Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs

Although liposomes have proven useful for the delivery of drugs and gene therapy vectors, their potencies are often compromised by poor unloading following uptake into their target cells. We have consequently explored the properties of a novel 29-residue amphipathic peptide that was designed by arrangement of hydrophobic and hydrophilic residues to disrupt liposomes at lower peptide concentrations than previously tested peptides. The peptide was indeed found to promote pH-dependent liposome unloading with improved efficiency. A peptide of the same sequence, but half the length, however, promoted pH-dependent permeabilization only at much higher concentrations. Further characterization of the longer peptide revealed that release of liposome contents (i) occurred at a pH of ∼6, (ii) became less efficient as the size of the encapsulated cargo increased, and (iii) was moderately suppressed in cholesterol-containing liposomes. Use of this peptide to enhance the cytotoxicity of cytosine arabinoside encapsulated in folate-targeted liposomes demonstrated an increase in drug potency of ∼30-fold. Gene expression by a serum-stable folate-targeted liposomal vector was also measurably enhanced by inclusion of the peptide. We conclude that intracellular unloading of liposomal contents can be significantly improved by co-encapsulation of an optimally designed, pH-sensitive peptide.     

Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs

Although liposomes have proven useful for the delivery of drugs and gene therapy vectors, their potencies are often compromised by poor unloading following uptake into their target cells. We have consequently explored the properties of a novel 29-residue amphipathic peptide that was designed by arrangement of hydrophobic and hydrophilic residues to disrupt liposomes at lower peptide concentrations than previously tested peptides. The peptide was indeed found to promote pH-dependent liposome unloading with improved efficiency. A peptide of the same sequence, but half the length, however, promoted pH-dependent permeabilization only at much higher concentrations. Further characterization of the longer peptide revealed that release of liposome contents (i) occurred at a pH of approximately 6, (ii) became less efficient as the size of the encapsulated cargo increased, and (iii) was moderately suppressed in cholesterol-containing liposomes. Use of this peptide to enhance the cytotoxicity of cytosine arabinoside encapsulated in folate-targeted liposomes demonstrated an increase in drug potency of approximately 30-fold. Gene expression by a serum-stable folate-targeted liposomal vector was also measurably enhanced by inclusion of the peptide. We conclude that intracellular unloading of liposomal contents can be significantly improved by co-encapsulation of an optimally designed, pH-sensitive peptide.     

Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine-tagged proteins

Arginine-rich peptide-mediated protein delivery into living cells is a novel technology for controlling cell functions with therapeutic potential. In this report, a novel approach for the intracellular delivery of histidine-tagged proteins was introduced where a Ni(II) chelate of octaarginine peptide bearing nitrilotriacetic acid [R8-NTA-Ni(II)] was used as a membrane-permeable carrier molecule. Significant internalization of histidine-tagged enhanced green fluorescent protein (EGFP) into HeLa cells was observed by confocal microscopic observation in the presence of R8-NTA-Ni(II). Nuclear condensation characteristic in apoptotic cell death was also induced in the cells treated with a histidine-tagged apoptosis-inducing peptide [pro-apoptotic domain peptide (PAD)], indicating that the cargo molecules really went through the membrane to reach the cytosol. The apoptosis-inducing activity of the peptide thus delivered was compared with that of the PAD peptide covalently connected with the octaarginine peptide.     

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to α_Vβ₃ integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the α_Vβ₃ integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI/10 nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.     

Co-Encapsulating the Fusogenic Peptide INF7 and Molecular Imaging Probes in Liposomes Increases Intracellular Signal and Probe Retention

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging.     

SiRNA delivery systems based on neutral cross-linked dendrimers

A neutral cross-linked dendritic system is described for use of delivering small interfering RNA (siRNA) for targeted gene silencing. By replacing the terminal amines with hydrazide groups and N-acetylgalactosamine (GalNAc) ligands, cationic polyamidoamine (PAMAM) dendrimers were transformed into neutral glycosylated carriers for siRNA delivery. Mainly owing to the pH sensitivity and the proton-absorption capability of the tertiary amines within the interior branches, these PAMAM derivatives showing neutrality under physiological conditions (pH 7.4) formed complexes with siRNA through electrostatic interactions at pH 5. Cross-linking procedures via reactions with glutaraldehyde were established, and cytocompatible cross-linked systems loaded with siRNA obtained, with the particulate properties variable with the cross-linking condition and the GalNAc level in the dendritic carrier. In vitro cellular uptake and RNAi experiments showed that the cross-linked dendritic systems with an appropriate level of GalNAc composition effectively interacted with HepG2 cells and inhibited the expression of a luciferase reporter gene. Neutral cross-linked dendritic systems provide a new paradigm for designing siRNA delivery systems with biocompatibility and targeting capability.     

Simultaneous membrane interaction of amphipathic peptide monomers,self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

pH-responsive poly(styrene-alt-maleic anhydride) alkylamide copolymers for intracellular drug delivery

Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.     

pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents

Abstract Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

Low pH-induced formation of ion channels by clostridium difficile toxin B in target cells

Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH 相似文献   

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC₅₀ value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.     

PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells

Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.     

pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents

Abstract

Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease.