High yield expression and purification of full-length Neurotensin with pyroglutamate modification.

Neurotensin (NT) is a 13-residue endogenous peptide found in mammals, with neurotransmission and hormonal roles in the central nervous system and gastrointestinal tract, respectively. The first residue of NT is a pyroglutamate (pGlu) that makes the expression and purification of large amounts of NT with native modification challenging. Here, we describe a simple and efficient procedure for expression and purification of large amounts of NT based on using the small ubiquitin-like modifier (SUMO) as a fusion partner and subsequent enzymatic conversion of the N-terminal glutamine to pGlu. Yields of 13 mg/L and 8 mg/L of pure peptide were obtained from expression in rich and minimal media, respectively. The method is adaptable to expression and purification of proteins and peptides with pGlu modification in a wide range of eukaryotic and prokaryotic expression hosts.

Messenger RNA expression and localization of xenin in the gastrointestinal tract in sheep.

The present study aimed to determine the primary sequence of ovine xenin and clarify the mRNA expression and peptide localization of xenin in the gastrointestinal tract in sheep. The colocalization of xenin and glucose-dependent insulinotropic polypeptide was also compared in the antrum and duodenum. Analysis of the nucleotide sequence of ovine xenin revealed a high degree (97.9%) of sequence homology of the sequence between sheep and cattle, and the amino acids sequence determined for ovine xenin coincided (100%) with that of other mammalian species. Real-time quantitative PCR for ovine xenin did not show regional difference in the mRNA expression ratio of xenin. In contrast to the real-time quantitative PCR results, anti-xenin positive cells were abundantly localized in the abomasal antrum (P < 0.01) and at a lesser amount in the duodenum, but no antixenin positive cells were observed in the other regions. Anti-xenin single-positive cells were in a majority in the abomasal antrum, whereas anti-xenin single-positive cells, and anti-GIP single-positive cells, and double-positive cells were even colocalized in the duodenum. These results suggest that abomasal antrum is a major source of xenin in the ovine gastrointestinal tract.

Optimizing the Profile of [99mTc]Tc-NT(7-13) Tracers in Pancreatic Cancer Models by Means of Protease Inhibitors.

BACKGROUND: The overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers specifically to cancer sites for theranostic purposes. We have recently shown that [99mTc]Tc-DT1 ([99mTc]Tc-[N4-Gly7]NT(7-13)) and [99mTc]Tc-DT5 ([99mTc]Tc-[N4-ßAla7,Dab9]NT(7-13)) show notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with untreated controls. Aiming toward translation of this promising approach in NTS1R-positive pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered NEP/ACE inhibitors on the performance of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 in pancreatic cancer models. METHODS: The cellular uptake of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 was tested in a panel of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto, lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency (SCID) mice bearing pancreatic AsPC-1 xenografts. RESULTS: The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing [99mTc]Tc-DT1 in mice, resulting in notably enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated controls at 4 h post-injection; p < 0.0001). CONCLUSIONS: This study has shown the feasibility of optimizing the uptake of [99mTc]Tc-DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.

Neurotensins and their therapeutic potential: research field study.

The natural tridecapeptide neurotensin has been emerged as a promising therapeutic scaffold for the treatment of neurological diseases and cancer. In this work, we aimed to identify the top 100 most cited original research papers as well as recent key studies related to neurotensins. The Web of Science Core Collection database was searched and the retrieved research articles were analyzed by using the VOSviewer software. The most cited original articles were published between 1973 and 2013. The top-cited article was by Carraway and Leeman reporting the discovery of neurotensin in 1973. The highly cited terms were associated with hypotension and angiotensin-converting-enzyme. The conducted analysis reveals the therapeutic potentials of neurotensin, and further impactful research toward its clinical development is warrantied.

Preclinical Evaluation of NHS-Activated Gold Nanoparticles Functionalized with Bombesin or Neurotensin-Like Peptides for Targeting Colon and Prostate Tumours.

Recent advances and large-scale use of hybrid imaging modalities like PET-CT have led to the necessity of improving nano-drug carriers that can facilitate both functional and metabolic screening in nuclear medicine applications. In this study, we focused on the evaluation of four potential imaging nanoparticle structures labelled with the 68Ga positron emitter. For this purpose, we functionalized NHS-activated PEG-gold nanoparticles with 68Ga-DOTA-Neuromedin B, 68Ga-DOTA-PEG(4)-BBN(7-14), 68Ga-DOTA-NT and 68Ga-DOTA-Neuromedin N. In vitro binding kinetics and specific binding to human HT-29 colon carcinoma cells and DU-145 prostate carcinoma cells respectively were assessed, over 75% retention being obtained in the case of 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP in prostate tumour cells and over 50% in colon carcinoma cells. Biodistribution in NU/J mice highlighted a three-fold uptake increase in tumours at 30 min post-injection of 68Ga-DOTA-NT-AuNP and 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP compared to 68Ga-DOTA-NT and 68Ga-DOTA-PEG(4)-BBN(7-14) respectively, therewith fast distribution in prostate and colon tumours and minimum accumulation in non-targeted tissues.

A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules.

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

The Influence of Different Spacers on Biological Profile of Peptide Radiopharmaceuticals for Diagnosis and Therapy of Human Cancers.

BACKGROUND: Cancer is the leading cause of death worldwide. Early detection can reduce the disadvantageous effects of diseases and the mortality in cancer. Nuclear medicine is a powerful tool that has the ability to diagnose malignancy without harming normal tissues. In recent years, radiolabeled peptides have been investigated as potent agents for cancer detection. Therefore, it is necessary to modify radiopeptides in order to achieve more effective agents. OBJECTIVE: This review describes modifications in the structure of radioconjugates with spacers who have improved the specificity and sensitivity of the peptides that are used in oncologic diagnosis and therapy. METHODS: To improve the biological activity, researchers have conjugated these peptide analogs to different spacers and bifunctional chelators. Many spacers of different kinds, such as hydrocarbon chain, amino acid sequence, and poly (ethyleneglycol) were introduced in order to modify the pharmacokinetic properties of these biomolecules. RESULTS: Different spacers have been applied to develop radiolabeled peptides as potential tracers in nuclear medicine. Spacers with different charge and hydrophilicity affect the characteristics of peptide conjugate. For example, the complex with uncharged and hydrophobic spacers leads to increased liver uptake, while the composition with positively charged spacers results in high kidney retention. Therefore, the pharmacokinetics of radio complexes correlates to the structure and total charge of the conjugates. CONCLUSION: Radio imaging technology has been successfully applied to detect a tumor in the earliest stage. For this purpose, the assessment of useful agents to diagnose the lesion is necessary. Developing peptide radiopharmaceuticals using spacers can improve in vitro and in vivo behavior of radiotracers leading to better noninvasive detection and monitoring of tumor growth.

Impact of polyphenols on receptor-ligand interactions by NMR: the case of neurotensin (NT)-neurotensin receptor fragment (NTS1) complex.

Ligand-receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand-receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand-receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT-NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.Communicated by Ramaswamy H. Sarma.

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples.

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

Gene-Embedded Nanostructural Biotic-Abiotic Optoelectrode Arrays Applied for Synchronous Brain Optogenetics and Neural Signal Recording.

Optogenetics is a recently established neuromodulation technique in which photostimulation is used to manipulate neurons with high temporal and spatial precision. However, sequential genetic and optical insertion with double brain implantation tends to cause excessive tissue damage. In addition, the incorporation of light-sensitive genes requires the utilization of viral vectors, which remains a safety concern. Here, by combining device fabrication design, nanotechnology, and cell targeting technology, we developed a new gene-embedded optoelectrode array for neural implantation to enable spatiotemporal electroporation (EP) for gene delivery/transfection, photomodulation, and synchronous electrical monitoring of neural signals in the brain via one-time implantation. A biotic-abiotic neural interface (called PG) composed of reduced graphene oxide and conductive polyelectrolyte 3,4-ethylenedioxythiophene-modified amphiphilic chitosan was developed to form a nanostructural hydrogel with assembled nanodomains for encapsulating nonviral gene vectors (called PEI-NT-pDNA) formulated by neurotensin (NT) and polyethylenimine (PEI)-coupled plasmid DNA (pDNA). The PG can maintain high charge storage ability to respond to a minimal current of 125 µA for controllable gene delivery. The in vitro analysis of PG-PEI-NT-pDNA on the microelectrode array chip showed that the microelectrodes provided electrically inductive electropermeabilization, which permitted gene transfection into localized rat adrenal pheochromocytoma cells with a strong green fluorescent protein expression that was up to 8-fold higher than that in nontreated cells. Furthermore, the in vivo implantation enabled on-demand spatiotemporal gene transfection to neurons with 10-fold enhancement of targeting ability compared with astrocytes. Finally, using the real optogenetic opsin channelrhodopsin-2, the flexible neural probe incorporated with an optical waveguide fiber displayed photoevoked extracellular spikes in the thalamic ventrobasal region after focal EP for only 7 days, which provided a proof of concept for the use of photomodulation to facilitate neural therapies.

Dimeric Prodrug Self-Delivery Nanoparticles with Enhanced Drug Loading and Bioreduction Responsiveness for Targeted Cancer Therapy.

Efficient drug accumulation in tumor cells is essential for cancer therapy. Herein, we developed dimeric prodrug self-delivery nanoparticles (NPs) with enhanced drug loading and bioreduction responsiveness for triple negative breast cancer (TNBC) therapy. Specially designed camptothecin dimeric prodrug (CPTD) containing a disulfide bond was constructed to realize intracellular redox potential controlled drug release. Direct conjugation of hydrophobic CPTD to poly(ethylene glycol) PEG5000, a prodrug-based amphiphilic CPTD-PEG5000 co-polymer was synthesized, which could encapsulate parental CPTD prodrug spontaneously and form ultrastable NPs due to the highly analogous structure. Such dimeric prodrug self-delivery nanoparticles showed ultrahigh stability with critical micelle concentration as low as 0.75 µg/mL and remained intact during endocytosis. In addition, neurotensin (NT), a 13 amino acid ligand, was further modified on the nanoparticles for triple negative breast cancer (TNBC) targeting. Optimized NT-CPTD NPs showed improved pharmacokinetics profile and increased drug accumulation in TNBC lesions than free CPT, which largely reduced the systemic toxicity and presented an improved anticancer efficacy in vivo. In summary, with advantages of extremely high drug loading capacity, tumor microenvironmental redox responsiveness, and targeted TNBC accumulation, NT-CPTD NPs showed their potential for effective triple negative breast cancer therapy.

Evidence of 68Ga-DOTA-NT-20.3 Uptake in Pancreatic Adenocarcinoma AsPC-1 Cell Line - in vitro Study.

BACKGROUND: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. OBJECTIVE: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. METHOD: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 µg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. RESULTS: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. CONCLUSION: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.

Development of [18F]AlF-NOTA-NT as PET Agents of Neurotensin Receptor-1 Positive Pancreatic Cancer.

Several studies have suggested that neurotensin receptors (NTRs) and neurotensin (NT) greatly affect the growth and survival of pancreatic ductal adenocarcinoma (PDAC). Developing NTR-targeted PET probes could therefore be important for the management of a pancreatic cancer patient by providing key information on the NTR expression profile noninvasively. Despite the initial success on the synthesis of 18F-labeled NT PET probes, the labeling procedure generally requires lengthy steps including azeotropic drying of 18F. Using a straightforward chelation method, here we report the simple preparation of aluminum-18F-NOTA-NT starting from aqueous 18F. The cell binding test demonstrated that [19F]AlF-NOTA-NT maintained high receptor-binding affinity to NTR1. This probe was then further evaluated in NTR1 positive pancreatic tumor models (AsPC-1 and PANC-1). After the administration of [18F]AlF-NOTA-NT, small animal PET studies showed a high contrast between tumor and background in both models at 1 and 4 h time points. A blocking experiment was performed to demonstrate the receptor specificity: the tumor uptake in AsPC1 without and with blocking agent was 1.0 ± 0.2 and 0.1 ± 0.0%ID/g, respectively, at 4 h post injection. In summary, a NTR specific PET agent, [18F]AlF-NOTA-NT, was prepared through the simple chelation method. This NTR-targeted PET probe may not only be used to detect NTR1 positive pancreatic tumors (diagnosis), but also it may be fully integrated to NTR target therapy leading to personalized medicine (theranostic).

Iontophoresis-stimulated silk fibroin films as a peptide delivery system for wound healing.

Silk fibroin (SF) films containing a peptide, neurotensin (NT), stimulated by iontophoresis were developed aiming to modulate the inflammatory process and prevent the growth of microorganisms typical of wounds. NT-loaded SF films composition shows predominance of ß-sheet structures that conferred adequate mechanical properties, transparency, moderate roughness and low swelling index to fibroin films. Infrared spectroscopy and thermal analysis suggested the presence of non-covalent interactions between NT and fibroin. Using the MALDI imaging technique, it was possible to visualize the homogeneous NT distribution throughout the film surface, in addition to its prolonged release for up to 72 h. In vitro studies in E. coli liposaccharide-stimulated macrophages showed a significant reduction of interleukins production after NT-loaded film application, whereas NT solution did not reduce them. Bi-laminated NT-loaded fibroin films containing silver electrodes provided a burst release of NT when anodic iontophoresis was applied, enabling a rapid onset of drug action. In addition, anodic iontophoresis presented a bacteriostatic effect against gram-positive microorganisms. Different iontophoresis densities, from 0.2 to 0.6 mA/cm2, did not significantly reduce fibroblast viability after 30 min of application. In conclusion, iontophoretic-stimulated peptide-loaded fibroin films could be a promising platform for the treatment of wounds.

A novel GLP-1/xenin hybrid peptide improves glucose homeostasis, circulating lipids and restores GIP sensitivity in high fat fed mice.

Combined modulation of peptide hormone receptors including, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and xenin, have established benefits for the treatment of diabetes. The present study has assessed the biological actions and therapeutic efficacy of a novel exendin-4/xenin-8-Gln hybrid peptide, both alone and in combination with the GIP receptor agonist (DAla2)GIP. Exendin-4/xenin-8-Gln was enzymatically stable and exhibited enhanced insulin secretory actions when compared to its parent peptides. Exendin-4/xenin-8-Gln also possessed ability to potentiate the in vitro actions of GIP. Acute administration of exendin-4/xenin-8-Gln in mice induced appetite suppressive effects, as well as significant and protracted glucose-lowering and insulin secretory actions. Twice daily administration of exendin-4/xenin-8-Gln, alone or in combination with (DAla2)GIP, for 21-days significantly reduced non-fasting glucose and increased circulating insulin levels in high fat fed mice. In addition, all exendin-4/xenin-8-Gln treated mice displayed improved glucose tolerance, insulin sensitivity and metabolic responses to GIP. Combination therapy with (DAla2)GIP did not result in any obvious further benefits. Metabolic improvements in all treatment groups were accompanied by reduced pancreatic beta-cell area and insulin content, suggesting reduced insulin demand. Interestingly, body weight, food intake, circulating glucagon, metabolic rate and amylase activity were unaltered by the treatment regimens. However, all treatment groups, barring (DAla2)GIP alone, exhibited marked reductions in total- and LDL-cholesterol. Furthermore, exendin-4 therapy also reduced circulating triacylglycerol. This study highlights the positive antidiabetic effects of exendin-4/xenin-8-Gln, and suggests that combined modulation of GLP-1 and xenin related signalling pathways represents an exciting treatment option for type 2 diabetes.

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin.

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLNE475Q in complex with the products of neurotensin cleavage at 2.7Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-ß peptide, Aß1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aß35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

Regio-Selective Intramolecular Hydrogen/Deuterium Exchange in Gas-Phase Electron Transfer Dissociation.

Protein backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) typically utilizes enzymatic digestion after the exchange reaction and before MS analysis to improve data resolution. Gas-phase fragmentation of a peptic fragment prior to MS analysis is a promising technique to further increase the resolution. The biggest technical challenge for this method is elimination of intramolecular hydrogen/deuterium exchange (scrambling) in the gas phase. The scrambling obscures the location of deuterium. Jørgensen's group pioneered a method to minimize the scrambling in gas-phase electron capture/transfer dissociation. Despite active investigation, the mechanism of hydrogen scrambling is not well-understood. The difficulty stems from the fact that the degree of hydrogen scrambling depends on instruments, various parameters of mass analysis, and peptide analyzed. In most hydrogen scrambling investigations, the hydrogen scrambling is measured by the percentage of scrambling in a whole molecule. This paper demonstrates that the degree of intramolecular hydrogen/deuterium exchange depends on the nature of exchangeable hydrogen sites. The deuterium on Tyr amide of neurotensin (9-13), Arg-Pro-Tyr-Ile-Leu, migrated significantly faster than that on Ile or Leu amides, indicating the loss of deuterium from the original sites is not mere randomization of hydrogen and deuterium but more site-specific phenomena. This more precise approach may help understand the mechanism of intramolecular hydrogen exchange and provide higher confidence for the parameter optimization to eliminate intramolecular hydrogen/deuterium exchange during gas-phase fragmentation. Graphical Abstract ᅟ.

Regulation of human GDNF gene expression in nigral dopaminergic neurons using a new doxycycline-regulated NTS-polyplex nanoparticle system.

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease.

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine.

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na+, K+-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [3H]-ouabain binding (to analyze K+ site of Na+, K+-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na+, K+-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [3H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2mg/kg) and clozapine (3, 10 and 30mg/kg). Animals were sacrificed 18h later, cerebral cortices harvested, membrane fractions prepared and high affinity [3H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10mg/kg clozapine. Rats were administered with neurotensin (3, 10y 30µg, i.c.v.) and 60min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [3H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30µg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [3H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2mg/kg, i.p.) or clozapine (10mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na+, K+-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na+, K+-ATPase at central synapses.

Neurotensin-Conjugated Reduced Graphene Oxide with Multi-Stage Near-Infrared-Triggered Synergic Targeted Neuron Gene Transfection In Vitro and In Vivo for Neurodegenerative Disease Therapy.

Delivery efficiency with gene transfection is a pivotal point in achieving maximized therapeutic efficacy and has been an important challenge with central nervous system (CNS) diseases. In this study, neurotensin (NT, a neuro-specific peptide)-conjugated polyethylenimine (PEI)-modified reduced graphene oxide (rGO) nanoparticles with precisely controlled two-stage near-infrared (NIR)-laser photothermal treatment to enhance the ability to target neurons and achieve high gene transfection in neurons. First-stage NIR laser irradiation on the cells with nanoparticles attached on the surface can increase the permeability of the cell membrane, resulting in an apparent increase in cellular uptake compared to untreated cells. In addition, second-stage NIR laser irradiation on the cells with nanoparticles inside can further induce endo/lysosomal cavitation, which not only helps nanoparticles escape from endo/lysosomes but also prevents plasmid DNA (pDNA) from being digested by DNase I. At least double pDNA amount can be released from rGO-PEI-NT/pDNA under NIR laser trigger release compared to natural release. Moreover, in vitro differentiated PC-12 cell and in vivo mice (C57BL/6) brain transfection experiments have demonstrated the highest transfection efficiency occurring when NT modification is combined with external multi-stage stimuli-responsive NIR laser treatment. The combination of neuro-specific targeting peptide and external NIR-laser-triggered aid provides a nanoplatform for gene therapy in CNS diseases.