Brain-derived neurotrophic factor delivered to the brain using poly (lactide-co-glycolide) nanoparticles improves neurological and cognitive outcome in mice with traumatic brain injury

Currently, traumatic brain injury (TBI) is the leading cause of death or disabilities in young individuals worldwide. The multi-complexity of its pathogenesis as well as impermeability of the blood–brain barrier (BBB) makes the drug choice and delivery very challenging. The brain-derived neurotrophic factor (BDNF) regulates neuronal plasticity, neuronal cell growth, proliferation, cell survival and long-term memory. However, its short half-life and low BBB permeability are the main hurdles to be an effective therapeutic for TBI. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles coated by surfactant can enable the delivery of a variety of molecules across the BBB by receptor-mediated transcytosis. This study examines the ability of PLGA nanoparticles coated with poloxamer 188 (PX) to deliver BDNF into the brain and neuroprotective effects of BNDF in mice with TBI. C57bl/6 mice were subjected to weight-drop closed head injuries under anesthesia. Using enzyme-linked immunosorbent assay, we demonstrated that the intravenous (IV) injection of nanoparticle-bound BDNF coated by PX (NP-BDNF-PX) significantly increased BDNF levels in the brain of sham-operated mice (p?<?0.001) and in both ipsi- (p?<?0.001) and contralateral (p?<?0.001) parts of brain in TBI mice compared to controls. This study also showed using the passive avoidance (PA) test, that IV injection of NP-BDNF-PX 3?h post-injury prolonged the latent time in mice with TBI thereby reversing cognitive deficits caused by brain trauma. Finally, neurological severity score test demonstrated that our compound efficiently reduced the scores at day 7 after the injury indicating the improvement of neurological deficit in animals with TBI. This study shows that PLGA nanoparticles coated with PX effectively delivered BDNF into the brain, and improved neurological and cognitive deficits in TBI mice, thereby providing a neuroprotective effect.     

Permeating the blood brain barrier and abrogating the inflammation in stroke: implications for stroke therapy

Cell therapy has been shown as a potential treatment for stroke and other neurological disorders. Human umbilical cord blood (HUCB) may be a promising source of stem cells for cell therapy. The most desired outcomes occur when stem cells cross the blood brain barrier (BBB) and eventually reach the injured brain site. We propose, from our previous studies, that mannitol is capable of disrupting the BBB, allowing the transplanted cells to enter the brain from the periphery. However, when the BBB is compromised, the inflammatory response from circulation may also be able to penetrate the brain and thus may actually exacerbate the stroke rather than afford therapeutic effects. We discuss how an NF-kB decoy can inhibit the inflammatory responses in the stroke brain thereby reducing the negative effects associated with BBB disruption. In this review, we propose the combination of mannitol-induced BBB permeation and NF-kB decoy for enhancing the therapeutic benefits of cell therapy in stroke.     

Blood-brain barrier permeation and efflux exclusion of anticholinergics used in the treatment of overactive bladder

Overactive bladder (OAB) is a common condition, particularly in the elderly. Anticholinergic agents are the mainstay of pharmacological treatment of OAB; however, many anticholinergics can cross the blood-brain barrier (BBB) and may cause central nervous system (CNS) effects, including cognitive deficits, which can be especially detrimental in older patients. Many anticholinergics have the potential to cause adverse CNS effects due to muscarinic (M(1)) receptor binding in the brain. Of note, permeability of the BBB increases with age and can also be affected by trauma, stress, and some diseases and medications. Passive crossing of a molecule across the BBB into the brain is dependent upon its physicochemical properties. Molecular characteristics that hinder passive BBB penetration include a large molecular size, positive or negative ionic charge at physiological pH, and a hydrophilic structure. Active transport across the BBB is dependent upon protein-mediated transporter systems, such as that of permeability-glycoprotein (P-gp), which occurs only for P-gp substrates, such as trospium chloride, darifenacin and fesoterodine. Reliance on active transport can be problematic since genetic polymorphisms of P-gp exist, and many commonly used drugs and even some foods are P-gp inhibitors or are substrates themselves and, due to competition, can reduce the amount of the drug that is actively transported out of the CNS. Therefore, for drugs that are preferred not to cross into the CNS, such as potent anticholinergics intended for the bladder, it is optimal to have minimal passive crossing of the BBB, although it may also be beneficial for the drug to be a substrate for an active efflux transport system. Anticholinergics demonstrate different propensities to cross the BBB. Darifenacin, fesoterodine and trospium chloride are substrates for P-gp and, therefore, are actively transported away from the brain. In addition, trospium chloride has not been detected in cerebrospinal fluid assays and does not appear to have significant CNS penetration. This article reviews the properties of anticholinergics that affect BBB penetration and active transport out of the CNS, discusses issues of increased BBB permeability in patients with OAB, and examines the clinical implications of BBB penetration on adverse events associated with anticholinergics.     

Lipid-based nanocarrier efficiently delivers highly water soluble drug across the blood–brain barrier into brain

Delivering highly water soluble drugs across blood–brain barrier (BBB) is a crucial challenge for the formulation scientists. A successful therapeutic intervention by developing a suitable drug delivery system may revolutionize treatment across BBB. Efforts were given here to unravel the capability of a newly developed fatty acid combination (stearic acid:oleic acid:palmitic acid?=?8.08:4.13:1) (ML) as fundamental component of nanocarrier to deliver highly water soluble zidovudine (AZT) as a model drug into brain across BBB. A comparison was made with an experimentally developed standard phospholipid-based nanocarrier containing AZT. Both the formulations had nanosize spherical unilamellar vesicular structure with highly negative zeta potential along with sustained drug release profiles. Gamma scintigraphic images showed both the radiolabeled formulations successfully crossed BBB, but longer retention in brain was observed for ML-based formulation (MGF) as compared to soya lecithin (SL)-based drug carrier (SYF). Plasma and brain pharmacokinetic data showed less clearance, prolonged residence time, more bioavailability and sustained release of AZT from MGF in rats compared to those data of the rats treated with SYF/AZT suspension. Thus, ML may be utilized to successfully develop drug nanocarrier to deliver drug into brain across BBB, in a sustained manner for a prolong period of time and may provide an effective therapeutic strategy for many diseases of brain. Further, many anti-HIV drugs cannot cross BBB sufficiently. Hence, the developed formulation may be a suitable option to carry those drugs into brain for better therapeutic management of HIV.     

Transport of Human Recombinant Brain-Derived Neurotrophic Factor (BDNF) Through the Rat Blood−Brain Barrier in Vivo Using Vector-Mediated Peptide Drug Delivery

The blood–brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA–OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA–OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA–OX26 conjugate.     

Transport of brain-derived neurotrophic factor across the blood–brain barrier

Brain-derived neurotrophic factor (BDNF) is a potential therapeutic agent for degenerative disorders of the central nervous system. In this report, we investigated the ability of BDNF to cross the blood–brain barrier (BBB). BDNF was stable in blood up to 60 min after i.v. injection, with evidence for aggregation, and had an early, rapid influx into brain. By 10 min, most of the BDNF sequestered by the cerebral cortex was associated with the parenchyma rather than with the endothelial cells, demonstrating complete passage across the BBB. A small dose of unlabeled BDNF enhanced the entry of ¹²⁵I-BDNF from blood to brain after an i.v. bolus injection, whereas larger doses had no effect. In contrast, a large dose of unlabeled BDNF inhibited the influx of ¹²⁵I-BDNF during in situ brain perfusion. After intracerebroventricular injection, the efflux of BDNF from brain to blood occurred at a rate similar to that for reabsorption of cerebrospinal fluid, and no evidence for self-inhibition was found. Therefore, we conclude that intact BDNF in the peripheral circulation crosses the BBB by a high-capacity, saturable transport system.     

脑损伤过程中血脑屏障通透性的变化及其调节机制

血脑屏障是由脑微血管内皮细胞、星形神经胶质细胞、外膜细胞、血管周围巨噬细胞和基底膜组成的一个复杂系统,对维持中枢神经系统的正常功能非常重要。脑损伤如脑缺血、脑缺氧、脑外伤和蛛网膜下腔出血过程中伴随血脑屏障通透性的变化。脑缺血及其再灌注后可通过花生四烯酸代谢途径、嘌呤核苷酸代谢途径及一氧化氮途径产生自由基,可能是血脑屏障通透性增加的重要机制。血脑屏障的破坏可加重脑损伤程度;脑血管疾病时,保护血脑屏障的完整性可能是减轻脑损伤的重要措施。     

Blood-Brain Barrier Permeability of Asiaticoside,Madecassoside and Asiatic Acid in Porcine Brain Endothelial Cell Model

Neurotherapeutic potentials of Centella asiatica and its reputation to boost memory, prevent cognitive deficits and improve brain functions are widely acknowledged. The plant's bioactive compounds, i.e. asiaticoside, madecassoside and asiatic acid were reported to have central nervous system (CNS) actions, particularly in protecting the brain against neurodegenerative disorders. Hence, it is important for these compounds to cross the blood-brain barrier (BBB) to be clinically effective therapeutics. This study aimed to explore the capability of asiaticoside, madecassoside and asiatic acid to cross the BBB using in vitro BBB model from primary porcine brain endothelial cells (PBECs). Our findings showed that asiaticoside, madecassoside and asiatic acid are highly BBB permeable with apparent permeability (P_app) of 70.61 ± 6.60, 53.31 ± 12.55 and 50.94 ± 10.91 × 10^?6 cm/s respectively. No evidence of cytotoxicity and tight junction disruption of the PBECs were observed in the presence of these compounds. Asiatic acid showed cytoprotective effect towards the PBECs against oxidative stress. This study reported for the first time that Centella asiatica compounds demonstrated high capability to cross the BBB, comparable to central nervous system drugs, and therefore warrant further development as therapeutics for the treatment of neurodegenerative diseases.     

Transport of the delta-opioid receptor agonist [D-penicillamine2,5] enkephalin across the blood-brain barrier involves transcytosis1

The delta opioid receptor antagonist [D-penicillamine2,5]enkephalin (DPDPE) is an enzymatically stable peptide analogue of Met-enkephalin. DPDPE uses a saturable transport mechanism to cross the blood-brain barrier (BBB), though the exact mechanism is not fully understood. The aim of the present study was to identify the mechanism by which DPDPE enters the brain. The effect of phenylarsine oxide (PAO), an endocytosis inhibitor, on the transport of [3H]DPDPE was investigated using both in vitro and in situ transport studies. Two in vitro models of the BBB utilizing primary bovine brain microvascular endothelial cells (BBMEC) were studied. [3H]DPDPE permeability across monolayers of BBMEC grown on polycarbonate filters was studied. PAO significantly reduced the permeability of [3H]DPDPE across the monolayer. PAO also reduced the uptake of [3H]DPDPE into BBMEC cells, without affecting binding to the cells. The in situ perfusion model of the BBB was also studied, PAO reduced DPDPE uptake by the brain in a dose-dependent manner. These studies indicate that DPDPE enters the brain via an energy-dependent transcytotic mechanism.     

Transport of a GABAA receptor modulator and its derivatives from Valeriana officinalis L. s. l. across an in vitro cell culture model of the blood-brain barrier

The roots and rhizome of Valeriana officinalis L . s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA (A) receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA (A) modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 +/- 0.29 microm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 +/- 0.63 microm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 +/- 0.58 microm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. S. L. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.     

Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier.

A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens.     

Assessment of blood-brain barrier penetration: in silico,in vitro and in vivo

The amount of drug achieved and maintained in the brain after systemic administration is determined by the agent's permeability at blood-brain barrier (BBB), potential involvement of transport systems, and the distribution, metabolism and elimination properties. Passive diffusion permeability may be predicted by an in silico method based on a molecule's structure property. In vitro cell culture is another useful tool for the assessment of passive permeability and BBB transports (e.g. PGP, MRP). In situ or in vivo techniques like carotid artery single injection or perfusion, brain microdialysis, autoradiography, and others are used at various stages of drug discovery and development to estimate CNS penetration and PK/PD correlation. Each technique has its own application with specific advantages and limitations.     

Neuroprotective effects of a biodegradable poly(lactic-co-glycolic acid)-ginsenoside Rg3 nanoformulation: a potential nanotherapy for Alzheimer’s disease?

It is well established that overproduction and accumulation of the β-amyloid (Aβ) peptide 1–42 (Aβ(1–42)) is a trigger of the pathological cascade in Alzheimer’s disease (AD) that manifests as cognitive impairment. Ginsenoside Rg3 is an important constituent of ginseng, plays an essential role in memory and improved cognition, and is known to produce antioxidant effects via the reduction of free radicals. Therefore, ginsenoside Rg3 may be a promising candidate as a neuroprotective agent for the treatment of AD. A novel nanotherapeutic strategy that enhances delivery of ginsenosides to the brain by increasing its transport across the blood brain barrier (BBB) would facilitate neuroprotection and limit the accumulation of Aβ plaques and subsequent neurodegeneration. In this current study, we formulated and characterised biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that encapsulate ginsenoside Rg3 and Thioflavin T, an Aβ diagnostic; examine its neuroprotective effects; investigate key mechanisms that may underlie its neuroprotective effects; and evaluate its ability to cross the BBB using an in vitro BBB model. Our PLGA-Rg3 NPs offers an exciting new theranostic material capable of encapsulating natural nutraceuticals for the detection and treatment of AD. In addition, this nanotechnology strategy can be adapted to treat other neurological diseases, utilising many natural therapeutic agents which are limited by their solubility and/or poor pharmacokinetics.     

Correlation between breakdown of the blood-brain barrier and disease outcome of viral encephalitis in mice

Changes in the permeability of the blood-brain barrier (BBB) were evaluated in two mouse models of viral encephalitis. The ability of sodium fluorescein (NaFl) to cross the BBB from the serum into the central nervous system was assayed in animals inoculated with virulent strains of either Banzi or Semliki Forest viruses. To test the hypothesis that increases in BBB permeability were associated with poor disease outcome subsequent experiments measured BBB permeability in conjunction with treatment with the interferon inducer Ampligen (poly I:poly C(12)U). A single intraperitoneal injection of Ampligen (1 mg/kg) administered either 24 h or 4-6 h before, but not 24 h after, virus inoculation with Banzi virus provided significant improvements in survival, viral brain titers, weight change and BBB permeability. In comparison, a similar treatment with Ampligen administered either 24 h or 4-6 h before inoculation with Semliki Forest virus was able to significantly improve weight change, and BBB permeability, but only animals receiving Ampligen 4-6 h pre-virus showed a significantly improved mortality. In general, it was found that evaluation of BBB permeability was a more sensitive indicator of disease outcome and the antiviral efficacy Ampligen than either weight change or brain viral titers.     

Cerebral open flow microperfusion: A new in vivo technique for continuous measurement of substance transport across the intact blood–brain barrier

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Nanomaterial-mediated CNS delivery of diagnostic and therapeutic agents

Research into the diagnosis and treatment of central nervous system (CNS) diseases has been enhanced by rapid advances in nanotechnology and an expansion in the library of nanostructured carriers. This review discusses the latest applications of nanomaterials in the CNS with an emphasis on brain tumors. Novel administration routes and transport mechanisms for nanomaterial-mediated CNS delivery of diagnostic and therapeutic agents to bypass or cross the blood brain barrier (BBB) are also discussed. These include temporary disruption of the BBB, use of impregnated polymers (polymer wafers), convection-enhanced delivery (CED), and intranasal delivery. Moreover, an in vitro BBB model capable of mimicking geometrical, cellular and rheological features of the human cerebrovasculature has been developed. This is a useful tool that can be used for screening CNS nanoparticles or therapeutics prior to in vivo and clinical investigation. A discussion of this novel model is included.     

Methods to assess drug permeability across the blood-brain barrier

Much research has focussed on the development of novel therapeutic agents to target various central nervous system disorders, however less attention has been given to determining the potential of such agents to permeate the blood-brain barrier (BBB), a factor that will ultimately govern the effectiveness of these agents in man. In order to assess the potential for novel compounds to permeate the BBB, various in-vitro, in-vivo and in-silico methods may be employed. Although in-vitro models (such as primary cell culture and immortalized cell lines) are useful as a screening method and can appropriately rank compounds in order of BBB permeability, they often correlate poorly to in-vivo brain uptake due to down-regulation of some BBB-specific transporters. In-vivo models (such as the internal carotid artery single injection or perfusion, intravenous bolus injection, brain efflux index and intracerebral microdialysis) provide more accurate information regarding brain uptake, and these can be complemented with novel imaging techniques (such as magnetic resonance imaging and positron emission tomography), although such methods are not suited to high-throughput permeability assessment. This paper reviews current methods used for assessing BBB permeability and highlights the particular advantages and disadvantages associated with each method, with a particular focus on methods suitable for moderate- to high-throughput screening.     

Drug Targeting to the Brain

The goal of brain drug targeting technology is the delivery of therapeutics across the blood–brain barrier (BBB), including the human BBB. This is accomplished by re-engineering pharmaceuticals to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Certain endogenous peptides, such as insulin or transferrin, undergo receptor-mediated transport (RMT) across the BBB in vivo. In addition, peptidomimetic monoclonal antibodies (MAb) may also cross the BBB via RMT on the endogenous transporters. The MAb may be used as a molecular Trojan horse to ferry across the BBB large molecule pharmaceuticals, including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene medicines. Fusion proteins of the molecular Trojan horse and either neurotrophins or single chain Fv antibodies have been genetically engineered. The fusion proteins retain bi-functional properties, and both bind the BBB receptor, to trigger transport into brain, and bind the cognate receptor inside brain to induce the pharmacologic effect. Trojan horse liposome technology enables the brain targeting of non-viral plasmid DNA. Molecular Trojan horses may be formulated with fusion protein technology, avidin–biotin technology, or Trojan horse liposomes to target to brain virtually any large molecule pharmaceutical.