Strategies for cytosolic delivery of liposomal macromolecules

Potential approaches to achieve cytosolic delivery of liposomal macromolecules are presented. These approaches include: (1) the co-encapsulation of fusogenic peptides into targeted drug-containing liposomes (2) coupling of the HIV-1-derived cell-penetrating peptide TAT to the surface of liposomes and (3) photochemical internalization, based on photochemically inducible permeabilization of endocytic vesicles.     

A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.     

Synergistic effects of cell-penetrating peptide Tat and fusogenic peptide HA2-enhanced cellular internalization and gene transduction of organosilica nanoparticles

The nonviral gene delivery system is an attractive alternative to cancer therapy. A new kind of gelatin-silica nanoparticles (GSNPs) was developed through a two-step sol-gel procedure. To improve the transfection efficacy, GSNPs modified with different fusion peptides (Tat, HA2, R8, Tat/HA2, and Tat/R8) were prepared for particle size, zeta potential, cellular uptake, hemolysis activity at physiological pH (7.0) or acidic pH (5.0), and condensation of plasmid DNA. The results suggest that the sizes and zeta potentials of GS-peptide conjugates were 147 - 161 nm and 19 - 33 mV, respectively; GS-peptide conjugates exhibited low cytotoxicity; the plasmid DNA was readily entrapped at a GS-peptide/pDNA weight ratio of 50 - 200. The in vitro and in vivo studies demonstrated that the synergistic effects of cell-penetrating peptide Tat and fusogenic peptide HA2 could promote the efficient cellular internalization, endosome escape, and nucleus targeting, hence delivering the therapeutic nucleic acid efficiently.     

Characterisation of cell-penetrating peptide-mediated peptide delivery

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia相似文献   

Effect of synthetic cell-penetrating peptides on TrkA activity in PC12 cells

As TrkA, a high-affinity receptor of nerve growth factor (NGF), is a potential target for relieving uncontrolled inflammatory pain, an effective inhibitor of TrkA has been required for pain management. To identify a specific inhibitor of TrkA activity, we designed cell-penetrating peptides combined with amino-acid sequences in the activation loop of TrkA to antagonize tyrosine kinase activity. To select a peptide inhibiting TrkA activity, we examined the effect of cell-penetrating peptides on tyrosine kinase activity of recombinant TrkA in vitro and studied their effects on NGF-stimulated neurite outgrowth and protein phosphorylation in PC12 cells. Thereafter we investigated the effect of the selected peptide on NGF-stimulated TrkA activity and the expression of transient receptor potential channel 1 in PC12 cells. The selected peptide inhibited TrkA activity, but did not inhibit tyrosine kinase activities of other receptor-type tyrosine kinases in vitro. It also suppressed NGF-stimulated responses in PC12 cells. The selected synthetic cell-penetrating peptide antagonizing TrkA function would be a candidate for inflammatory pain therapy.     

Biological responses towards cationic peptides and drug carriers

In drug development, major resources are invested into the development of cellular delivery systems to increase the effectiveness of a large array of potential therapeutics, such as proteins and oligonucleotides. These carriers comprise cell-penetrating peptides (CPPs), cationic lipids and cationic polymers. In recent years, evidence has been accumulating that these carriers not only act as mere pharmacokinetic modifiers but also interfere with cellular processes in various ways. In this review, we present an overview of the biological side effects associated with carrier systems. The focus will be on CPPs, which have been explored for a diverse set of cargos. Reported activities range from an induction of receptor internalization to the generation of reactive oxygen species. Ultimately, cell-penetrating molecules with such biological side effects might evolve into new bioactive agents that combine delivery capacity and pharmacophore in a single molecular entity. First examples for such molecules will be presented.     

Self-assembled peptide (CADY-1) improved the clinical application of doxorubicin

CADY-1 is an amphipathic peptide that possesses cell-penetrating activity. As an amphipathic peptide, CADY-1 is capable of forming complexes by self-assembly, and they are these complexes that possess cell-penetrating activity. This distinct characteristic of CADY-1 makes it a potent cell-penetrating drug delivery system. Doxorubicin is a widely used cytotoxic anti-cancer drug but is limited by its toxicity. Although the liposomal formulation of doxorubicin ameliorates its toxicity, its complicated synthesis remains an obstacle to its wide clinical use. In this study, our findings revealed that CADY-1 and doxorubicin form a stable complex at optimised molar ratios in a self-assembling manner. Formation of the complex extended the blood residence time of doxorubicin in a similar fashion to that of liposomal doxorubicin. In addition, the complex was capable of carrying doxorubicin across the cell membrane, which increased the therapeutic index of doxorubicin. Experimental animals treated with a CADY-1/doxorubicin complex exhibited better tolerance and anti-tumour activity than animals treated with either liposomal doxorubicin or the free form of doxorubicin. Collectively, the findings in this study support the advantages of using complexes formed by the self-assembled peptide CADY-1 and suggest that CADY-1 is a potent drug delivery system.     

Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells

The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biological functions into cells has gained much attention in recent years. However, the internalization mechanisms and delivery properties of CPP-cargo remains controversial. In this study, low- and high-molecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluorescein isothiocyanate-labeled nona-arginine (CPP-FITC), and the latter was the fluorescently labeled nona-arginine-avidin complex (CPP-avidin). We measured the intracellular trafficking of CPP-FITC and CPP-avidin in four cancer cell lines in a series of microenvironments altered by the presence or absence of serum, different temperatures and different incubation times. The results revealed that CPP-cargo delivery exhibited no specificity toward any cell line, but the levels were found to be related to cell type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with related endocytic inhibitors. Two different types of CPP-cargo were required to cross the plasma membrane to bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs and small cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocytic route. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in an energy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependent pathway is not essential to the translocation of either type of CPP-cargo.     

Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor

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The vimentin-tubulin binding site peptide (Vim-TBS.58-81) crosses the plasma membrane and enters the nuclei of human glioma cells

Cell-penetrating peptides (CPPs) can translocate through the plasma membrane and localize in different cell compartments providing a promising delivery system for peptides, proteins, nucleic acids, and other products. Here we describe features of a novel cell-penetrating peptide derived from the intermediate filament protein vimentin, called Vim-TBS.58-81. We show that it enters cells from a glioblastoma line via endocytosis where it distributes throughout the cytoplasm and nucleus. Moreover, when coupled to the pro-apoptogenic peptide P10, it localizes to the nucleus inhibiting cell proliferation. Thus, the Vim-TBS.58-81 peptide represents an effective vector for delivery of peptides and potentially a broad range of cargos to the nucleus.     

Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains

High mole ratio BR96 immunoconjugates were synthesized using branched peptide-doxorubicin linkers designed to liberate doxorubicin following antigen-specific internalization into lysosomes. However, these immunoconjugates are highly prone to noncovalent, dimeric aggregation. We hypothesize that this is due to (1) the hydrophobic nature of the peptides, (2) the loss of positive charge upon amide formation at the 3'-amino group of doxorubicin, and (3) the proximity of the peptide hydrophobic residues to form efficient intermolecular stacking interactions. By introducing a hydrophilic methoxytriethylene glycol chain onto the doxorubicin portion of the branched peptide linkers, aggregation has been eliminated or greatly reduced in the immunoconjugate products. The methoxytriethylene glycol chain was linked to the doxorubicin moiety of the linker via a hydrazone bond that is stable at pH 7 but hydrolyzes rapidly at pH 5 to release free drug. BR96 immunoconjugates synthesized from methoxytriethylene glycol-modified branched peptide-doxorubicin linkers are highly potent and immunospecific in vitro. The data suggest that the methoxytriethylene glycol chain hydrolyzes as designed upon antigen-specific internalization into tumor lysosomes in vitro, where enzymatic degradation of the peptide linker releases free doxorubicin.     

The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis

Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (0050 and 0040). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.     

In silico identification and experimental validation of cellular uptake and intracellular labeling by a new cell penetrating peptide derived from CDN1

Bioactive therapeutic molecules are generally impermeable to the cell membrane, hindering their utility and efficacy. A group of peptides called cell-penetrating peptides (CPPs) were found to have the capability of transporting different types of cargo molecules across the cell membrane. Here, we identified a short peptide named P2, which has a higher proportion of basic residues than the CDN1 (cyclin-dependent kinase inhibitor 1) protein it is derived from, and we used bioinformatic analysis and experimental validation to confirm the penetration property of peptide P2. We found that peptide P2 can efficiently enter different cell lines in a concentration-dependent manner. The endocytosis pathway, especially receptor-related endocytosis, may be involved in the process of P2 penetration. Our data also showed that peptide P2 is safe in cultured cell lines and red blood cells. Lastly, peptide P2 can efficiently deliver self-labeling protein HaloTag into cells for imaging. Our study illustrates that peptide P2 is a promising imaging agent delivery vehicle for future applications.     

Effect of DC/mDC iontophoresis and terpenes on transdermal permeation of methotrexate: in vitro study

We have reported previously that a basic peptide, arginine peptide, can be used as an efficient system for delivery of foreign genes. In this work, to better understand the mechanism of arginine peptide-mediated gene delivery, we further evaluated the process of cellular uptake and nuclear localization of the peptide/DNA complex. To investigate the effect of cellular proteoglycans on arginine peptide/DNA complexes, interactions between polyanionic glycosaminoglycans (GAGs) and peptide/DNA complexes were examined by the ethidium bromide interaction assay. Sulfated GAGs were found to relax the complexed DNA at low peptide/DNA charge ratios. Condensed peptide/DNA complexes facilitate cellular uptake, but their mechanism of uptake is poorly understood. Studies of various endocytosis inhibitors suggested that the peptide/DNA complex internalization involved the caveolar-related endocytosis pathway. A critical step in the gene delivery is the cytosol-to-nucleus transport of exogenous DNA following initial complex uptake. Nuclear localization of peptide/DNA complex was confirmed by confocal laser scanning microscopic observation. Further, we show that transfections with peptides result in an early accumulation of plasmid DNA in the nucleus of growth-arrested cells, which suggest nuclear transport. To assess the potential for arginine peptide as an agent for therapeutic gene delivery, in vivo complexed DNA transduction studies were performed. Mice were injected subcutaneously with the reporter gene beta-galactosidase, resulting in high levels of gene expression in dermal tissue.     

TAT介导的几种纳米药物载体的研究概况

TAT是一种应用最早且现今最为常用的细胞穿膜肽,已经成功与多种纳米载体如脂质体、胶束、纳米粒等连结形成可内化进入细胞内的载药体系。文章主要对TAT介导的几种纳米载药体系的穿透机制、体内外抗肿瘤活性及靶向病变部位能力等方面的研究进行综述,同时对微环境pH敏感的TAT靶向药物载体的研究进展作了简单介绍。     

Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives

Cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, are increasingly drawing attention as a non-invasive delivery technology for macromolecules. Delivery of a diverse set of cargo in terms of size and nature ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides (CPPs) in vitro and in vivo. However, the internalization mechanism of CPPs is an unresolved issue to date, with dramatic changes in view regarding the involvement of endocytosis as a pathway of internalization. A key reason for the lack of consensus on the mechanism can be attributed to the methodology in deciphering the internalization mechanism. In this review, we highlight some of the methodology concerns, focus more on the internalization pathway and also provide a novel perspective about the intracellular processing of CPPs, which is a crucial aspect to consider when selecting a cell penetrating peptide as a drug delivery system. In addition, recent applications of cell penetrating peptides for the delivery of small molecules, peptides, proteins, oligonucleotides, nanoparticles and liposomes have been reviewed.     

Delineation of the motilin domain involved in desensitization and internalization of the motilin receptor by using full and partial antagonists

Studies with fragments of the gastrointestinal peptide, motilin, indicate that the C-terminal region of this peptide plays an important role in the desensitization of the motilin receptor (MTLR). AIM: To verify this hypothesis we studied the desensitization, phosphorylation and internalization induced by motilin analogues of different chain length with agonistic and antagonistic properties in CHO-MTLR cells. METHODS: We studied motilin [1-22], the [1-14] fragment, the analogues Phe(3)[1-22] and Phe(3)[1-14], and two putative antagonists, GM-109 and MA-2029 (modified 1-4 and 1-3 fragments). Activation and desensitization (2h preincubation with the motilin analogues 10muM) were studied in CHO-MTLR cells by an aequorin based luminescence assay. Phosphorylation was studied by immunoprecipitation and internalization was visualized in CHO-MTLR cells containing an enhanced green fluorescent protein (CHO-MTLR-EGFP). RESULTS: Motilin [1-22] and [1-14] were more potent than Phe(3)[1-22] and Phe(3)[1-14] (pEC(50): 9.77, 8.78, 7.36 and 6.65, respectively) to induce Ca(2+) release. GM-109 and MA-2029 were without agonist activity. [1-22] and Phe(3)[1-22] decreased the second response to motilin from 78+/-2% to 11+/-3% and 34+/-3% (P<0.001), respectively, whereas [1-14], Phe(3)[1-14], GM-109 and MA-2029 had no desensitizing effect (68+/-5%, 78+/-3%, 78+/-6% and 78+/-5%, respectively, P>0.05). The rank order of MTLR-phosphorylation was: [1-22]>[1-14]>Phe(3)[1-22]=Phe(3)[1-14]>GM-109=MA-2029. Only motilin [1-22] and [1-14] induced receptor MTLR-EGFP internalization as shown by a decrease in membrane fluorescence: 20+/-3% and 7+/-3%, respectively. CONCLUSION: The C-terminus of motilin enhances desensitization, phosphorylation and internalization of the MTLR while modifications of the N-terminus can favor a conformation of the receptor that is less susceptible to phosphorylation and internalization.     

Antisense c-myc Oligodeoxyribonucleotide Cellular Uptake

Antisense oligonucleotides have therapeutic potential as inhibitors of gene expression. However, the mechanism by which an intact oligonucleotide reaches the intracellular site of action is unknown. In this study, we use an Oligodeoxyribonucleotide 21-mer complementary to the translation initiation codon of the c-myc proto-oncogene to study the mechanism of oligonucleotide uptake and internalization into Rauscher Red 5-1.5 cells. We find trypsin-sensitive and trypsin-insensitive surface binding, in addition to internalization. Uptake is partially energy dependent and inhibited by charged molecules, including DNA, ATP, a random sequence oligonucleotide, and dextran sulfate. Uptake does not appear to occur via a traditional receptor-mediated uptake pathway because chloro-quine, monensin, and phenylarsine oxide pretreatment does not significantly decrease internalization. An anion channel inhibitor, SITS, and the salts, NaCl, Na₂SO₄, and NH₄Cl, significantly decrease oligonucleotide uptake. Whether uptake occurs via a channel or a novel uptake mechanism is still unknown. A model is proposed which reasonably simulates the experimental data.