Mitochondrial targeting functional peptides as potential devices for the mitochondrial delivery of a DF-MITO-Porter

To achieve mitochondrial therapy, we previously reported on the use of an octaarginine (R8) modified Dual Function (DF)-MITO-Porter for delivering molecules to mitochondria in living cells. In this study, using isolated mitochondria, homogenates and living cells, we evaluated the utility of mitochondrial targeting functional peptides as a ligand for delivering carriers. The S2 peptide modified carrier showed a high mitochondrial targeting activity in homogenates and living cells. In addition, the S2 peptide had a lower cell toxicity compared to R8 modified liposomes. The S2 peptide represents a potentially useful moiety for constructing an efficient and safe mitochondrial delivery system.     

Octaarginine-modified liposomes enhance the anti-oxidant effect of Lecithinized superoxide dismutase by increasing its cellular uptake

The anti-oxidant enzyme superoxide dismutase (SOD) has the potential for use as a therapeutic agent in the treatment of various diseases caused by reactive oxygen species. However, achieving this would be difficult without a suitable delivery system for SOD. We previously reported that PC-SOD, in which four molecules of a phosphatidylcholine (PC) derivative were covalently bound to each dimer of recombinant human CuZnSOD, was a high affinity for the cell membrane [14]. Here, we show that an octaarginine (R8) modified liposome equipped with PC-SOD (R8-LP (PC-SOD)) enhances its anti-oxidant effect. High-density R8-modified liposomes can stimulate macropinocytosis and are taken up efficiently by cells as demonstrated in a previous study [21]. Flow cytometry analyses showed that R8-LP (PC-SOD) was taken up by cells more efficiently than PC-SOD. Moreover, R8-LP (PC-SOD) liposomes were found to scavenge superoxide anions (O₂⁻) very efficiently. These results suggest that the efficient cytosolic delivery of PC-SOD by R8-modified liposomes would enhance the anti-oxidant effects of PC-SOD.     

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.     

Arginine-rich,cell penetrating peptide–anti-microRNA complexes decrease glioblastoma migration potential

MicroRNAs (miRNAs) are a class of gene regulators originating from non-coding endogenous RNAs. Altered expression, both up- and down-regulation, of miRNAs plays important roles in many human diseases. Correcting miRNA dysregulation by either inhibiting or restoring miRNA function may provide therapeutic benefit. However, efficient, nontoxic miRNA delivery systems are in need. Cell penetrating peptides (CPPs) have been widely exploited for protein, DNA, and RNA delivery. Few have examined CPP transfection efficiency with single stranded anti-miRNA. The R₈ peptide condensed both siRNA and anti-miRNA. Greater than 50% of cells had anti-miRNA/R₈ complexes associated and in these cells 68% of anti-miRNA escapes the endosome/lysosome. Single-stranded antisense miR-21 inhibitor (anti-miR-21) administered using the R₈ peptide elicited efficient downstream gene upregulation. Glioblastoma cell migration was inhibited by 25% compared to the negative control group. To our knowledge, this is the first demonstration of miRNA modulation with anti-miR-21/R₈ complexes, which has laid the groundwork for further exploring octaarginine as intracellular anti-miRNAs carrier.     

Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.     

Zinc(II)-coordinated oligotyrosine: a new class of cell penetrating peptide

A new series of cell penetrating peptides (CPPs) are described. The peptides are oligomers of Tyr-ZnDPA, a tyrosine derivative with an appended 2,2'-dipicolylamine unit that forms a very stable coordination complex with a zinc (II) cation. This in turn allows reversible association with a chelating oxyanion such as a carboxylate or phosphate derivative. The peptide oligomers (Tyr-ZnDPA) n where n = 1, 2, 4, 8, are highly water soluble, but upon association with fatty acids or phospholipids they partition into an organic octanol phase. Furthermore, a fluorescent, fluorescein-labeled version of the octamer, (Tyr-ZnDPA) 8-Fl, can enter living mammalian cells via endocytosis and a biotin derivative can deliver fluorescein-labeled streptavidin. Fluorescence microscopy and flow cytometry experiments show that cell uptake is diminished by conditions that inhibit endocytosis. Additionally, uptake of (Tyr-ZnDPA) 8-Fl is greater than fluorescein labeled octaarginine (Arg 8-Fl) in all cell lines tested (CHO, COS-7, HeLa). Another difference with Arg 8-Fl is that cell uptake of (Tyr-ZnDPA) 8-Fl does not require the presence of heparan sulfate proteoglycans on the cell surface. This difference may eventually be of practical value because drug delivery systems that employ alternative endocytic mechanisms may be optimal for different cell lines or they may deliver selectively to different organelles within a cell.     

Membrane permeability commonly shared among arginine-rich peptides

Delivery of proteins and other macromolecules using membrane-permeable carrier peptides is a recently developed novel technology, which enables us to modulate cellular functions for biological studies with therapeutic potential. One of the most often used carrier peptides is the arginine-rich basic peptide derived from HIV-1 Tat protein [HIV-1 Tat (48-60)]. Using this peptide, efficient intracellular delivery of molecules including proteins, oligonucleic acids and liposomes has been achieved. We have demonstrated that these features were commonly shared among many arginine-rich peptides such as HIV-1 Rev (34-50) and octaarginine. Not only the linear peptides but also branched-chain peptides showed efficient internalization with an optimum number of arginines (approximately eight residues). The structural and mechanistic features of the translocation of these membrane-permeable arginine-rich peptides are reviewed.     

Apolipoprotein B binding domains: evidence that they are cell-penetrating peptides that efficiently deliver antigenic peptide for cross-presentation of cytotoxic T cells

Low-density lipoproteins (LDLs) are a good source of cholesterol, which is important in cellular homeostasis and production of steroids. Apolipoprotein B-100 (ApoB-100), the sole protein component of LDL, is known to bind to cell surface LDL receptor (LDLR) or cell surface-bound proteoglycans and to be internalized into cells. We found that APCs, consisting of macrophages and dendritic cells, upregulate LDLR on culture in vitro without obvious stimulation. In contrast, T cell populations only upregulate LDLR on activation. Thus, we strategized that tagging immunogens to ApoB-100 might be a useful means to target Ag to APCs. We generated fusion proteins consisting of receptor binding sites in ApoB-100, coupled to OVA peptide (ApoB-OVA), as Ag delivery vehicles and demonstrated that this novel delivery method successfully cross-presented OVA peptides in eliciting CTL responses. Surprisingly, internalization of ApoB-OVA peptide occurred via cell surface proteoglycans rather than LDLRs, consistent with evidence that structural elements of ApoB-100 indicate it to have cell-penetrating peptide properties. Finally, we used this strategy to assess therapeutic vaccination in a tumor setting. OVA-expressing EL-4 tumors grew progressively in mice immunized with ApoB-100 alone but regressed in mice immunized with ApoB-OVA fusion protein, coinciding with development of OVA-specific CTLs. Thus, to our knowledge, this is the first article to describe the cell-penetrating properties of a conserved human origin cell penetrating peptide that may be harnessed as a novel vaccination strategy as well as a therapeutics delivery device.     

Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents.     

Upregulation of IGF-IIRα intensifies doxorubicin-induced cardiac damage

Cardiotoxicity by doxorubicin hampers its therapeutic potential as an anticancer drug, but mechanisms leading to cardiotoxicity remain contentious. Through this study, the functional contribution of insulin-like growth factor receptor type II α (IGF-IIRα) which is a novel stress-inducible protein was explored in doxorubicin-induced cardiac stress. Employing both in vitro H9c2 cells and in vivo transgenic rat models (SD-TG [IGF-IIRα]) overexpressing IGF-IIRα specifically in heart, we found that IGF-IIRα leads to cardiac structural abnormalities and functional perturbations that were severely aggravated by doxorubicin-induced cardiac stress. Overexpression of IGF-IIRα leads to cumulative elevation of stress associated cardiac hypertrophy and apoptosis factors. There was a significant reduction of survival associated proteins p-Akt and estrogen receptor β/α, and abnormal elevation of cardiac hypertrophy markers such as atrial natriuretic peptide, cardiac troponin-I, and apoptosis-inducing agents such as p53, Bax, and cytochrome C, respectively. IGF-IIRα also altered the expressions of AT1R, ERK1/2, and p38 proteins. Besides, IGF-IIRα also increased the reactive oxygen species production in H9c2 cells which were markedly aggravated by doxorubicin treatment. Together, we showed that IGF-IIRα is a novel stress-induced protein that perturbed cardiac homeostasis and cumulatively exacerbated the doxorubicin-induced cardiac injury that perturbed heart functions and ensuing cardiomyopathy.     

Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape

The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.     

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.     

Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.     

Possible existence of common internalization mechanisms among arginine-rich peptides.

Basic peptides such as human immunodeficiency virus type 1 (HIV-1) Tat-(48-60) and Drosophila Antennapedia-(43-58) have been reported to have a membrane permeability and a carrier function for intracellular protein delivery. We have shown that not only Tat-(48-60) but many arginine-rich peptides, including HIV-1 Rev-(34-50) and octaarginine (Arg(8)), efficiently translocated through the cell membranes and worked as protein carriers (Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 276, 5836-5840). Quantification and time course analyses of the cellular uptake of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa, and simian kidney COS-7 cells revealed that Rev-(34-50) and Arg(8) had a comparable translocation efficiency to Tat-(48-60). Internalization of Tat-(48-60) and Rev-(34-50) was saturable and inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic inhibitors had little effect on the internalization. The uptake of these peptides was significantly inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the cells with the anti-heparan sulfate antibody or heparinase III also lowered the translocation of these peptides. These results strongly suggest that the arginine-rich basic peptides share a certain part of the internalization pathway.     

Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis

Background

Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.

Methodology/Principal Findings

In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.

Conclusions/Significance

Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.     

Vasopressin V2R-targeting peptide carrier mediates siRNA delivery into collecting duct cells

Internalization of receptor proteins after interacting with specific ligands has been proposed to facilitate siRNA delivery into the target cells via receptor-mediated siRNA transduction. In this study, we demonstrated a novel method of vasopressin V2 receptor (V2R)-mediated siRNA delivery against AQP2 in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We synthesized the dDAVP conjugated with nine D-arginines (dDAVP-9r) as a peptide carrier for siRNA delivery. The structure of synthetic peptide carrier showed two regions (i.e., ligand domain to V2R (dDAVP) and siRNA carrying domain (nine D-arginine)) bisected with a spacer of four glycines. The results revealed that 1) synthesized dDAVP-9r peptides formed a stable polyplex with siRNA; 2) siRNA/dDAVP-9r polyplex could bind to the V2R of IMCD cells and induced AQP2 phosphorylation (Ser 256); 3) siRNA/dDAVP-9r polyplex was stable in response to the wide range of different osmolalities, pH levels, or to the RNases; 4) fluorescein-labeled siRNA was delivered into V2R-expressing MDCK and LLC-PK1 cells by siRNA/dDAVP-9r polyplex, but not into the V2R-negative Cos-7 cells; and 5) AQP2-siRNA/dDAVP-9r polyplex effectively delivered siRNA into the IMCD cells, resulting in the significant decrease of protein abundance of AQP2, but not AQP4. Therefore, for the first time to our knowledge, we demonstrated that V2R-mediated siRNA delivery could be exploited to deliver specific siRNA to regulate abnormal expression of target proteins in V2R-expressing kidney cells. The methods could be potentially used in vivo to regulate abnormal expression of proteins associated with disease conditions in the V2R-expressing kidney cells.     

Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.     

The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis

Specific recognition of pathogens is mediated by plant disease resistance (R) genes and translated into a successful defense response. The extent of associated hypersensitive cell death varies from none to an area encompassing cells surrounding an infection site, depending on the R gene activated. We constructed double mutants in Arabidopsis between positive regulators of R function and a negative regulator of cell death, LSD1, to address whether genes required for normal R function also regulate the runaway cell death observed in lsd1 mutants. We report here that EDS1 and PAD4, two signaling genes that mediate some but not all R responses, also are required for runaway cell death in the lsd1 mutant. Importantly, this novel function of EDS1 and PAD4 is operative when runaway cell death in lsd1 is initiated through an R gene that does not require EDS1 or PAD4 for disease resistance. NDR1, another component of R signaling, also contributes to the control of plant cell death. The roles of EDS1 and PAD4 in regulating lsd1 runaway cell death are related to the interpretation of reactive oxygen intermediate-derived signals at infection sites. We further demonstrate that the fate of superoxide at infection sites is different from that observed at the leading margins of runaway cell death lesions in lsd1 mutants.