Cell-penetrating Peptide TAT Research Articles

Caspase 3 Targeted Cargo Delivery in Apoptotic Cells Using Capped Mesoporous Silica Nanoparticles.

Excessive apoptotic cell death is at the origin of several pathologies, such as degenerative disorders, stroke or ischemia-reperfusion damage. In this context, strategies to improve inhibition of apoptosis and other types of cell death are of interest and may represent a pharmacological opportunity for the treatment of cell-death-related disorders. In this scenario new peptide-containing delivery systems (solids S1 -P1 and S1 -P2 ) are described based on mesoporous silica nanoparticles (MSNs) loaded with a dye and capped with the KKGDEVDKKARDEVDK (P1 ) peptide that contains two repeats of the DEVD target sequence that are selectively hydrolyzed by caspase 3 (C3). This enzyme plays a central role in the execution-phase of apoptosis. HeLa cells electroporated with S1 -P1 are able to deliver the cargo in the presence of staurosporin (STS), which induces apoptosis with the consequent activation of the cytoplasmic C3 enzyme. Moreover, the nanoparticles S1 -P2 , containing both a cell-penetrating TAT peptide and P1 also entered in HeLa cells and delivered the cargo preferentially in cells treated with the apoptosis inducer cisplatin.

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties.

Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

Targeted delivery of transferrin and TAT co-modified liposomes encapsulating both paclitaxel and doxorubicin for melanoma

The purpose of this study was to develop an efficient dual-ligand based liposomal drug delivery system with targeting specificity as well as properties that would kill melanoma cells. Liposomes modified with transferrin (Tf) and cell-penetrating peptide TAT was prepared, which encapsulated two kinds of chemotherapy drugs, paclitaxel and doxorubicin (Tf/TAT-PTX/DOX-LP). The Tf ligands specifically bind to the overexpressed Tf receptors on the surface of melanoma cells, while the TAT ligands functioned as a classical cell penetrating peptide, helping dual-ligand liposomes be internalized by melanoma cells. The effect of dual-targeting system and “double-drug” combination therapy were evaluated both in vitro and in vivo. In vitro, cellular uptake, intracellular distribution and tumor spheroids penetration studies demonstrated that the system could not only be selectively and efficiently penetrate melanoma cells. Besides, apoptosis staining assay and cytotoxicity showed effective anti-tumor capability and obvious synergistic effect of combination therapy of PTX and DOX. In vivo imaging and fluorescent images of tumor section further demonstrated that Tf/TAT-PTX/DOX-LP had the highest tumor distribution. The results of these experiments demonstrated that double-drug liposomal drug delivery systems (DDS) had both enhanced targeting efficiency and increased therapeutic efficacy.

Chimeric peptides as modulators of CK2-dependent signaling: Mechanism of action and off-target effects

Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α′) and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127–7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Synergy of BID with doxorubicin in the killing of cancer cells

Overexpression of the BH3-interacting domain death agonist (BID) protein sensitizes certain cancer cell lines to apoptosis induced by anticancer agents, particularly by those acting through death receptors (e.g. TRAIL). Previously, we showed that recombinant BID fused with TAT cell penetrating peptide (TAT-BID) allowed for controlled delivery of BID to different cancer cell lines and moderately sensitized some of them to TRAIL or slightly to camptothecin. In the present study, we showed that TAT-BID delivered to HeLa cells strongly sensitized them to doxorubicin, as identified by cell viability and apoptosis assays. Another cell line sensitized to doxorubicin was PC3, whereas A549 and LNCaP cells were sensitized moderately or not at all, respectively. Sensitization was more pronounced at 1 μM doxorubicin administered for 48 h than for lower doses and shorter treatments. TAT-BID and doxorubicin may thus be considered as a potential therapeutic combination for cervical carcinoma and advanced prostate cancer treatment.

The CCL2 chemokine is a negative regulator of autophagy and necrosis in luminal B breast cancer cells.

Luminal A and B breast cancers are the most prevalent forms of breast cancer diagnosed in women. Compared to luminal A breast cancer patients, patients with luminal B breast cancers experience increased disease recurrence and lower overall survival. The mechanisms that regulate the luminal B subtype remain poorly understood. The chemokine CCL2 is overexpressed in breast cancer, correlating with poor patient prognosis. The purpose of this study was to determine the role of CCL2 expression in luminal B breast cancer cells. Breast tissues, MMTV-PyVmT and MMTV-Neu transgenic mammary tumors forming luminal B-like lesions, were immunostained for CCL2 expression. To determine the role of CCL2 in breast cancer cells, CCL2 gene expression was silenced in mammary tumor tissues and cells using TAT cell-penetrating peptides non-covalently cross linked to siRNAs (Ca-TAT/siRNA). CCL2 expression was examined by ELISA and flow cytometry. Cell growth and survival were analyzed by flow cytometry, immunocytochemistry, and fluorescence microscopy. CCL2 expression was significantly increased in luminal B breast tumors, MMTV- PyVmT and MMTV-Neu mammary tumors, compared or normal breast tissue or luminal A breast tumors. Ca-TAT delivery of CCL2 siRNAs significantly reduced CCL2 expression in PyVmT mammary tumors, and decreased cell proliferation and survival. CCL2 gene silencing in PyVmT carcinoma cells or BT474 luminal B breast cancer cells decreased cell growth and viability associated with increased necrosis and autophagy. CCL2 expression is overexpressed in luminal B breast cancer cells and is important for regulating cell growth and survival by inhibiting necrosis and autophagy.

Dual-function nanosystem for synergetic cancer chemo-/radiotherapy through ROS-mediated signaling pathways

Radioresistance and limitation of irradiative dosage usually lead to failure in depletion of hypoxic tumors. Herein we developed multifunctional mesoporous silica nanoparticles (MSNs) as a carrier of a novel anticancer selenoamino acid (selenocystine, SeC), to achieve synergistic chemo-/radiotherapy. This multifunctional nanosystem effectively sensitizes cancer cells to X-ray radiotherapy. Conjugation of TAT cell penetrating peptide and transferrin to the surface of MSNs significantly enhances its internalization in cancer cells through receptor-mediated endocytosis. SeC@MSNs-Tf/TAT significantly enhanced X-ray-induced growth inhibition in cervical cancer cells by induction of apoptosis, mainly through death receptor-mediated extrinsic apoptotic pathway. Upon radiation, SeC@MSNs-Tf/TAT promoted intracellular ROS overproduction, which induced apoptotic cell death by affecting p53, AKT and MAPKs pathways. Furthermore, SeC@MSNs-Tf/TAT also significantly inhibited HeLa tumor growth in nude mice model through suppression of cell proliferation and induction of apoptosis. In vivo toxicity of the SeC@MSNs-Tf/TAT nanoparticles was investigated using the mouse model. The results of histological analysis revealed that, the nanoparticles did not show any obvious damage to these major organs under the experimental conditions, including heart, liver, spleen, lung and kidney. Taken together, this study demonstrates an effective and safe strategy for cancer-targeted chemo-/radiotherapy of human cancers.

Optimization of the cyclotide framework to improve cell penetration properties.

Cell penetrating peptides have been regarded as promising vectors to deliver hydrophilic molecules inside cells. Although they are great tools for research and have high potential as drug delivery systems, their application as drugs is impaired by their low stability in serum. Cyclotides, cyclic disulfide-rich peptides from plants, are ultra-stable molecules that have inspired applications in drug design as they can be used as scaffolds to stabilize linear bioactive sequences. Recently, they have also been shown to possess cell-penetrating properties. The combination of their remarkable stability and cell-penetrating properties opens new avenues for the application of peptides to bind to and inhibit intracellular proteins. Nevertheless, for a broader application of these molecules as vectors is of utmost importance to improve their cellular internalization efficiency. In this study we successfully modified MCoTI-II, one of the most widely studied cyclotide scaffolds in drug design, and improved its internalization properties. The internalization of the newly designed MCoTI-II is as efficient as the gold standard cell-penetrating peptide (CPP) TAT and maintains all the required features as a template to graft desired bioactivities.

Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer.

Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.

Cell penetrating peptide TAT can kill cancer cells via membrane disruption after attachment of camptothecin

Attachment of traditional anticancer drugs to cell penetrating peptides is an effective strategy to improve their application in cancer treatment. In this study, we designed and synthesized the conjugates TAT-CPT and TAT-2CPT by attaching camptothecin (CPT) to the N-terminus of the cell penetrating peptide TAT. Interestingly, we found that TAT-CPT and especially TAT-2CPT could kill cancer cells via membrane disruption, which is similar to antimicrobial peptides. This might be because that CPT could perform as a hydrophobic residue to increase the extent of membrane insertion of TAT and the stability of the pores. In addition, TAT-CPT and TAT-2CPT could also kill cancer cells by the released CPT after they entered cells. Taken together, attachment of CPT could turn cell penetrating peptide TAT into an antimicrobial peptide with a dual mechanism of anticancer action, which presents a new strategy to develop anticancer peptides based on cell penetrating peptides.

Efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer.

This study was mainly focused on the development of a dual-ligand liposomal delivery system for targeting the delivery of paclitaxel (PTX) to lung cancer. The specific ligand peptide HAIYPRH (T7) and the cationic cell-penetrating peptide TAT were connected with phospholipid via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (T7/TAT-LP-PTX). Physicochemical characterizations of T7/TAT-LP-PTX, such as particle size, ζ potential, morphology, encapsulation efficiency, and in vitro PTX release, were also evaluated. In the cellular uptake study, the T7/TAT-LP endocytosed by the A549 cells was 2.26-, 3.48- and 8.56-fold higher than TAT-LP, T7-LP and LP, respectively. The IC50 values of TAT-LP-PTX, T7-LP-PTX and LP-PTX were much higher than those of T7/TAT-LP-PTX, respectively. The homing specificity of T7/TAT-LP was evaluated on the tumor spheroids, which revealed that T7/TAT-LP was more efficaciously internalized in tumor cells than TAT-LP, T7-LP and LP, respectively. Compared to LP, TAT-LP and T7-LP, T7/TAT-LP showed the strongest cell uptake property, and the highest accumulation ability in tumor spheroids in vitro. In the in vivo study, the T7/TAT-LP-PTX exhibited the best inhibitory effect of tumor growth for A549-bearing mice. Collectively, these results suggested that T7/TAT-LP-PTX is a promising drug delivery system for the treatment of lung cancer.

Cell penetrating peptide (CPP)-conjugated desferrioxamine for enhanced neuroprotection: synthesis and in vitro evaluation.

Iron overload causes progressive and sometimes irreversible damage due to accelerated production of reactive oxygen species. Desferrioxamine (DFO), a siderophore, has been used clinically to remove excess iron. However, the applications of DFO are limited because of its inability to access intracellular labile iron. Cell penetrating peptides (CPPs) have become an efficient delivery vector for the enhanced internalization of drugs into the cytosol. We describe, herein, an efficient method for covalently conjugating DFO to the CPPs TAT(47-57) and Penetratin. Both conjugates suppressed the redox activity of labile plasma iron in buffered solutions and in iron-overloaded sera. Enhanced access to intracellular labile iron compared to the parent siderophore was achieved in HeLa and RBE4 (a model of blood-brain-barrier) cell lines. Iron complexes of both conjugates also had better permeability in both cell models. DFO antioxidant and iron binding properties were preserved and its bioavailability was increased upon CPP conjugation, which opens new therapeutic possibilities for neurodegenerative processes associated with brain iron overload.

Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis.

BackgroundLow cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study.MethodsUptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay.ResultsTAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID.ConclusionsTAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-771) contains supplementary material, which is available to authorized users.

Interactions of skin with gold nanoparticles of different surface charge, shape, and functionality.

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP-OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R7 are found in the skin in larger quantities than polyethylene glycol-functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.

Preparation of TAT peptide-modified poly(N-isopropylacrylamide) microgel particles and their cellular uptake, intracellular distribution, and influence on cytoviability in response to temperature change

Thermo-sensitive microgel particles may exert a swelling force inside cells and influence on cell viability due to their volume transition in response to external temperature change. In this study, cross-linked poly(N-isopropylacrylamide) (PNIPAM) microgel particles with a thermo-responsive volume expansion ability were prepared by precipitation polymerization of NIPAM, poly(ethylene glycol) diacrylate and acrylic acid. To endow the microgel particles with enhanced cellular uptake and visualization, cell penetrating peptide TAT and fluorescent probe were further covalently immobilized. The cellular uptake, intracellular distribution and thermo-responsive cytotoxicity of the microgel particles were studied by co-culture with lung adenocarcinoma (A549) cells. The PNIAPM microgel particles were largely ingested by A549 cells and mainly located in lysosomes. TAT modification enhanced the cellular internalization of particles but did not alter their intracellular distribution. While the PNIPAM microgel particles did not show significant impact on cell viability at 37°C, they caused cytotoxicity to some extent when being cultured at 25°C for 4h. Doxorubicin loaded PNIPAM microgel particles showed the strongest cytotoxicity when being cultured at 25°C for 4h, suggesting the combinational effect of intracellular volume expansion and drug release on cells.

Systemic delivery of P42 peptide: a new weapon to fight Huntington's disease.

BackgroundIn Huntington’s disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice.ResultsTo this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections.ConclusionsThese data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0086-x) contains supplementary material, which is available to authorized users.

Increased delivery of doxorubicin into tumor cells using extracellularly activated TAT functionalized liposomes: in vitro and in vivo study.

The development of highly efficient tumor-targeted delivery systems is crucial for successful tumor treatment. Previously, a novel cell-penetrating peptide TAT and cleavable polyethylene glycol (PEG) co-modified liposome delivery system (C-TAT-Lipo) showed enhanced accumulation in tumor regions. Under the control of cysteine (Cys), the liposomes were activated extracellularly and achieved increased delivery of their cargo into tumor cells efficiently. In this study, we developed an optimal formulation for the encapsulation of Doxorubicin (DOX) by this delivery system for tumor treatment. The in vitro study showed that the C-TAT-Lipo with Cys delivery system not only enhanced the amount of DOX delivered by at least 100% compared to other DOX-containing formulations, but also displayed high cytotoxicity against tumorigenic cell lines. Compared to other groups, the DOX-loaded C-TAT-Lipo formulation in the presence of cysteine enhanced treatment efficacy by lowering the IC50 (1.67 +/- 0.14 microM) and increasing the cancer cell apoptosis percentage (37.10%). Moreover, the in vivo antitumor activity also showed that DOX-loaded C-TAT-Lipo with injection of cysteine achieved the best tumor growth inhibition with a tumor growth rate of only 58.40 +/- 16.33% (% of initial volume/day), which was significant less than that achieved by other DOX formulations.

Preparation and evaluation of RGD and TAT co-modified paclitaxel loaded liposome

To prepare Arg-Gly-Asp (RGD) and cell penetrating peptide TAT co-modified paclitaxel loaded liposome (RGD/TAT-LP-PTX) for MCF-7 cell inhibition. The co-modified liposome was prepared by film-ultrasonic method. The appearance, particle size and zeta potential were evaluated. The cellular uptake by MCF-7 cells in vitro was used to evaluate the targeting efficiency. The anti-proliferation efficiency of RGD/TAT-LP-PTX was evaluated by MTT assay. Tumor spheroids were used to evaluate anti-tumor ability of RGD/TAT-LP-PTX in vitro. The particle diameter of the co-modified liposome was (138.8 ± 12.4) nm with the Zeta potential of (25.85 ± 2.75) mV. The entrapment efficiency of PTX was 88.3%. The RGD/TAT-LP uptaken by MCF-7 cells at 4 h was 1.79 times higher than that at 2 h. The co-modified liposome uptaken by MCF-7 cells was 2.25 and 2.72 times higher than that of TAT-LP and RGD-LP, respectively. The anti-proliferation rate of RGD/TAT-LP-PTX increased with time. The inhibition rate of RGD/TAT-LP-PTX for MCF-7 cells at 48 h was 1.78 times higher than that at 24 h. The MTT assay demonstrated the cell viability of RGD/TAT-LP-PTX was 1.65, 1.82 and 2.55 times higher than that of TAT-LP-PTX, RGD-LP-PTX and LP-PTX, respectively. Co-modified liposome may serve as a promising breast cancer delivery system for antitumor drugs.

Novel peptide VIP-TAT with higher affinity for PAC1 inhibited scopolamine induced amnesia

A novel peptide VIP-TAT with a cell penetrating peptide TAT at the C-terminus of VIP was constructed and prepared using intein mediated purification with an affinity chitin-binding tag (IMPACT) system to enhance the brain uptake efficiency for the medical application in central nervous system. It was found by labeling VIP-TAT and VIP with fluorescein isothiocyanate (FITC) that the extension with TAT increased the brain uptake efficiency of VIP-TAT significantly. Then short-term and long-term treatment with scopolamine (Scop) was used to evaluate the effect of VIP-TAT or VIP on Scop induced amnesia. Both short-term and long-term administration of VIP-TAT inhibited the latent time reduction in step-through test induced by Scop significantly, but long-term administration of VIP aggravated the Scop induced amnesia. Long-term i.p. injection of VIP-TAT was shown to have positive effect by inhibiting the oxidative damage, apoptosis and the cholinergic system activity reduction that induced by Scop, while VIP exerted negative effect in brain opposite to that in periphery system. The in vitro data showed that VIP-TAT had not only protective but also proliferative effect on Neuro2a cells which was inhibited by PAC1 antagonist PACAP(6-38). Competition binding assay and cAMP assay confirmed that VIP-TAT had higher affinity and activation for PAC1 than VIP. So it was concluded that the significantly stronger protective effect of VIP-TAT against Scop induced amnesia than VIP was due to (1) the enhanced brain uptake efficiency of VIP-TAT and (2) the increased affinity and activation of VIP-TAT for receptor PAC1.