrug activation, by exogenously administered enzymes, for cancer therapy is an approach to achieve better selectivity and less systemic toxicity than conventional chemotherapy. However, the short half-lives of the activating enzymes in the bloodstream has limited its success. Demonstrated here is that a tyrosinase-MOF nanoreactor activates the prodrug paracetamol in cancer cells in a long-lasting manner.

Linearmycins are lytic membrane-targeting antibiotics.

The linearmycin family of polyketides was originally classified as antifungal metabolites. However, in addition to antifungal activity, we previously found that linearmycins cause cellular lysis and colony degradation of the Gram-positive bacterium Bacillus subtilis. We recently showed that Streptomyces sp.

High efficiency and long-term intracellular activity of an enzymatic nanofactory based on metal-organic frameworks.

Enhancing or restoring enzymatic function in cells is highly desirable in applications ranging from ex vivo cellular manipulations to enzyme replacement therapies in humans. However, because enzymes degrade in biological milieus, achieving long-term enzymatic activities can be challenging. Herein we report on the in cellulo properties of nanofactories that consist of antioxidative enzymes encapsulated in metal-organic frameworks (MOFs).

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated.

Membrane Oxidation in Cell Delivery and Cell Killing Applications.

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargoes into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides.

An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape.

Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications.

Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

Cell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined.