Cell-penetrating Peptide Octa-arginine Research Articles

Octa-Arginine-Conjugated Liposomal Nimodipine Incorporated in a Temperature-Responsive Gel for Nasoencephalic Delivery.

Nimodipine is the primary clinical drug used to treat cerebral vasospasm following subarachnoid hemorrhage. Currently, tablets have low bioavailability when taken orally, and injections contain ethanol. Therefore, we investigated a new method of nimodipine administration, namely, nasoencephalic administration. Nasal administration of nimodipine was carried out by attaching the cell-penetrating peptide octa-arginine (R8) to liposomes of nimodipine and incorporating it into a temperature-sensitive in situ gel. The prepared liposomes and gels underwent separate evaluations for in vitro characterization. In vitro release exhibited a significant slow-release effect. In vitro toad maxillary cilia model, RPMI 2650 cytotoxicity, and in vivo SD rat pathological histotoxicity experiments showed that all the dosage from the groups had no significant toxicity to toad maxillary cilia, RPMI 2650 cells, and SD rat tissues and organs, and the cilia continued to oscillate up to 694 ± 10.15 min, with the survival rate of the cells being above 85%. A transwell nasal mucosa cell model and an isolated porcine nasal mucosa model were established, and the results showed that the osmolality of the R8-modified nimodipine liposomal gel to nasal mucosal cells and isolated porcine nasal mucosa was 30.41 ± 2.14 and 65.9 ± 7.34 μg/mL, respectively, which was significantly higher than that of the NM-Solution and PEGylated nimodipine liposome gel groups. Animal fluorescence imaging studies revealed that the R8-modified nimodipine liposomal gel displayed increased brain fluorescence intensity compared to the normal liposomal gel. Pharmacokinetic results showed that after transnasal administration, the AUC(0-∞) of the R8-modified nimodipine liposomal gel was 11.662 ± 1.97 μg·mL-1, which was significantly higher than that of the plain nimodipine liposomal gel (5.499 ± 2.89 μg·mL-1). Brain-targeting experiments showed that the brain-targeting efficiencies of the PEGylated nimodipine liposome gel and R8-modified PEGylated nimodipine liposome gels were 20.44 and 33.45, respectively, suggesting that R8/PEG/Lip-NM-TSG significantly increased the brain-targeting of the drug.

A novel octa-arginine-modified injectable self-assembling peptide hydrogel for multidrug-resistant cancer therapy

Surgery combined with systemic chemotherapy is currently the main modality of cancer treatment. However, the development of cancer multidrug resistance and inevitable surgical residual lesions lead to high rates of cancer recurrence and mortality. Meanwhile, systemic drug toxic side effects and surgical bleeding complications seriously reduce the quality of life of patients. To avoid cancer multidrug resistance and comprehensively optimize cancer treatment, this work provides a novel injectable cell penetrating peptide octa-arginine (R8)-modified RADA16 (RR) self-assembling peptide nanofiber hydrogel as an anticancer drug carrier for multidrug-resistant cancer therapy. In RR hydrogel, highly compatible RADA16 functions as the β-sheet-based self-assembling backbone which tightly adheres to cancer tissue, prevents postoperative bleeding, and sustainedly releases anticancer drugs for long-term effects. The introduced R8 motifs endow RR hydrogel with the capacity to selectively kill cancer cells and reverse cancer multidrug resistance, which minimally impact normal tissue cells. Specifically, R8 motifs on the hydrogel scaffold selectively interfere with cancer cell membranes, increase drug uptake and penetration into cancer cells and tissues by enhancing multiple internalization pathways, and reduce drug efflux by inhibiting P-gp and BCRP multidrug-resistant transporters. Therefore, this study provides a multifunctional hydrogel material with strong potential for multidrug-resistant cancer clinical translation.

Intracellular Bacterial Targeting by a Thiazolyl Benzenesulfonamide and Octaarginine Peptide Complex.

A brominated thiazolyl benzenesulfonamide (BTB) derivative is conjugated with the cell-penetrating peptide octaarginine (R8) in an effort to construct innovative antibacterial products. The noncovalent complex of BTB and R8 is characterized by Fourier transform infrared (FTIR) spectroscopy, which indicates hydrogen bonding between the two constituents. Attachment of the peptide moiety renders aqueous solubility to the hydrophobic benzenesulfonamide drug and bestows bactericidal activity. Confocal imaging in conjunction with dye probes shows successful clearance of intracellular Staphylococcus aureus bacteria by the BTB-R8 complex. Scanning electron micrographs and studies with a set of fluorescent dyes suggest active disruption of the bacterial cell membrane by the BTB-R8 complex. In contrast, the complex of BTB with octalysine (K8) fails to cause membrane damage and displays a modest antibacterial effect. A complex of BTB with the water-soluble hydrophilic polymer poly(vinylpyrrolidone) (PVP) does not display any antibacterial effect, indicating the distinctive role of the cell-penetrating peptide (CPP) R8 in the cognate complex. The leakage of the encapsulated dye from giant unilamellar vesicles upon interaction with the BTB-R8 complex further highlights the membrane activity of the complex, which cannot be accomplished by bare sulfonamide alone. This work broadens the scope of use of CPPs with respect to eliciting antibacterial activity and potentially expands the limited arsenal of membrane-targeting antibiotics.

Intracellular delivery of oxaliplatin conjugate via cell penetrating peptide for the treatment of colorectal carcinoma in vitro and in vivo

Pt-based drugs are one of the main active agents in colorectal cancer treatment. However, drug resistance and dose-dependent side effects are the main barriers that restrict their clinical applications. As an alternative approach to these issues, we designed and synthesized a cell penetrating peptide (CPP) octaarginine-oxaliplatin conjugate that quickly and successfully delivered oxaliplatin into colon cancer cells. The CPP octaarginine is a well-studied cationic peptide that can play a role as a drug delivery vector. In this work, an octaarginine CPP (RRRRRRRR) was conjugated with oxaliplatin via a specific heterobifunctional linker. The in vitro studies showed the conjugate had affinity toward mitochondria inside cells and the MTT assay confirmed that conjugate is active in low micromolar range against colon cancer cells, requiring much lower concentrations than the oxaliplatin alone to reach IC50. More importantly, in the in vivo mouse study, the conjugate effectively inhibited tumor growth and showed considerably high antitumor activity, demonstrating the conjugate can perform well in vivo. This strategy may offer a new approach for designing oxaliplatin derivatives or prodrugs with remarkable therapeutic capabilities.

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes

ABSTRACT A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (μM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.

Complying with the physiological functions of Golgi apparatus for secretory exocytosis facilitated oral absorption of protein drugs.

Intestinal epithelial cells are the primary biological barriers for orally administrated nano-formulations and the delivered protein drugs. Thereinto, besides the cellular uptake, intracellular trafficking pathway and the related exocytosis are of great importance to the trans-epithelial transport of drug-loaded NPs. Herein, inspired by the physiological functions of Golgi apparatus for secreting proteins out of cells, Golgi localization-related amino acid l-cysteine (Cys) was modified on the surface of NPs to see whether and how this modification could guide the Golgi pathway-related transport and facilitate the exocytosis of drug-loaded NPs. Meanwhile, cell-penetrating peptide octa-arginine (R8) was co-modified to increase the cellular uptake. The proportion of R8 and Cys modification was explored to get the best effect of endocytosis and exocytosis of NPs. As a result, 25%R8 + 75%Cys NPs with most Cys modification showed efficient transcytosis with the highest transcytosis/endocytosis ratio (0.87). Interestingly, exocytosis mechanism studies indicated that they trafficked through the Golgi secretory pathway and bypassed lysosomes due to Cys modification. The detailed Golgi position mechanism studies further suggested that the thiol group from Cys was important for mediating Golgi transport. In particular, competitive inhibition studies demonstrated that Cys-modified NPs were more conducive to their exocytosis after being transported through the Golgi secretory pathway. We proved that cargos transported via Golgi apparatus tended to be trafficked out of the cells and avoid degradation, which contributed to the transcytosis of 25%R8 + 75%Cys NPs in vitro. Inspiringly, compared with unmodified NPs, 25%R8 + 75%Cys NPs also exhibited promoted intestinal penetration and oral absorption in vivo. Oral delivery of insulin-loaded 25%R8 + 75%Cys NPs showed stronger hypoglycemic effects in diabetic rats. In summary, this work provides a strategy for complying with the physiological functions of Golgi apparatus for secreting to facilitate the exocytosis of NPs, thus further improving the oral absorption of loaded protein drugs.

Enhancing Aqueous Solubility and Antibacterial Activity of Curcumin by Complexing with Cell-Penetrating Octaarginine.

Bacterial resistance to antimicrobial drugs is one of the biggest threats to human health and novel drugs, and strategies are needed to obviate this resistance crisis. An innovative strategy for designing novel antimicrobial drugs is based on the hybridization of an antimicrobial agent with a second functional entity. Here, we use a cell-penetrating peptide–octaarginine (R8) as the second functional entity and develop a complex or hybrid of R8 and curcumin that possibly targets the bacterial cell membrane. Minimum inhibitory concentration assays show that the antibacterial activity of the complex is enhanced in a synergistic manner and rapid killing kinetics are obtained, emphasizing a bactericidal mode of action. In addition, electron microscopy images reveal bacterial membrane disruption by the complex. The R8–curcumin complex also displays activity against HeLa cells.

A novel mitochondrial targeted hybrid peptide modified HPMA copolymers for breast cancer metastasis suppression

Primary tumor metastasis remains to be a tough obstacle for clinical breast cancer treatment. Since evidences have shown that mitochondria play a crucial role in tumor metastasis, we designed a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to simultaneously inhibit breast cancer progression and metastasis. A novel mitochondrial targeted hybrid peptide R8MTS, which consists of a cell penetrating peptide octaarginine (R8) and a mitochondrial targeting sequence ALD5MTS, was used as targeting ligand and attached to doxorubicin (DOX) as model drug (DOX-R8MTS). After entering into the tumor cells, DOX-R8MTS was pH-responsibly released from HPMA copolymer backbone in acidic lysosome and efficiently targeted to mitochondria, resulting in enhanced reactive oxygen species (ROS) generation and apoptosis initiation. By destroying mitochondria, P-D-R8MTS not only inhibited cell proliferation but also suppressed migration and invasion of breast cancer 4T1 and MDA-MB-231 cells in vitro. Moreover, P-D-R8MTS exhibited superior inhibition of tumor growth and showed no apparent lung metastatic nodules on 4T1-bearing mice in vivo, which was partially via down-regulation of typical proteins associated with tumor metastasis and invasion: matrix metalloproteinases-2 (MMP-2), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β). Collectively, our work provided a prospectively potential strategy for metastatic cancer treatment through mitochondrial targeted drug delivery.

A simple and efficient transfection protocol for Cryptosporidium parvum using Polyethylenimine (PEI) and Octaarginine.

The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.

EJP18 peptide derived from the juxtamembrane domain of epidermal growth factor receptor represents a novel membrane-active cell-penetrating peptide.

Membrane-active peptides have been extensively studied to probe protein-membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, non-toxic concentrations the peptides outperformed the well-characterised CPP octaarginine in cellular delivery capacity for a fluorophore or proteins that were associated with the peptide covalently or via ionic interactions. EJP18 thus represents a novel membrane-active peptide that may be used as a naturally derived model for biophysical protein-membrane interactions or for delivery of cargo into cells for therapeutic or diagnostic applications.

Redox-Responsive Multifunctional Polypeptides Conjugated with Au Nanoparticles for Tumor-Targeting Gene Therapy and Their 1 + 1 > 2 Synergistic Effects.

Gene therapy is regarded as one of the most potential technologies for tumor therapy. Gene delivery systems with high specificity and good biocompatibility are urgently demanded. Hence, in this research, we designed and synthesized a series of tumor targeting and redox-responsive gold nanoparticles conjugated with three kinds of functional polypeptides (AuNPPs) that consisted of targeting peptide GE11, cell-penetrating peptide octaarginine (R8), and polyhistidine. All the AuNPPs exhibited superior cancer cellular internalization ability and targeting gene transfection efficiency compared with commercial agent BPEI 25K. It is interesting to find that different relative positions of GE11 and R8 can cause the change of target ability and gene transfection efficiency, and the suitable relative position of R8 and GE11 can not only endow the gene vector with functions that peptides previously own but also bring the synergistic effects. The best-performed AuNPP6-1 was chosen to transport the epidermal growth factor receptor (EGFR)-shRNA into A549 tumor-bearing BALB/c nude mice, and in vivo fluorescence imaging showed AuNPP6-1 mainly accumulated in tumor sites and achieved a great targeting therapy effect. These results provide significantly important information on understanding and constructing the tumor-targeting gene vector.

Coated microneedles mediated intradermal delivery of octaarginine/BRAF siRNA nanocomplexes for anti-melanoma treatment

BRAF is the most frequently mutated gene in skin melanoma. Applying BRAF siRNA (siBraf) to silencing BRAF gene is a current frontline therapy for melanoma. However, delivery of macromolecular siRNA into the tumor site and introduction of siRNA into the tumor cells remain as challenges. In this study, we for the first time developed a siBraf delivery system based on cell penetrating peptide octaarginine (R8) nanocomplexes combined with coated microneedles (MNs), i.e. R8/siBraf coated MNs, for targeted anti–melanoma treatment. The R8/siBraf nanocomplexes were optimized based on the internalization of siBraf by A375 cells. In vitro A375 cell experiments presented that R8/siBraf can enhance siBraf transfection, silence BRAF gene, and inhibit tumor cells growth, comparable to polyethylenimine (PEI)/siBraf. R8/siBraf coated MNs can effectively deliver R8/siBraf into the disease site. In vivo anti-melanoma experiments indicated that R8/siBraf coated MNs can significantly inhibit the melanoma development, induce the tumor cells apoptosis, and suppress their proliferation. The BRAF gene in tumor were also significantly silenced in vivo. SiBraf intradermal delivery via combining MNs and R8 nanocomplexes is a promising approach for skin melanoma treatment, which exploited both virtues of MNs and cell penetrating peptide to obtain the targeting inhibition efficacy on skin melanoma.

Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.

Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy.

Cell penetrating peptides (CPPs) were widely used for drug delivery to tumor. However, the nonselective in vivo penetration greatly limited the application of CPPs-mediated drug delivery systems. And the treatment of malignant tumors is usually followed by poor prognosis and relapse due to the existence of extravascular core regions of tumor. Thus it is important to endue selective targeting and stronger intratumoral diffusion abilities to CPPs. In this study, an RGD reverse sequence dGR was conjugated to a CPP octa-arginine to form a CendR (R/KXXR/K) motif contained tandem peptide R8-dGR (RRRRRRRRdGR) which could bind to both integrin αvβ3 and neuropilin-1 receptors. The dual receptor recognizing peptide R8-dGR displayed increased cellular uptake and efficient penetration ability into glioma spheroids in vitro. The following in vivo studies indicated the active targeting and intratumoral diffusion capabilities of R8-dGR modified liposomes. When paclitaxel was loaded in the liposomes, PTX-R8-dGR-Lip induced the strongest anti-proliferation effect on both tumor cells and cancer stem cells, and inhibited the formation of vasculogenic mimicry channels in vitro. Finally, the R8-dGR liposomal drug delivery system prolonged the medium survival time of intracranial C6 bearing mice by 2.1-fold compared to the untreated group, and achieved an exhaustive anti-glioma therapy including anti-tumor cells, anti-vasculogenic mimicry and anti-brain cancer stem cells. To sum up, all the results demonstrated that R8-dGR was an ideal dual receptor recognizing CPP with selective glioma targeting and efficient intratumoral diffusion, which could be further used to equip drug delivery system for effective glioma therapy.

Development of a novel nanoparticle by dual modification with the pluripotential cell‐penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex

Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery.

Improved Efficacy of Fosmidomycin against Plasmodium and Mycobacterium Species by Combination with the Cell-Penetrating Peptide Octaarginine

Cellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot permeate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate intracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octaarginine host cell permeability and its ability to improve the efficacy of fosmidomycin. Plasmodium berghei liver-stage parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly, Toxoplasma gondii was only susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin greatly enhanced efficacy against blood-stage Plasmodium falciparum. This complex and a covalently linked conjugate of octaarginine and fosmidomycin also reverted resistance of Mycobacteria to fosmidomycin. These findings provide chemical genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically relevant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomycin formulations for malaria and tuberculosis drug development.

SiRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.

Chemical Synthesis of Cell-Permeable Apoptotic Peptides from in Vivo Produced Proteins

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.

Gene delivery to tumor cells by cationic polymeric nanovectors coupled to folic acid and the cell-penetrating peptide octaarginine

Target ligand folic acid (FA) and cell-penetrating peptide octaarginine (R8) were coupled with the gene vectors (PEI 600-CyD, PC) composed of β-cyclodextrin (β-CyD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form nanovectors for highly efficient gene delivery to tumor cells. The resultant ternary nanocomplexes of FA-PC/R8-PC/pDNA produced excellent gene transfaction abilities in the folate receptor (FR)-positive tumor cells in vitro and in vivo. The FR-mediated endocytosis and the R8-mediated transmembrane functionality together contributed to the high transfection levels. This study provides a promising means to produce gene nanovectors for in vivo applications.

Click chemistry for the generation of cell permeable apoptotic peptides

he use of proteins and peptides as drug molecules has been held back by their proteolytic instability and inability to cross-cellular membranes. Proteins and long peptides are often produced by expression in E. coli rather than by solid phase peptide synthesis. A drawback of in vivo protein and peptide synthesis is the difficulty to selectively modify the product peptide by the attachment of fluorescent dyes or ligation to other macromolecules like polysaccharides, lipids or peptides. Here we present a facile method to modify an expressed protein or peptide to create a C-terminal alkyne group. This functionality is then used inter alia for conjugation to the cell-penetrating peptide octa-arginine. This will provide a vector for delivery across the plasma membrane of cells. To demonstrate our method, we have produced in E. coli a peptide derived from the Bak protein; one of the key regulators of apoptosis in eukaryotic cells. In the cell it is usually found bound to Bcl-xL at the outer mitochondrial membrane. If this interaction is disrupted, Bak oligomerizes and forms pores which trigger mitochondria dependent apoptosis through cytochrome c release. Small peptides derived from the BH3 helix of Bak have been shown to induce apoptosis. We have expressed such a peptide in E. coli as a fusion protein. The ketosteroid isomerase fusion protein is insoluble and readily purified from cell extracts. The peptide is then cleaved from the fusion protein by reaction with cyanogen bromide at a strategically inserted methionine residue to generate a homoserine lactone at the C-terminus of the Bak peptide. This lactone is then used for direct amide formation with inexpensive propargylamine. The resulting alkynyl peptide serves as a reagent for highly efficient ‘click’ reactions to couple to a wide range of azides. Since the Bak peptide is not able to cross the cell membrane, the well-known octa-arginine cell penetrating peptide sequence was added as a delivery vector. Here we discuss the synthesis of this semi-synthetic peptide and its interaction with, and uptake into, cancer cell lines.