Human serum albumin fusion protein as therapeutics for targeting amyloid beta in Alzheimer's diseases.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.

Targeted iron oxide nanoparticles for the enhancement of radiation therapy.

To increase the efficacy of radiation, iron oxide nanoparticles can be utilized for their ability to produce reactive oxygen species (ROS). Radiation therapy promotes leakage of electrons from the electron transport chain and leads to an increase in mitochondrial production of the superoxide anion which is converted to hydrogen peroxide by superoxide dismutase. Iron oxide nanoparticles can then catalyze the reaction from hydrogen peroxide to the highly reactive hydroxyl radical. Therefore, the overall aim of this project was to utilize iron oxide nanoparticles conjugated to a cell penetrating peptide, TAT, to escape lysosomal encapsulation after internalization by cancer cells and catalyze hydroxyl radical formation. It was determined that TAT functionalized iron oxide nanoparticles and uncoated iron oxide nanoparticles resulted in permeabilization of the lysosomal membranes. Additionally, mitochondrial integrity was compromised when A549 cells were treated with both TAT-functionalized nanoparticles and radiation. Pre-treatment with TAT-functionalized nanoparticles also significantly increased the ROS generation associated with radiation. A long term viability study showed that TAT-functionalized nanoparticles combined with radiation resulted in a synergistic combination treatment. This is likely due to the TAT-functionalized nanoparticles sensitizing the cells to subsequent radiation therapy, because the nanoparticles alone did not result in significant toxicities.

Intraperitoneal Administration of a Novel TAT-BDNF Peptide Ameliorates Cognitive Impairments via Modulating Multiple Pathways in Two Alzheimer's Rodent Models.

Although Alzheimer's disease (AD) has been reported for more than 100 years, there is still a lack of effective cures for this devastating disorder. Among the various obstacles that hold back drug development, the blood-brain barrier (BBB) is one of them. Here, we constructed a novel fusion peptide by linking the active domain of brain-derived neurotrophic factor (BDNF) with an HIV-encoded transactivator of transcription (TAT) that has a strong membrane-penetrating property. After intraperitoneal injection, the eGFP-TAT could be robustly detected in different brain regions. By using scopolamine-induced rats and APPswe mice representing AD-like cholinergic deficits and amyloidosis, respectively, we found that intraperitoneal administration of the peptide significantly improved spatial memory with activation of the TrkB/ERK1/2/Akt pathway and restoration of several memory-associated proteins in both models. Administration of the peptide also modulated ß-amyloid and tau pathologies in APPswe mice, and it increased the amount of M receptor with modulation of acetylcholinesterase in scopolamine-induced rats. We conclude that intraperitoneal administration of our TAT-BDNF peptide could efficiently target multiple molecular pathways in the brain and improve the cognitive functions in AD-like rodent models.

Attenuating HIV Tat/TAR-mediated protein expression by exploring the side chain length of positively charged residues.

RNA is a drug target involved in diverse cellular functions and viral processes. Molecules that inhibit the HIV TAR RNA-Tat protein interaction may attenuate Tat/TAR-dependent protein expression and potentially serve as anti-HIV therapeutics. By incorporating positively charged residues with mixed side chain lengths, we designed peptides that bind TAR RNA with enhanced intracellular activity. Tat-derived peptides that were individually substituted with positively charged residues with varying side chain lengths were evaluated for TAR RNA binding. Positively charged residues with different side chain lengths were incorporated at each Arg and Lys position in the Tat-derived peptide to enhance TAR RNA binding. The resulting peptides showed enhanced TAR RNA binding affinity, cellular uptake, nuclear localization, proteolytic resistance, and inhibition of intracellular Tat/TAR-dependent protein expression compared to the parent Tat-derived peptide with no cytotoxicity. Apparently, the enhanced inhibition of protein expression by these peptides was not determined by RNA binding affinity, but by proteolytic resistance. Despite the high TAR binding affinity, a higher binding specificity would be necessary for practical purposes. Importantly, altering the positively charged residue side chain length should be a viable strategy to generate potentially useful RNA-targeting bioactive molecules.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Evaluation of the safety and brain-related tissues distribution characteristics of TAT-HaFGF via intranasal administration.

Disabilities triggered by neurodegeneration mainly result in mortality in the elderly, and patients with neurodegenerative disease also display deficits in olfactory function. Therefore drug distribution to the brain through intranasal administration has become one of the most difficult challenges in the treatment of central nervous system (CNS) diseases. TAT-human acidic fibroblast growth factor (HaFGF) is a new fused protein retaining the neuroprotective activities of HaFGF, and is a promising prospect in the treatment of neurodegenerative diseases. TAT (a cell-penetrating peptide) contains a high relative abundance of positively charged amino acids such as lysine and arginine, which have a powerful attraction to the negatively charge on the nasal epithelial membrane. The present study focused on the evaluation of the safety and absorption characteristics of TAT-HaFGF following intranasal administration. After TAT-HaFGF intranasal administration (100, 300, 600 µg/kg) for 5 weeks, hematoxylin-eosin (HE) staining showed no pathology in any of the investigated tissues and organs. The expression of olfactory marker protein (OMP) was observed with immunohistochemical staining, which showed no altered expression in the sensory neurons of the nasal epithelium. Nasal ciliotoxicity studies carried out using an in situ palate model and optical microscope showed that TAT-HaFGF had no nasal ciliotoxicity. The distribution of the TAT-HaFGF following intranasal administration was assessed using a radioisotopic tracing method. Radioactivity was observed in the brain after 15 min. This became stronger at 30 min and weaker at 1 h. All of the results confirmed the in vivo safety of TAT-HaFGF via intranasal administration.

TAT-LHRH conjugated low molecular weight chitosan as a gene carrier specific for hepatocellular carcinoma cells.

To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC). TLC/DNA nanoparticles (TLCDNPs) were characterized by agarose gel retardation, atomic force microscopy, and dynamic light scattering analysis. In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System. The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution. The in vitro study demonstrated TLC was highly selective for hepatoma cells and essentially nontoxic.

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model.

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.

Transdermal absorption and stability enhancement of salmon calcitonin by Tat peptide.

CONTEXT: Highly organized structure of stratum corneum (SC) is the major barrier of the delivery of macromolecules such as proteins and peptides across the skin. Recently, cell penetrating peptides (CPPs) such as HIV1-trans-activating transcriptional (Tat) have been used to enhance the topical delivery of proteins and peptides. OBJECTIVE: This study aimed to enhance the transdermal absorption and chemical stability of salmon calcitonin (sCT) by co-incubation with Tat. MATERIALS AND METHODS: Tat-sCT mixture at 1:1 molar ratio was prepared. Transdermal absorption and chemical stability of the mixture was evaluated in comparing with free sCT. RESULTS: Tat-sCT mixture gave higher cumulative amounts and fluxes of sCT than free sCT. The maximum percentage of sCT of 58.36 ± 12.33% permeated into the receiving chamber was found in Tat-sCT mixture at 6 h which was 3.50 folds of free sCT. Tat-sCT mixture demonstrated better sCT stability than sCT solution after 1 month storage at 4°C, 25°C and 45°C. DISCUSSION: The positively-charged arginine groups in Tat might be responsible for the binding of peptide complexes to negatively charged cell surfaces by electrostatic interactions and also the translocation of sCT through the excised skin. CONCLUSION: This study demonstrated the enhancements of transdermal absorption and stability of sCT by Tat peptide with potential for further application in transdermal delivery of other therapeutic peptides.

Synthesis of TAT peptide-tagged PEGylated chitosan nanoparticles for siRNA delivery targeting neurodegenerative diseases.

Delivery of therapeutic molecules to the brain for the treatment of Neurodegenerative diseases (ND) is a challenging task. This manuscript introduces a novel scheme of synthesizing peptide-tagged polyethylene glycol (PEG)ylated chitosan polymer to develop nanoparticles for siRNA delivery for use in ND. Specifically, this manuscript proposes a facile chemoselective conjugation of monomethoxy PEG, at the C2 hydroxyl group of chitosan polymer, with conjugation of PEG to a cell-penetrating peptide, Trans-Activator of Transcription. The synthesized Chitosan-PEG-TAT polymer was used to form the nanoparticles of approximately 5 nm, complexing siRNA to be delivered in neuronal cells (Neuro 2a), with no/minimal toxicity. The various intermediates and the final product formed during the synthesis were characterized using (1)H Nuclear Magnetic Resonance and Fourier Transform Infrared Spectroscopy spectra. The morphological details of the nanoparticles were studied using Transmission Electron Microscopy. The nanoparticles were tested to deliver a functional siRNA against the Ataxin-1 gene in an in-vitro established model of a ND Spinocerebellar ataxia (SCA1) over-expressing ataxin protein. The results indicate successful suppression of the SCA1 protein following 48 h of transfection. Result of this study has potential in ND like SCA, Parkinson's, Alzheimer's and others.

The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.

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

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

Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain glioma in animals.

The treatment of central nervous system diseases such as brain glioma is a major challenge due to the presence of the blood-brain barrier (BBB). A cell-penetrating peptide TAT (AYGRKKRRQRRR), which appears to enter cells with alacrity, was employed to enhance the delivery efficiency of normal drug formulation to the brain. Targeting liposomal formulations often apply modified phospholipids as anchors. However, cholesterol, another liposomal component more stable and cheaper, has not been fully investigated as an alternative anchor. In our study, TAT was covalently conjugated with cholesterol for preparing doxorubicin-loaded liposome for brain glioma therapy. Cellular uptake by brain capillary endothelial cells (BCECs) and C6 glioma cells was explored. The anti-proliferative activity against C6s confirmed strong inhibitory effect of the liposomes modified with doxorubicin-loaded TAT. The bio-distribution findings in brains and hearts were evident of higher efficiency of brain delivery and lower cardiotoxic risk. The results on survival of the brain glioma-bearing animals indicate that survival time of the glioma-bearing rats treated with TAT-modified liposome was much longer than in the other groups. In conclusion, the potency of the TAT-modified liposome to enter the BBB appears to be related with the TAT on the liposome's surface. The TAT-modified liposome may improve the therapeutic efficacy on brain glioma in vitro and in vivo.

[Potential of cell penetrating peptides for cell drug delivery]. / Administration de molécules actives dans les cellules: le potentiel des peptides de pénétration cellulaire.

The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes… This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions.

Imaging DNA damage in vivo using gammaH2AX-targeted immunoconjugates.

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.

[Study of TAT-PEG loaded magnetic liposomes crossing blood spinal cord barrier after spinal cord injury in rats].

OBJECTIVE: To evaluate the ability of a kind of novel magnetic liposomes modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) to cross the blood spinal cord barrier (BSCB) so as to demonstrate whether or not they can accumulate at the lesions of injured spinal cord. METHODS: The novel liposomes were made through reverse-phase evaporation method modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) with an iron core. Thirty-six Wistar rats subject to spinal cord injury (SCI) at T10 were randomly divided into three groups (Groups I, II and III). The rats of Group III were injected with TAT-PEG loaded magnetic liposomes (4.55 mg/kg). The rats of GroupII received an injection of the equivalent PEG loaded magnetic liposomes while those of control group (GroupI) the equivalent normal saline. The accumulation of liposomes was observed by MRI (magnetic resonance imaging), Prussian blue staining, electron microscope and flame atomic absorption spectrophotometer. RESULTS: This kind of TAT-PEG loaded magnetic liposomes could cross the BSCB and enter into the cells around the injured tissue. A low signal of T2WI on MRI could also be found in Group III. The results of flame atomic absorption spectrophotometer showed that the iron content accumulated around the lesion site in Group III was obviously higher than the other two groups (P < 0.05). CONCLUSION: The TAT-PEG loaded magnetic liposomes may be employed as one kind of novel drug carrier to cross the BSCB and accumulate at tissue cells of spinal cord. It is likely to become a new therapy for SCI.

Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells.

FOXO proteins are Akt-regulated transcription factors involved in the control of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression. We reported that plasmid-based overexpression of constitutively active FOXO3 in cells from chronic lymphocytic leukemia (CLL) reduced their survival, suggesting that increasing FOXO3 activity in hematologic malignancies may represent a promising therapeutic strategy. The transactivating transcription factor (TAT) protein transduction domain (PTD) derived from the HIV TAT protein was shown to efficiently deliver macromolecular cargo in various cell types. In this study, wild-type FOXO3 and FOXO3 mutated on Akt sites [FOXO3 T32A/S253A/S315A or TM (triple mutant)] were fused to the TAT-PTD. Using biochemical techniques, flow cytometry, and microscopy analysis, we found a rapid and dose-dependent cell penetration into leukemic cells of unlabeled and fluorescein isothiocyanate-labeled TAT-FOXO3 fusion proteins followed by their accumulation within nuclear and cytoplasmic compartments. Treatment with TAT-FOXO3 TM-but not wild-type TAT-FOXO3-proteins induced Jurkat and K562 leukemic cell death and affected cell viability of other hematologic malignancies including primary cells from CLL. Cell transduction with TAT-FOXO3 TM induced apoptotic cell death as shown by morphologic changes, Annexin V/7-AAD (7-amino-actinomycin D) staining, activation of effector caspases, and PARP cleavage, caspase blockade through the use of the inhibitor Z-VAD, and expression of Bim and p27(KIP1). By contrast, TAT-FOXO3 TM blocked cell proliferation of primary T cells, without affecting their viability. Together, our data show that cell penetrating TAT-FOXO3 TM fusion proteins constitute novel potential therapeutic agents in the treatment of lymphoproliferative disorders and hematologic malignancies.

Transdermal absorption enhancement of N-terminal Tat-GFP fusion protein (TG) loaded in novel low-toxic elastic anionic niosomes.

Elastic anionic niosomes (Tween 61/cholesterol/dicetyl phosphate at 1:1:0.05 molar ratio of 20 mM) with various concentrations of ethanol and edge activators sodium cholate (NaC) and sodium deoxycholate (NaDC) showed larger vesicular size (171.94 ± 63.52 - 683.17 ± 331.47 nm) and higher negative zeta potential (-6.45 ± 2.76 to - 17.40 ± 2.51 mV) than the nonelastic anionic niosomes. The elasticity (deformability index) and entrapment efficiency of all elastic vesicles except the NaDC vesicles were higher than the nonelastic vesicles. The morphology, under transmission electron microscope, of elastic and nonelastic niosomes loaded and not loaded with Tat-green fluorescent protein fusion protein (TG) were in large unilamellar structure. TG loaded in elastic (1 mol% NaC) anionic niosomes gave the highest cell viability both in HT-29 (92.32 ± 3.82%) and KB cells (96.62 ± 5.96%), the highest cumulative amounts (62.75 ± 2.68 µg/cm(2) ) and fluxes (10.46 ± 3.45 µg/cm(2) h) in receiving chamber in rat skin transdermal study by Franz diffusion cells. This study has not only indicated the synergistic enhancement effects of the Tat peptide and the niosomal delivery system on the cellular uptake and transdermal absorption of TG but also 1 mol% NaC as an edge activator to obtain a novel low-toxic elastic anionic niosomes for topical use of therapeutic macromolecules such as proteins, as well.

TAT is not capable of transcellular delivery across an intact endothelial monolayer in vitro.

Developing delivery vehicles capable of penetrating cell barriers is critical for drug delivery to the brain due to the presence of the blood-brain barrier (BBB). Cell-penetrating peptides (CPPs) are one potential solution since they can enter cells; however, it is unclear whether CPPs can pass through cell barriers. In this study, the ability of the TAT CPP to cross an endothelial barrier without disrupting the integrity of its tight junctions was investigated. Endothelial cell monolayers (bEnd.3) were exposed to the TAT peptide, and cell integrity was quantified by zona occludens-1 immunofluorescence, trans-endothelial electrical resistance, and hydraulic conductivity. None of these parameters were significantly altered following exposure to TAT. To evaluate the passage of TAT through the monolayer, the permeability of a green fluorescent protein (GFP)-TAT fusion protein was not significantly different from the permeability of GFP or fluorescent dextrans of similar sizes. Furthermore, GFP-TAT was unable to significantly transduce astrocytes on the opposite side of the bEnd.3 monolayer. We conclude, therefore, that although TAT may not be an efficient delivery vehicle for trans-BBB delivery, our TAT construct may have utility in delivering therapeutic cargos to endothelial cells or to the brain parenchyma after BBB disruption.

[Pharmacokinetics of a fusion protein for human acidic fibroblast growth factor and transcriptional activator protein in rat and its penetration across blood-brain barrier].

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.