Cell-penetrating Peptide TAT Research Articles

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

SummaryThe effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT-Like Cell-Penetrating Peptide and the Lipid Bis(Monoacylglycerol)Phosphate

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Herein, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycerol)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of the CPP’s membrane leakage activity. These results therefore support a novel model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.

Multi-functional nanocomplex codelivery of Trp2 and R837 to activate melanoma-specific immunity.

Antigen-adjuvant combination could induce a protective and long-lasting anti-tumor immune response. However, exploiting system which could co-deliver melanoma antigen peptide Trp2 (Tyrosinase-related protein 2) and Toll-like-receptor-7 (TLR7) agonists imiquimod (R837) both are poor aqueous solubility is still challenging. Our new nanocomplex was explored for specific delivery of Trp2 and R837 into antigen-presenting cells (APCs). R837 was loaded into mannosylated-β-cyclodextrin (Man-CD) to target dendritic cells (DCs) by binding mannose receptors (MR) on DCs. A fusion peptide (WT) was constructed by incorporating the amino acid region of TAT (cell-penetrating peptide) into Trp2 to improve the TAT-mediated intracellular efficiency. Negatively charged sodium alginate (SA), a biocompatible polymer, which can induce adjuvant responses by affecting the functions of DCs, was complexed with Man-CD/R837 and WT via physical adsorption. The optimized nanocomplex promoted the cellular uptake and showed remarkable efficacy to enhance the secreting of Th1-cytokines. This multi-functional nanocomplex system may allow effective targeting-codelivery of multi-hydrophobic drugs and be a promising subunit vaccine candidate as a potential prevention action of tumor.

A Multiparametric Evaluation of Quantum Dot Size and Surface-Grafted Peptide Density on Cellular Uptake and Cytotoxicity.

Despite the progress in nanotechnology for biomedical applications, great efforts are still being employed in optimizing nanoparticle (NP) design parameters to improve functionality and minimize bionanotoxicity. In this study, we developed CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) that are compact ligand-coated and surface-functionalized with an HIV-1-derived TAT cell-penetrating peptide (CPP) analog to improve both biocompatibility and cellular uptake. Multiparametric studies were performed in different mammalian and murine cell lines to compare the effects of varying QD size and number of surface CPPs on cellular uptake, viability, generation of reactive oxygen species, mitochondrial health, cell area, and autophagy. Our results showed that the number of cell-associated NPs and their respective toxicity are higher for the larger QDs. Meanwhile, increasing the number of surface CPPs also enhanced cellular uptake and induced cytotoxicity through the generation of mitoROS and autophagy. Thus, here we report the optimal size and surface CPP combinations for improved QD cellular uptake.

Synergetic Tumor Probes for Facilitating Therapeutic Delivery by Combined-Functionalized Peptide Ligands.

Both targeting and penetrating ability are the key characteristics for tissue probing and precise delivery. To construct an efficient nano probing and delivery system toward human epidermal growth factor receptor 2 (HER2) positive cancer, we established a nano liposomal system functionalized with a newly screened HER2 targeting peptide (HP2, YDLKEPEH) and the cell-penetrating peptide TAT simultaneously. Compared with the monofunctionalized liposomal probes, the dual-functional ones demonstrated a synergetic effect in cell uptake, drug delivery, and in vivo imaging. The improved efficacy of the synergetic system provides a prospective strategy for cancer diagnosis and therapy.

MrgX2 is a promiscuous receptor for basic peptides causing mast cell pseudo-allergic and anaphylactoid reactions.

Activation of MrgX2, an orphan G protein‐coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine‐like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug‐like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug‐like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl‐Arg9‐amide) and TAT (49‐60), a fragment of HIV‐1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo‐allergic reactions known as red man syndrome, is an agonist of MrgX2.

Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

Abstract 3616: Towards intracellular targeting: Cytosolic delivery using TAT-trimers

Abstract Large therapeutic biomolecules, particularly antibodies, are generally directed against cancer cell surface antigens to facilitate antibody-antigen interaction. This limitation excludes a rich array of potential intracellular cancer targets, which has prompted research to solve the problem of transport of large molecules across the cell membrane and their release into the cytoplasm. However, current efforts suffer from endosomal entrapment and lack of efficacy due to low intracellular concentration ranges. Using the archetypal cell penetrating peptide (CPP) TAT, we report the design, synthesis and evaluation of two novel trimeric TAT clusters with enhanced efficiency in the low micromolar range. In addition, we demonstrate that geometry and conformation of TAT clusters affects internalization properties and mechanism. TAT-trimers were synthesized using copper catalyzed 2+3 cycloaddition between tetrakis core structures and azide / alkyne modified TAT (49-57) peptide. The trimeric clusters were furnished with AlexaFluor488 (AF488) for in vitro detection. Tri-TAT A is designed around a smaller, more strained core structure than tri-TAT B, which is designed around a larger core structure and more flexible. Both trimers, as well as monomeric AF488 labelled TAT (mono-TAT), were evaluated for their cell penetrating properties using live-cell fluorescence confocal microscopy in HeLa and CHO cells. The uptake of tri-TAT A and tri-TAT B into HeLa and CHO was found to be significantly higher than that of mono-TAT (1 μM; 60 min); in addition, the uptake of the trimers (1 μM) was found to be significantly higher than that of 10 μM mono-TAT, indicating that clustering TAT peptides improves their efficacy. Interestingly, we found that tri-TAT A shows homogenous cytosolic uptake and nucleolar staining at 1 μM, while tri-TAT B shows no evidence of cytosolic uptake or nucleolar staining in either HeLa or CHO cells. The fluorescence signal in cells treated with tri-TAT B was focal in nature, which is typical of endosomal entrapment. These results suggest that the geometry of the core structure and the conformational flexibility of TAT trimers is of decisive importance to the efficacy of cytosolic uptake. Time course experiments with tri-TAT A indicate that the homogenous cytosolic signal spreads evenly from the membrane into the cytosol, suggesting that the trimer translocates directly across the membrane, rather than or in addition to entry into the cytosol via endosomal escape. TAT-trimers were successfully conjugated to antibodies and antibody fragments (Fab). These constructs showed favourable internalisation properties in preliminary cell uptake studies in comparison single TAT constructs. Trimeric arrangement of CPPs on a rigid scaffold represents a promising approach to promoting cell uptake of anticancer drugs directed against intracellular targets and a significant improvement over recently reported CPP approaches. Citation Format: Ole Tietz, Rod Chalk, Katherine A. Vallis. Towards intracellular targeting: Cytosolic delivery using TAT-trimers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3616.

A novel therapeutic peptide targeting myocardial reperfusion injury.

Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction. An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application.

PH-Sensitive Ratiometric Fluorescent Probe for Evaluation of Tumor Treatments.

Determining therapeutic efficacy is critical for tumor precision theranostics. In order to monitor the efficacy of anti-cancer drugs (e.g., Paclitaxel), a pH-sensitive ratiometric fluorescent imaging probe was constructed. The pH-sensitive ratiometric fluorescent dye ANNA was covalently coupled to the N-terminal of the cell-penetrating TAT peptide through an amidation reaction (TAT-ANNA). The in vitro cellular experiments determined that the TAT-ANNA probe could penetrate the cell membrane and image the intracellular pH in real time. The in vivo experiments were then carried out, and the ratiometric pH response to the state of the tumor was recorded immediately after medication. The TAT-ANNA probe was successfully used to monitor the pharmacodynamics of anti-cancer drugs in vivo.

Enhanced cellular uptake of near-infrared triggered targeted nanoparticles by cell-penetrating peptide TAT for combined chemo/photothermal/photodynamic therapy

Recently, the emergence of cell-penetrating peptides (CPPs) like TAT has greatly improved the efficiency of cancer therapy by enhancing cellular uptake of nanomaterials. Here, we designed a near-infrared (NIR) triggered TAT-based targeted nanoplatform (cRGD@TAT-DINPs), which co-delivered anticancer drug doxorubicin (DOX) and biocompatible dye indocyanine green (ICG) to realize combined chemo/photothermal/photodynamic therapy of cancer in vitro. The resulting nanoparticles showed favorable monodispersity and colloidal stability. Impressively, the DOX could be released in a promoted manner once the nanoparticles were exposed to NIR light. Confocal laser scanning microscopy (CLSM) and flow cytometry analysis demonstrated an immensely enhanced cellular accumulation of DOX after the simultaneous introduction of targeted ligand cRGD and CPP TAT. In addition, the obtained nanoparticles exhibited explosive temperature elevation and reactive oxygen species (ROS) generation mediated by encapsulated ICG under NIR irradiation, and in vitro cytotoxicity assay confirmed the cRGD@TAT-DINPs had an increasing cytotoxicity and excellent synergistic inhibition capacity. Thus, TAT-based nanosystems provide a high-efficient drug delivery strategy for optimizing combined therapy efficiency of cancer.

Cardioprotective effects of a synthetic peptide targeting the extrinsic apoptotic pathway in a mouse model of ischemia-reperfusion

Apoptosis is a main contributor of myocardial reperfusion injury during acute infarction. In a previous study, we showed that reperfusion injury was mediated by the FAS-dependent apoptotic signal mobilizing the DAXX (death-associated protein) adaptor protein. To evaluate the cardioprotective effects of a synthetic peptide that uncouples FAS to the DAXX downstream pathway. The SPOT technology was used to design a synthetic peptide interfering with FAS:DAXX interaction. This peptide was coupled to the Tat cell penetrating peptide (Tat-DAXXp). Infarct size (TTC staining) and apoptosis (DNA fragmentation) were evaluated in the left ventricle of mice subjected to a surgical protocol of reversible coronary artery ligation and treated by the peptide at the onset of reperfusion. Cellular internalization of the peptide was measured using a fluorescent peptide both in primary cultures of cardiomyocytes or in left ventricles after surgery. Anti-apoptotic properties of the synthetic peptide were demonstrated in primary cardiomyocytes. In vivo, one bolus of Tat-DAXXp (1 mg/kg), injected intravenously 5 min before reperfusion in a murine myocardial ischemia-reperfusion model decreased infarct size by 48% after 24 hours of reperfusion. After a 30-min delayed administration, Tat-DAXXp was still able to protect against reperfusion-induced apoptosis and was completely degraded and eliminated within 24 hours thereby reducing risks of potential side effects. Importantly, post-infarction mortality was reduced by 67% by Tat-DAXXp treatment. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting ischemia-reperfusion injury.

Microenvironment-Induced In Situ Self-Assembly of Polymer-Peptide Conjugates That Attack Solid Tumors Deeply.

In cancer treatment, the unsatisfactory solid-tumor penetration of nanomaterials limits their therapeutic efficacy. We employed an in vivo self-assembly strategy and designed polymer-peptide conjugates (PPCs) that underwent an acid-induced hydrophobicity increase with a narrow pH-response range (from 7.4 to 6.5). In situ self-assembly in the tumor microenvironment at appropriate molecular concentrations (around the IC50 values of PPCs) enabled drug delivery deeper into the tumor. A cytotoxic peptide KLAK, decorated with the pH-sensitive moiety cis-aconitic anhydride (CAA), and a cell-penetrating peptide TAT were conjugated onto poly(β-thioester) backbones to produce PT-K-CAA, which can penetrate deeply into solid tumors owing to its small size as a single chain. During penetration in vivo, CAA responds to the weak acid, leading to the self-assembly of PPCs and the recovery of therapeutic activity. Therefore, a deep-penetration ability for enhanced cancer therapy is provided by this in vivo assembly strategy.

Enhanced Cell Killing by Paclitaxel-Loaded Recombinant Protein Micelles Bearing Integrin-Binding and Cell-Penetrating Peptides.

Peptide ligands are effective and specific vectors that can target cell surface receptors, and have shown great potential for targeting drug delivery vehicles. Often, materials used as drug delivery matrices are chemically synthesized and difficult to functionalize, which compromises their development as smart drug carriers. Here, we assemble carriers from a recombinant protein as a novel approach to overcome these limitations. We have previously shown that oleosin, a natural surfactant protein, can be engineered to self-assemble into spherical micelles, and that functionalizing oleosin with RGDS can increase cellular uptake in integrin bearing cells. Here, we investigated whether we could further enhance cellular by incorporating either a RGDS synergy peptide PHSRN or a cell-penetrating Tat peptide derived from human immunodeficiency virus (HIV). The resulting modified oleosins self-assemble into spherical micelles in aqueous environments. Spherical micelles made from oleosin can effectively encapsulate the hydrophobic chemotherapeutic drug paclitaxel (PX). After 15 hours, 350 nM PX loaded oleosin micelles equipped with both RGDS and Tat increased cell killing by twofold compared to free paclitaxel, and 1.2-fold compared to micelles made from RGD-oleosin alone. Micelles equipped with PHSRN alone does not facilitate cell killing compared to free paclitaxel, whereas micelles equipped with both PHSRN and RGDS increased cell killing by 1.1 fold compared to micelles with RGDS alone in 15 hours. Therefore, incorporating multiple motifs into oleosin is an approach for candidate for making a versatile drug delivery carrier.

A novel Granzyme B nanoparticle delivery system simulates immune cell functions for suppression of solid tumors.

Cell-based immunotherapy for the treatment of hematologic malignancies, such as leukemia and lymphoma, has seen much success and played an increasingly important role in clinical studies. Nevertheless, the efficacy of immunotherapy in solid tumors still needs improvements due to the immunosuppressive properties of tumor cells and the microenvironment. To overcome these limitations, we prepared a novel tumor-targeting delivery system based on the underlying mechanism of immune-targeted cell death that encapsulated granzyme B protein within a porous polymeric nanocapsule.Methods: A cell-penetrating peptide TAT was attached onto granzyme B (GrB) to enhance its transmembrane transport efficiency and potency to induce cell apoptosis. The endocytosis and internalization pathways of GrB-TAT (GrB-T) were analyzed in comparison with perforin by confocal microscopy and flow cytometry. Furthermore, the positively charged GrB-T was wrapped into nanoparticles by p-2-methacryloyloxy ethyl phosphorylcholine (PMPC)-modified HA (hyaluronic acid). The nanoparticles (called TCiGNPs) were characterized in terms of zeta potential and by transmission electron microscopy (TEM). The in vitro anti-tumor effects of GrB-T were examined by cell apoptosis assay and Western blotting analysis. The in vivo anti-tumor therapeutic efficacy of TCiGNPs was evaluated in a mouse tumor model.Results: The TAT peptide could play a role similar to perforin to mediate direct transmembrane transfer of GrB and improve GrB-induced cell apoptosis. The TCiGNPs were successfully synthesized and accumulated in the solid tumor through enhanced permeability and retention (EPR) effect. In the tumor microenvironment, TCiGNPs could be degraded by hyaluronidase and triggered the release of GrB-T. The TAT peptide enabled the translocation of GrB across the plasma membrane to induce tumor cell apoptosis in vivo.Conclusion: We successfully developed a granzyme B delivery system with a GrB-T core and a PMPC/HA shell that simulated CTL/NK cell-mediated cancer immunotherapy mechanism. The GrB delivery system holds great promise for cancer treatment analogous to the CTL/NK cell-induced immunotherapy.

Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells

Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.

Retinoprotective Effects of TAT-Bound Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase Activating Polypeptide

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.

MiR-122 and hepatocellular carcinoma: from molecular biology to therapeutics.

miR-122 and hepatocellular carcinoma: from molecular biology to therapeutics.

Liposomal codelivery of an SN38 prodrug and a survivin siRNA for tumor therapy.

PurposeA liposome-based siRNA–drug combination was evaluated as a potential therapeutic strategy to improve the curative effect.MethodsA topoisomerase inhibitor SN38 prodrug was combined with a survivin siRNA through codelivery using transferrin (Tf)-L-SN38/P/siRNA. In this combination, SN38 was conjugated to the cell penetrating peptide TAT through a polyethylene glycol (PEG) linker to synthesize TAT-PEG-SN38. The amphiphilic TAT-PEG-SN38 was used as an ingredient of liposomes to improve the cellular uptake. Protamine was added to form an electrostatic complex with siRNA in the core of the liposomes. Tf was introduced to enable tumor cell targeting of liposomes (Tf-L-SN38/P/siRNA).ResultsTf-L-SN38/P/siRNA exhibited a particle size of 148 nm and a ζ-potential of +7.8 mV. The cellular uptake and antitumor activity were dependent on Tf receptor targeting, TAT-PEG-SN38, and siRNA codelivery. Tf-L-SN38/P/siRNA was shown to be considerably more effective than liposomes carrying individual components. This combination induced potent tumor inhibition (76.8%) in HeLa cell xenograft tumor-bearing nude mice.ConclusionThese data indicated that Tf-L-SN38/P/siRNA was an effective system for codelivery of SN38 and a survivin siRNA and that its therapeutic potential deserved further evaluation.