Isotopic labeling

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Isotopic_labeling 

Isotopic labeling (or isotopic labelling) is a technique used to track the passage of an isotope (an atom with a detectable variation in neutron count) through chemical reaction, metabolic pathway, or a biological cell. The reactant is 'labeled' by replacing one or more specific atoms with their isotopes. The reactant is then allowed to undergo the reaction. The position of the isotopes in the products is measured to determine the sequence the isotopic atom followed in the reaction or the cell's metabolic pathway. The nuclides used in isotopic labeling may be stable nuclides or radionuclides. In the latter case, the labeling is called radiolabeling.

In isotopic labeling, there are multiple ways to detect the presence of labeling isotopes; through their mass, vibrational mode, or radioactive decay. Mass spectrometry detects the difference in an isotope's mass, while infrared spectroscopy detects the difference in the isotope's vibrational modes. Nuclear magnetic resonance detects atoms with different gyromagnetic ratios. The radioactive decay can be detected through an ionization chamber or autoradiographs of gels.

An example of the use of isotopic labeling is the study of phenol (C₆H₅OH) in water by replacing common hydrogen (protium) with deuterium (deuterium labeling). Upon adding phenol to deuterated water (water containing D₂O in addition to the usual H₂O), the substitution of deuterium for the hydrogen is observed in phenol's hydroxyl group (resulting in C₆H₅OD), indicating that phenol readily undergoes hydrogen-exchange reactions with water. Only the hydroxyl group is affected, indicating that the other 5 hydrogen atoms do not participate in the exchange reactions.

Isotopic tracer

A carbon-13 label was used to determine the mechanism in the 1,2- to 1,3-didehydrobenzene conversion of the phenyl substituted aryne precursor 1 to acenaphthylene.

An isotopic tracer, (also "isotopic marker" or "isotopic label"), is used in chemistry and biochemistry to help understand chemical reactions and interactions. In this technique, one or more of the atoms of the molecule of interest is substituted for an atom of the same chemical element, but of a different isotope (like a radioactive isotope used in radioactive tracing). Because the labeled atom has the same number of protons, it will behave in almost exactly the same way as its unlabeled counterpart and, with few exceptions, will not interfere with the reaction under investigation. The difference in the number of neutrons, however, means that it can be detected separately from the other atoms of the same element.

Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are used to investigate the mechanisms of chemical reactions. NMR and MS detects isotopic differences, which allows information about the position of the labeled atoms in the products' structure to be determined. With information on the positioning of the isotopic atoms in the products, the reaction pathway the initial metabolites utilize to convert into the products can be determined. Radioactive isotopes can be tested using the autoradiographs of gels in gel electrophoresis. The radiation emitted by compounds containing the radioactive isotopes darkens a piece of photographic film, recording the position of the labeled compounds relative to one another in the gel.

Isotope tracers are commonly used in the form of isotope ratios. By studying the ratio between two isotopes of the same element, we avoid effects involving the overall abundance of the element, which usually swamp the much smaller variations in isotopic abundances. Isotopic tracers are some of the most important tools in geology because they can be used to understand complex mixing processes in earth systems. Further discussion of the application of isotopic tracers in geology is covered under the heading of isotope geochemistry.

Isotopic tracers are usually subdivided into two categories: stable isotope tracers and radiogenic isotope tracers. Stable isotope tracers involve only non-radiogenic isotopes and usually are mass-dependent. In theory, any element with two stable isotopes can be used as an isotopic tracer. However, the most commonly used stable isotope tracers involve relatively light isotopes, which readily undergo fractionation in natural systems. See also isotopic signature. A radiogenic isotope tracer involves an isotope produced by radioactive decay, which is usually in a ratio with a non-radiogenic isotope (whose abundance in the earth does not vary due to radioactive decay).

Stable isotope labeling

Isotopic tracing through reactions in the pentose phosphate pathway. The blue circles indicate a labeled carbon atom, while white circles are an unlabeled carbon atom.

Stable isotope labeling involves the use of non-radioactive isotopes that can act as a tracers used to model several chemical and biochemical systems. The chosen isotope can act as a label on that compound that can be identified through nuclear magnetic resonance (NMR) and mass spectrometry (MS). Some of the most common stable isotopes are ²H, ¹³C, and ¹⁵N, which can further be produced into NMR solvents, amino acids, nucleic acids, lipids, common metabolites and cell growth media. The compounds produced using stable isotopes are either specified by the percentage of labeled isotopes (i.e. 30% uniformly labeled ¹³C glucose contains a mixture that is 30% labeled with ¹³ carbon isotope and 70% naturally labeled carbon) or by the specifically labeled carbon positions on the compound (i.e. 1-¹³C glucose which is labeled at the first carbon position of glucose).

A network of reactions adopted from the glycolysis pathway and the pentose phosphate pathway is shown in which the labeled carbon isotope rearranges to different carbon positions throughout the network of reactions. The network starts with fructose 6-phosphate (F6P), which has 6 carbon atoms with a label ¹³C at carbon position 1 and 2. 1,2-¹³C F6P becomes two glyceraldehyde 3-phosphate (G3P), one 2,3-¹³C T3P and one unlabeled T3P. The 2,3-¹³C T3P can now be reacted with sedoheptulose 7-phosphate (S7P) to form an unlabeled erythrose 4-phosphate(E4P) and a 5,6-¹³C F6P. The unlabeled T3P will react with the S7P to synthesize unlabeled products. The figure demonstrates the use of stable isotope labeling to discover the carbon atom rearrangement through reactions using position specific labeled compounds.

Metabolic flux analysis using stable isotope labeling

Determining the percent of isotope labeling throughout a reaction. If a 50% labeled and 50% unlabeled metabolite is split in the manner shown, the expected percent of each outcome can be found. The blue circles indicate a labeled atom, while a white circle indicates an unlabeled atom.

Metabolic flux analysis (MFA) using stable isotope labeling is an important tool for explaining the flux of certain elements through the metabolic pathways and reactions within a cell. An isotopic label is fed to the cell, then the cell is allowed to grow utilizing the labeled feed. For stationary metabolic flux analysis the cell must reach a steady state (the isotopes entering and leaving the cell remain constant with time) or a quasi-steady state (steady state is reached for a given period of time). The isotope pattern of the output metabolite is determined. The output isotope pattern provides valuable information, which can be used to find the magnitude of flux, rate of conversion from reactants to products, through each reaction.

The figure demonstrates the ability to use different labels to determine the flux through a certain reaction. Assume the original metabolite, a three carbon compound, has the ability to either split into a two carbon metabolite and one carbon metabolite in one reaction then recombine or remain a three carbon metabolite. If the reaction is provided with two isotopes of the metabolite in equal proportion, one completely labeled (blue circles), commonly known as uniformly labeled, and one completely unlabeled (white circles). The pathway down the left side of the diagram does not display any change in the metabolites, while the right side shows the split and recombination. As shown, if the metabolite only takes the pathway down the left side, it remains in a 50–50 ratio of uniformly labeled to unlabeled metabolite. If the metabolite only takes the right side new labeling patterns can occur, all in equal proportion. Other proportions can occur depending on how much of the original metabolite follows the left side of the pathway versus the right side of the pathway. Here the proportions are shown for a situation in which half of the metabolites take the left side and half the right, but other proportions can occur. These patterns of labeled atoms and unlabeled atoms in one compound represent isotopomers. By measuring the isotopomer distribution of the differently labeled metabolites, the flux through each reaction can be determined.

MFA combines the data harvested from isotope labeling with the stoichiometry of each reaction, constraints, and an optimization procedure resolve a flux map. The irreversible reactions provide the thermodynamic constraints needed to find the fluxes. A matrix is constructed that contains the stoichiometry of the reactions. The intracellular fluxes are estimated by using an iterative method in which simulated fluxes are plugged into the stoichiometric model. The simulated fluxes are displayed in a flux map, which shows the rate of reactants being converted to products for each reaction. In most flux maps, the thicker the arrow, the larger the flux value of the reaction.

Isotope labeling measuring techniques

Any technique in measuring the difference between isotopomers can be used. The two primary methods, nuclear magnetic resonance (NMR) and mass spectrometry (MS), have been developed for measuring mass isotopomers in stable isotope labeling.

Proton NMR was the first technique used for ¹³C-labeling experiments. Using this method, each single protonated carbon position inside a particular metabolite pool can be observed separately from the other positions. This allows the percentage of isotopomers labeled at that specific position to be known. The limit to proton NMR is that if there are n carbon atoms in a metabolite, there can only be at most n different positional enrichment values, which is only a small fraction of the total isotopomer information. Although the use of proton NMR labeling is limiting, pure proton NMR experiments are much easier to evaluate than experiments with more isotopomer information.

In addition to Proton NMR, using ¹³C NMR techniques will allow a more detailed view of the distribution of the isotopomers. A labeled carbon atom will produce different hyperfine splitting signals depending on the labeling state of its direct neighbors in the molecule. A singlet peak emerges if the neighboring carbon atoms are not labeled. A doublet peak emerges if only one neighboring carbon atom is labeled. The size of the doublet split depends on the functional group of the neighboring carbon atom. If two neighboring carbon atoms are labeled, a doublet of doublets may degenerate into a triplet if the doublet splittings are equal.

The drawbacks to using NMR techniques for metabolic flux analysis purposes is that it is different from other NMR applications because it is a rather specialized discipline. An NMR spectrometer may not be directly available for all research teams. The optimization of NMR measurement parameters and proper analysis of peak structures requires a skilled NMR specialist. Certain metabolites also may require specialized measurement procedures to obtain additional isotopomer data. In addition, specially adapted software tools are needed to determine the precise quantity of peak areas as well as identifying the decomposition of entangled singlet, doublet, and triplet peaks.

As opposed to nuclear magnetic resonance, mass spectrometry (MS) is another method that is more applicable and sensitive to metabolic flux analysis experiments. MS instruments are available in different variants. Different from two-dimensional nuclear magnetic resonance (2D-NMR), the MS instruments work directly with hydrolysate.

In gas chromatography-mass spectrometry (GC-MS), the MS is coupled to a gas chromatograph to separate the compounds of the hydrolysate. The compounds eluting from the GC column are then ionized and simultaneously fragmented. The benefit in using GC-MS is that not only are the mass isotopomers of the molecular ion measured but also the mass isotopomer spectrum of several fragments, which significantly increases the measured information.

In liquid chromatography-mass spectrometry (LC-MS), the GC is replaced with a liquid chromatograph. The main difference is that chemical derivatization is not necessary. Applications of LC-MS to MFA, however, are rare.

In each case, MS instruments divide a particular isotopomer distribution by its molecular weight. All isotopomers of a particular metabolite that contain the same number of labeled carbon atoms are collected in one peak signal. Because every isotopomer contributes to exactly one peak in the MS spectrum, the percentage value can then be calculated for each peak, yielding the mass isotopomer fraction. For a metabolite with n carbon atoms, n+1 measurements are produced. After normalization, exactly n informative mass isotopomer quantities remain.

The drawback to using MS techniques is that for gas chromatography, the sample must be prepared by chemical derivatization in order to obtain molecules with charge. There are numerous compounds used to derivatize samples. N,N-Dimethylformamide dimethyl acetal (DMFDMA) and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) are two examples of compounds that have been used to derivatize amino acids.

In addition, strong isotope effects observed affect the retention time of differently labeled isotopomers in the GC column. Overloading of the GC column also must be prevented.

Lastly, the natural abundance of other atoms than carbon also leads to a disturbance in the mass isotopomer spectrum. For example, each oxygen atom in the molecule might also be present as a ¹⁷O isotope and as a ¹⁸O isotope. A more significant impact of the natural abundance of isotopes is the effect of silicon with a natural abundance of the isotopes ²⁹Si and ³⁰Si. Si is used in derivatizing agents for MS techniques.

Radioisotopic labeling

Main article: Radioactive tracer

Radioisotopic labeling is a technique for tracking the passage of a sample of substance through a system. The substance is "labeled" by including radionuclides in its chemical composition. When these decay, their presence can be determined by detecting the radiation emitted by them. Radioisotopic labeling is a special case of isotopic labeling.

For these purposes, a particularly useful type of radioactive decay is positron emission. When a positron collides with an electron, it releases two high-energy photons traveling in diametrically opposite directions. If the positron is produced within a solid object, it is likely to do this before traveling more than a millimeter. If both of these photons can be detected, the location of the decay event can be determined very precisely.

Strictly speaking, radioisotopic labeling includes only cases where radioactivity is artificially introduced by experimenters, but some natural phenomena allow similar analysis to be performed. In particular, radiometric dating uses a closely related principle.

Applications

Applications in human mineral nutrition research

The use of stable isotope tracers to study mineral nutrition and metabolism in humans was first reported in the 1960s. While radioisotopes had been used in human nutrition research for several decades prior, stable isotopes presented a safer option, especially in subjects for which there is elevated concern about radiation exposure, e.g. pregnant and lactating women and children. Other advantages offered by stable isotopes include the ability to study elements having no suitable radioisotopes and to study long-term tracer behavior. Thus the use of stable isotopes became commonplace with the increasing availability of isotopically enriched materials and inorganic mass spectrometers. The use of stable isotopes instead of radioisotopes does have several drawbacks: larger quantities of tracer are required, having the potential of perturbing the naturally existing mineral; analytical sample preparation is more complex and mass spectrometry instrumentation more costly; the presence of tracer in whole bodies or particular tissues cannot be measured externally. Nonetheless, the advantages have prevailed making stable isotopes the standard in human studies.

Most of the minerals that are essential for human health and of particular interest to nutrition researchers have stable isotopes, some well-suited as biological tracers because of their low natural abundance. Iron, zinc, calcium, copper, magnesium, selenium and molybdenum are among the essential minerals having stable isotopes to which isotope tracer methods have been applied. Iron, zinc and calcium in particular have been extensively studied.

Aspects of mineral nutrition/metabolism that are studied include absorption (from the gastrointestinal tract into the body), distribution, storage, excretion and the kinetics of these processes. Isotope tracers are administered to subjects orally (with or without food, or with a mineral supplement) and/or intravenously. Isotope enrichment is then measured in blood plasma, erythrocytes, urine and/or feces. Enrichment has also been measured in breast milk and intestinal contents. Tracer experiment design sometimes differs between minerals due to differences in their metabolism. For example, iron absorption is usually determined from incorporation of tracer in erythrocytes whereas zinc or calcium absorption is measured from tracer appearance in plasma, urine or feces. The administration of multiple isotope tracers in a single study is common, permitting the use of more reliable measurement methods and simultaneous investigations of multiple aspects of metabolism.

The measurement of mineral absorption from the diet, often conceived of as bioavailability, is the most common application of isotope tracer methods to nutrition research. Among the purposes of such studies are the investigations of how absorption is influenced by type of food (e.g. plant vs animal source, breast milk vs formula), other components of the diet (e.g. phytate), disease and metabolic disorders (e.g. environmental enteric dysfunction), the reproductive cycle, quantity of mineral in diet, chronic mineral deficiency, subject age and homeostatic mechanisms. When results from such studies are available for a mineral, they may serve as a basis for estimations of the human physiological and dietary requirements of the mineral.

When tracer is administered with food for the purpose of observing mineral absorption and metabolism, it may be in the form of an intrinsic or extrinsic label. An intrinsic label is isotope that has been introduced into the food during its production, thus enriching the natural mineral content of the food, whereas extrinsic labeling refers to the addition of tracer isotope to the food during the study. Because it is a very time-consuming and expensive approach, intrinsic labeling is not routinely used. Studies comparing measurements of absorption using intrinsic and extrinsic labeling of various foods have generally demonstrated good agreement between the two labeling methods, supporting the hypothesis that extrinsic and natural minerals are handled similarly in the human gastrointestinal tract.

Enrichment is quantified from the measurement of isotope ratios, the ratio of the tracer isotope to a reference isotope, by mass spectrometry. Multiple definitions and calculations of enrichment have been adopted by different researchers. Calculations of enrichment become more complex when multiple tracers are used simultaneously. Because enriched isotope preparations are never isotopically pure, i.e. they contain all the element's isotopes in unnatural abundances, calculations of enrichment of multiple isotope tracers must account for the perturbation of each isotope ratio by the presence of the other tracers.

Due to the prevalence of mineral deficiencies and their critical impact on human health and well-being in resource-poor countries, the International Atomic Energy Agency has recently published detailed and comprehensive descriptions of stable isotope methods to facilitate the dissemination of this knowledge to researchers beyond western academic centers.

Applications in proteomics

In proteomics, the study of the full set of proteins expressed by a genome, identifying diseases biomarkers can involve the usage of stable isotope labeling by amino acids in cell culture (SILAC), that provides isotopic labeled forms of amino acid used to estimate protein levels. In protein recombinant, manipulated proteins are produced in large quantities and isotope labeling is a tool to test for relevant proteins. The method used to be about selectively enrich nuclei with ¹³C or ¹⁵N or deplete ¹H from them. The recombinant would be expressed in E.coli with media containing ¹⁵N-ammonium chloride as a source of nitrogen. The resulting ¹⁵N labeled proteins are then purified by immobilized metal affinity and their percentage estimated. In order to increase the yield of labeled proteins and cut down the cost of isotope labeled media, an alternative procedure primarily increases the cell mass using unlabeled media before introducing it in a minimal amount of labeled media. Another application of isotope labeling would be in measuring DNA synthesis, that is cell proliferation in vitro. Uses H₃-thymidine labeling to compare pattern of synthesis (or sequence) in cells.

Applications for ecosystem process analysis

Isotopic tracers are used to examine processes in natural systems, especially terrestrial and aquatic environments. In soil science ¹⁵N tracers are used extensively to study nitrogen cycling, whereas ¹³C and ¹⁴C, stable and radioisotopes of carbon respectively, are used for studying turnover of organic compounds and fixation of CO₂ by autotrophs. For example, Marsh et al. (2005) used dual labeled (¹⁵N- and ¹⁴C) urea to demonstrate utilization of the compound by ammonia oxidizers as both an energy source (ammonia oxidation) and carbon source (chemoautotrophic carbon fixation). Deuterated water is also used for tracing the fate and ages of water in a tree or in an ecosystem.

Applications for oceanography

Tracers are also used extensively in oceanography to study a wide array of processes. The isotopes used are typically naturally occurring with well-established sources and rates of formation and decay. However, anthropogenic isotopes may also be used with great success. The researchers measure the isotopic ratios at different locations and times to infer information about the physical processes of the ocean.

Particle transport

The ocean is an extensive network of particle transport. Thorium isotopes can help researchers decipher the vertical and horizontal movement of matter. ²³⁴Th has a constant, well-defined production rate in the ocean and a half-life of 24 days. This naturally occurring isotope has been shown to vary linearly with depth. Therefore, any changes in this linear pattern can be attributed to the transport of ²³⁴Th on particles. For example, low isotopic ratios in surface water with very high values a few meters down would indicate a vertical flux in the downward direction. Furthermore, the thorium isotope may be traced within a specific depth to decipher the lateral transport of particles.

Circulation

Circulation within local systems, such as bays, estuaries, and groundwater, may be examined with radium isotopes. ²²³Ra has a half-life of 11 days and can occur naturally at specific locations in rivers and groundwater sources. The isotopic ratio of radium will then decrease as the water from the source river enters a bay or estuary. By measuring the amount of ²²³Ra at a number of different locations, a circulation pattern can be deciphered. This same exact process can also be used to study the movement and discharge of groundwater.

Various isotopes of lead can be used to study circulation on a global scale. Different oceans (i.e. the Atlantic, Pacific, Indian, etc.) have different isotopic signatures. This results from differences in isotopic ratios of sediments and rocks within the different oceans. Because the different isotopes of lead have half-lives of 50–200 years, there is not enough time for the isotopic ratios to be homogenized throughout the whole ocean. Therefore, precise analysis of Pb isotopic ratios can be used to study the circulation of the different oceans.

Tectonic processes and climate change

Isotopes with extremely long half-lives and their decay products can be used to study multi-million year processes, such as tectonics and extreme climate change. For example, in rubidium–strontium dating, the isotopic ratio of strontium (⁸⁷Sr/⁸⁶Sr) can be analyzed within ice cores to examine changes over the earth's lifetime. Differences in this ratio within the ice core would indicate significant alterations in the earth's geochemistry.

Isotopes related to nuclear weapons

The aforementioned processes can be measured using naturally occurring isotopes. Nevertheless, anthropogenic isotopes are also extremely useful for oceanographic measurements. Nuclear weapons tests released a plethora of uncommon isotopes into the world's oceans. ³H, ¹²⁹I, and ¹³⁷Cs can be found dissolved in seawater, while ²⁴¹Am and ²³⁸Pu are attached to particles. The isotopes dissolved in water are particularly useful in studying global circulation. For example, differences in lateral isotopic ratios within an ocean can indicate strong water fronts or gyres. Conversely, the isotopes attached to particles can be used to study mass transport within water columns. For instance, high levels of Am or Pu can indicate downwelling when observed at great depths, or upwelling when observed at the surface.

Methods for isotopic labeling

• Chemical synthesis
• Enzyme-mediated exchange
• Recombinant protein expression in isotopic labeled media.

Nanoparticles for drug delivery to the brain

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Nanoparticles_for_drug_delivery_to_the_brain

Nanoparticles for drug delivery to the brain is a method for transporting drug molecules across the blood–brain barrier (BBB) using nanoparticles. These drugs cross the BBB and deliver pharmaceuticals to the brain for therapeutic treatment of neurological disorders. These disorders include Parkinson's disease, Alzheimer's disease, schizophrenia, depression, and brain tumors. Part of the difficulty in finding cures for these central nervous system (CNS) disorders is that there is yet no truly efficient delivery method for drugs to cross the BBB. Antibiotics, antineoplastic agents, and a variety of CNS-active drugs, especially neuropeptides, are a few examples of molecules that cannot pass the BBB alone. With the aid of nanoparticle delivery systems, however, studies have shown that some drugs can now cross the BBB, and even exhibit lower toxicity and decrease adverse effects throughout the body. Toxicity is an important concept for pharmacology because high toxicity levels in the body could be detrimental to the patient by affecting other organs and disrupting their function. Further, the BBB is not the only physiological barrier for drug delivery to the brain. Other biological factors influence how drugs are transported throughout the body and how they target specific locations for action. Some of these pathophysiological factors include blood flow alterations, edema and increased intracranial pressure, metabolic perturbations, and altered gene expression and protein synthesis. Though there exist many obstacles that make developing a robust delivery system difficult, nanoparticles provide a promising mechanism for drug transport to the CNS.

Background

The first successful delivery of a drug across the BBB occurred in 1995. The drug used was hexapeptide dalargin, an anti-nociceptive peptide that cannot cross the BBB alone. It was encapsulated in polysorbate 80 coated nanoparticles and intravenously injected. This was a huge breakthrough in the nanoparticle drug delivery field, and it helped advance research and development toward clinical trials of nanoparticle delivery systems. Nanoparticles range in size from 10 - 1000 nm (or 1 µm) and they can be made from natural or artificial polymers, lipids, dendrimers, and micelles. Most polymers used for nanoparticle drug delivery systems are natural, biocompatible, and biodegradable, which helps prevent contamination in the CNS. Several current methods for drug delivery to the brain include the use of liposomes, prodrugs, and carrier-mediated transporters. Many different delivery methods exist to transport these drugs into the body, such as peroral, intranasal, intravenous, and intracranial. For nanoparticles, most studies have shown increasing progression with intravenous delivery. Along with delivery and transport methods, there are several means of functionalizing, or activating, the nanoparticle carriers. These means include dissolving or absorbing a drug throughout the nanoparticle, encapsulating a drug inside the particle, or attaching a drug on the surface of the particle.

Types of nanoparticles for CNS drug delivery

Lipid-based

Diagram of liposome showing a phospholipid bilayer surrounding an aqueous interior.

One type of nanoparticle involves use of liposomes as drug molecule carriers. The diagram on the right shows a standard liposome. It has a phospholipid bilayer separating the interior from the exterior of the cell.

Liposomes are composed of vesicular bilayers, lamellae, made of biocompatible and biodegradable lipids such as sphingomyelin, phosphatidylcholine, and glycerophospholipids. Cholesterol, a type of lipid, is also often incorporated in the lipid-nanoparticle formulation. Cholesterol can increase stability of a liposome and prevent leakage of a bilayer because its hydroxyl group can interact with the polar heads of the bilayer phospholipids. Liposomes have the potential to protect the drug from degradation, target sites for action, and reduce toxicity and adverse effects. Lipid nanoparticles can be manufactured by high pressure homogenization, a current method used to produce parenteral emulsions. This process can ultimately form a uniform dispersion of small droplets in a fluid substance by subdividing particles until the desired consistency is acquired. This manufacturing process is already scaled and in use in the food industry, which therefore makes it more appealing for researchers and for the drug delivery industry.

Liposomes can also be functionalized by attaching various ligands on the surface to enhance brain-targeted delivery.

Cationic liposomes

Another type of lipid-nanoparticle that can be used for drug delivery to the brain is a cationic liposome. These are lipid molecules that are positively charged. One example of cationic liposomes uses bolaamphiphiles, which contain hydrophilic groups surrounding a hydrophobic chain to strengthen the boundary of the nano-vesicle containing the drug. Bolaamphiphile nano-vesicles can cross the BBB, and they allow controlled release of the drug to target sites. Lipoplexes can also be formed from cationic liposomes and DNA solutions, to yield transfection agents. Cationic liposomes cross the BBB through adsorption mediated endocytosis followed by internalization in the endosomes of the endothelial cells. By transfection of endothelial cells through the use of lipoplexes, physical alterations in the cells could be made. These physical changes could potentially improve how some nanoparticle drug-carriers cross the BBB.

Metallic

Metal nanoparticles are promising as carriers for drug delivery to the brain. Common metals used for nanoparticle drug delivery are gold, silver, and platinum, owing to their biocompatibility. These metallic nanoparticles are used due to their large surface area to volume ratio, geometric and chemical tunability, and endogenous antimicrobial properties. Silver cations released from silver nanoparticles can bind to the negatively charged cellular membrane of bacteria and increase membrane permeability, allowing foreign chemicals to enter the intracellular fluid.

Metal nanoparticles are chemically synthesized using reduction reactions. For example, drug-conjugated silver nanoparticles are created by reducing silver nitrate with sodium borohydride in the presence of an ionic drug compound. The drug binds to the surface of the silver, stabilizing the nanoparticles and preventing the nanoparticles from aggregation.

Metallic nanoparticles typically cross the BBB via transcytosis. Nanoparticle delivery through the BBB can be increased by introducing peptide conjugates to improve permeability to the central nervous system. For instance, recent studies have shown an improvement in gold nanoparticle delivery efficiency by conjugating a peptide that binds to the transferrin receptors expressed in brain endothelial cells.

Solid lipid

Diagram displays a solid lipid nanoparticle (SLN). There is only one phospholipid layer because the interior of the particle is solid. Molecules such as antibodies, targeting peptides, and drug molecules can be bound to the surface of the SLN.

Also, solid lipid nanoparticles (SLNs) are lipid nanoparticles with a solid interior as shown in the diagram on the right. SLNs can be made by replacing the liquid lipid oil used in the emulsion process with a solid lipid. In solid lipid nanoparticles, the drug molecules are dissolved in the particle's solid hydrophobic lipid core, this is called the drug payload, and it is surrounded by an aqueous solution. Many SLNs are developed from triglycerides, fatty acids, and waxes. High-pressure homogenization or micro-emulsification can be used for manufacturing. Further, functionalizing the surface of solid lipid nanoparticles with polyethylene glycol (PEG) can result in increased BBB permeability. Different colloidal carriers such as liposomes, polymeric nanoparticles, and emulsions have reduced stability, shelf life and encapsulation efficacy. Solid lipid nanoparticles are designed to overcome these shortcomings and have an excellent drug release and physical stability apart from targeted delivery of drugs.

Nanoemulsions

Another form for nanoparticle delivery systems is oil-in-water emulsions done on a nano-scale. This process uses common biocompatible oils such as triglycerides and fatty acids, and combines them with water and surface-coating surfactants. Oils rich in omega-3 fatty acids especially contain important factors that aid in penetrating the tight junctions of the BBB.

Polymer-based

Other nanoparticles are polymer-based, meaning they are made from a natural polymer such as polylactic acid (PLA), poly D,L-glycolide (PLG),

polylactide-co-glycolide (PLGA), and polycyanoacrylate (PCA). Some studies have found that polymeric nanoparticles may provide better results for drug delivery relative to lipid-based nanoparticles because they may increase the stability of the drugs or proteins being transported. Polymeric nanoparticles may also contain beneficial controlled release mechanisms.

Polymer Branch

Nanoparticles made from natural polymers that are biodegradable have the abilities to target specific organs and tissues in the body, to carry DNA for gene therapy, and to deliver larger molecules such as proteins, peptides, and even genes. To manufacture these polymeric nanoparticles, the drug molecules are first dissolved and then encapsulated or attached to a polymer nanoparticle matrix. Three different structures can then be obtained from this process; nanoparticles, nanocapsules (in which the drug is encapsulated and surrounded by the polymer matrix), and nanospheres (in which the drug is dispersed throughout the polymeric matrix in a spherical form).

One of the most important traits for nanoparticle delivery systems is that they must be biodegradable on the scale of a few days. A few common polymer materials used for drug delivery studies are polybutyl cyanoacrylate (PBCA), poly(isohexyl cyanoacrylate) (PIHCA), polylactic acid (PLA), or polylactide-co-glycolide (PLGA). PBCA undergoes degradation through enzymatic cleavage of its ester bond on the alkyl side chain to produce water-soluble byproducts. PBCA also proves to be the fastest biodegradable material, with studies showing 80% reduction after 24 hours post intravenous therapy injection. PIHCA, however, was recently found to display an even lower degradation rate, which in turn further decreases toxicity. PIHCA, due to this slight advantage, is currently undergoing phase III clinical trials for transporting the drug doxorubicin as a treatment for hepatocellular carcinomas.

Human serum albumin (HSA) and chitosan are also materials of interest for the generation of nanoparticle delivery systems. Using albumin nanoparticles for stroke therapy can overcome numerous limitations. For instance, albumin nanoparticles can enhance BBB permeability, increase solubility, and increase half-life in circulation. Patients who have brain cancer overexpress albumin-binding proteins, such as SPARC and gp60, in their BBB and tumor cells, naturally increasing the uptake of albumin into the brain. Using this relationship, researches have formed albumin nanoparticles that co-encapsulate two anticancer drugs, paclitaxel and fenretinide, modified with low weight molecular protamine (LMWP), a type of cell-penetrating protein, for anti-glioma therapy. Once injected into the patient's body, the albumin nanoparticles can cross the BBB more easily, bind to the proteins and penetrate glioma cells, and then release the contained drugs. This nanoparticle formulation enhances tumor-targeting delivery efficiency and improves the solubility issue of hydrophobic drugs. Specifically, cationic bovine serum albumin-conjugated tanshinone IIA PEGylated nanoparticles injected into a MCAO rat model decreased the volume of infarction and neuronal apoptosis. Chitosan, a naturally abundant polysaccharide, is particularly useful due to its biocompability and lack of toxicity. With its adsorptive and mucoadhesive properties, chitosan can overcome limitations of internasal administration to the brain. It has been shown that cationic chitosan nanoparticles interact with the negatively charged brain endothelium.

Coating these polymeric nanoparticle devices with different surfactants can also aid BBB crossing and uptake in the brain. Surfactants such as polysorbate 80, 20, 40, 60, and poloxamer 188, demonstrated positive drug delivery through the blood–brain barrier, whereas other surfactants did not yield the same results. It has also been shown that functionalizing the surface of nanoparticles with polyethylene glycol (PEG), can induce the "stealth effect", allowing the drug-loaded nanoparticle to circulate throughout the body for prolonged periods of time. Further, the stealth effect, caused in part by the hydrophilic and flexible properties of the PEG chains, facilitates an increase in localizing the drug at target sites in tissues and organs.

Mechanisms for delivery

Liposomes

A mechanism for liposome transport across the BBB is lipid-mediated free diffusion, a type of facilitated diffusion, or lipid-mediated endocytosis. There exist many lipoprotein receptors which bind lipoproteins to form complexes that in turn transport the liposome nano-delivery system across the BBB. Apolipoprotein E (apoE) is a protein that facilitates transport of lipids and cholesterol. ApoE constituents bind to nanoparticles, and then this complex binds to a low-density lipoprotein receptor (LDLR) in the BBB and allows transport to occur.

This diagram shows several ways in which transport across the BBB works. For nanoparticle delivery across the BBB, the most common mechanisms are receptor-mediated transcytosis and adsorptive transcytosis

Polymeric nanoparticles

The mechanism for the transport of polymer-based nanoparticles across the BBB has been characterized as receptor-mediated endocytosis by the brain capillary endothelial cells. Transcytosis then occurs to transport the nanoparticles across the tight junction of endothelial cells and into the brain. Surface coating nanoparticles with surfactants such as polysorbate 80 or poloxamer 188 was shown to increase uptake of the drug into the brain also. This mechanism also relies on certain receptors located on the luminal surface of endothelial cells of the BBB. Ligands coated on the nanoparticle's surface bind to specific receptors to cause a conformational change. Once bound to these receptors, transcytosis can commence, and this involves the formation of vesicles from the plasma membrane pinching off the nanoparticle system after internalization.

Additional receptors identified for receptor-mediated endocytosis of nanoparticle delivery systems are the scavenger receptor class B type I (SR-BI), LDL receptor (LRP1), transferrin receptor, and insulin receptor. As long as a receptor exists on the endothelial surface of the BBB, any ligand can be attached to the nanoparticle's surface to functionalize it so that it can bind and undergo endocytosis.

Another mechanism is adsorption mediated transcytosis, where electrostatic interactions are involved in mediating nanoparticle crossing of the BBB. Cationic nanoparticles (including cationic liposomes) are of interest for this mechanism, because their positive charges assist binding on the brain's endothelial cells. Using TAT-peptides, a cell-penetrating peptide, to functionalize the surface of cationic nanoparticles can further improve drug transport into the brain.

Magnetic and Magnetoelectric nanoparticles

In contrast to the above mechanisms, a delivery with magnetic fields does not strongly depend on the biochemistry of the brain. In this case, nanoparticles are literally pulled across the BBB via application of a magnetic field gradient. The nanoparticles can be pulled in as well as removed from the brain merely by controlling the direction of the gradient. For the approach to work, the nanoparticles must have a non-zero magnetic moment and have a diameter of less than 50 nm. Both magnetic and magnetoelectric nanoparticles (MENs) satisfy the requirements. However, it is only the MENs which display a non-zero magnetoelectric (ME) effect. Due to the ME effect, MENs can provide a direct access to local intrinsic electric fields at the nanoscale to enable a two-way communication with the neural network at the single-neuron level. MENs, proposed by the research group of Professor Sakhrat Khizroev at Florida International University (FIU), have been used for targeted drug delivery and externally controlled release across the BBB to treat HIV and brain tumors, as well as to wirelessly stimulate neurons deep in the brain for treatment of neurodegenerative diseases such as Parkinson's Disease and others.

Focused ultrasound

Studies have shown that focused ultrasound bursts can noninvasively be used to disrupt tight junctions in desired locations of BBB, allowing for the increased passage of particles at that location. This disruption can last up to four hours after burst administration. Focused ultrasound works by generating oscillating microbubbles, which physically interact with the cells of the BBB by oscillating at a frequency which can be tuned by the ultrasound burst. This physical interaction is believed to cause cavitation and ultimately the disintegration of the tight junction complexes which may explain why this effect lasts for several hours. However, the energy applied from ultrasound can result in tissue damage. Fortunately, studies have demonstrated that this risk can be reduced if preformed microbubbles are first injected before focused ultrasound is applied, reducing the energy required from the ultrasound. This technique has applications in the treatment of various diseases. For example, one study has shown that using focused ultrasound with oscillating bubbles loaded with a chemotherapeutic drug, carmustine, facilitates the safe treatment of glioblastoma in an animal model. This drug, like many others, normally requires large dosages to reach the target brain tissue diffusion from the blood, leading to systemic toxicity and the possibilities of multiple harmful side effects manifesting throughout the body. However, focused ultrasound has the potential to increase the safety and efficacy of drug delivery to the brain.

Toxicity

A study was performed to assess the toxicity effects of doxorubicin-loaded polymeric nanoparticle systems. It was found that doses up to 400 mg/kg of PBCA nanoparticles alone did not cause any toxic effects on the organism. These low toxicity effects can most likely be attributed to the controlled release and modified biodistribution of the drug due to the traits of the nanoparticle delivery system. Toxicity is a highly important factor and limit of drug delivery studies, and a major area of interest in research on nanoparticle delivery to the brain.

Metal nanoparticles are associated with risks of neurotoxicity and cytotoxicity. These heavy metals generate reactive oxygen species, which causes oxidative stress and damages the cells' mitochondria and endoplasmic reticulum. This leads to further issues in cellular toxicity, such as damage to DNA and disruption of cellular pathways. Silver nanoparticles in particular have a higher degree of toxicity compared to other metal nanoparticles such as gold or iron. Silver nanoparticles can circulate through the body and accumulate easily in multiple organs, as discovered in a study on the silver nanoparticle distribution in rats. Traces of silver accumulated in the rats' lungs, spleen, kidney, liver, and brain after the nanoparticles were injected subcutaneously. In addition, silver nanoparticles generate more reactive oxygen species compared to other metals, which leads to an overall larger issue of toxicity.

Research

In the early 21st century, extensive research is occurring in the field of nanoparticle drug delivery systems to the brain. One of the common diseases being studied in neuroscience is Alzheimer's disease. Many studies have been done to show how nanoparticles can be used as a platform to deliver therapeutic drugs to these patients with the disease. A few Alzheimer's drugs that have been studied especially are rivastigmine, tacrine, quinoline, piperine, and curcumin. PBCA, chitosan, and PLGA nanoparticles were used as delivery systems for these drugs. Overall, the results from each drug injection with these nanoparticles showed remarkable improvements in the effects of the drug relative to non-nanoparticle delivery systems. This possibly suggests that nanoparticles could provide a promising solution to how these drugs could cross the BBB. One factor that still must be considered and accounted for is nanoparticle accumulation in the body. With long-term and frequent injections that are often required to treat chronic diseases such as Alzheimer's disease, polymeric nanoparticles could potentially build up in the body, causing undesirable effects. This area for concern would have to be further assessed to analyze these possible effects and to improve them.

History of neuroimaging

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/History_of_neuroimaging

Neuroimaging is a medical technique that allows doctors and researchers to take pictures of the inner workings of the body or brain of a patient. It can show areas with heightened activity, areas with high or low blood flow, the structure of the patients brain/body, as well as certain abnormalities. Neuroimaging is most often used to find the specific location of certain diseases or birth defects such as tumors, cancers, or clogged arteries. Neuroimaging first came about as a medical technique in the 1880's with the invention of the human circulation balance and has since lead to other inventions such as the x-ray, air ventriculography, cerebral angiography, PET/SPECT scans, magnetoencephalography, and xenon CT scanning.

Neuroimaging Techniques

Human Circulation Balance

Angelo Mosso's 'human circulation balance.'

The 'human circulation balance' was a non-invasive way to measure blood flow to the brain during mental activities. This technique worked by placing patients on a table that was supported by a fulcrum, allowing the table to sway depending on activity levels. When patients were exposed to more cognitively complex stimuli, the table would sway towards the head. Invented in 1882 by Angelo Mosso, the 'human circulation balance' is said to be the first technique of neuroimaging created and is what Mosso is most known for.

Wilhelm Roentgen, creator of the X-ray.

X-ray

In the year of 1895, Wilhelm Roentgen developed the first radiograph, more commonly known as the X-ray. By 1901, Roentgen had been awarded a Nobel Peace Prize for his discovery. Immediately after its release, X-ray machines were being manufactured and used worldwide in medicine. However, this was only the first step in the development of neuroimaging. The brain is almost entirely composed of soft tissue that is not radio-opaque, meaning it remains essentially invisible to ordinary or plain X-ray examinations. This is also true of most brain abnormalities, though there are exceptions. For example, a calcified tumor (e.g.,meningioma, craniopharyngioma, and some types of glioma) can easily be seen.

Air Ventriculography

To combat this, in 1918, neurosurgeon Walter Dandy developed a technique called air ventriculography. This method injected filtered air directly into the lateral ventricles to better take pictures of the ventricle systems of the brain. Thanks to local anesthetics, this was not a painful procedure, but it was significantly risky. Hemorrhage, severe infection, and extreme changes in intrarenal pressure were all threats to the procedure. Despite this, Dandy did not stop there. In 1919, he proceeded to discover Encephalography, a medical procedure used to record the brain's electrical activity. This method involved attaching sensors to the brain that detect and measure the brain's electrical signals. These signals are then translated into a visual, showing the brain's activity patterns. With these early advances, neuroimaging was beginning to be used to diagnose conditions such as epilepsy, brain injuries, and sleep disorders. Providing invaluable information about brain function that would one day be added upon during the devolvement of modern neuroimaging.

Cerebral Angiography

Cerebral angiogram showing a transverse projection of the vertebrobasilar and posterior cerebral circulation.

Introduced in 1927, cerebral angiography enabled doctors to accurately detect and diagnose anomalies in the brain such as tumors and internal carotid artery occlusions. Over the course of a year, Egas Moniz, the inventor of cerebral angiography, ran experiments with various dye solution percentages that were injected into arteries to help better visualize the blood vessels in the brain before discovering that a solution consisting of 25% sodium iodide was the safest for patients, as well as the most effective in the visualization of blood vessels and arteries within the brain.

PET/SPECT Scans

Full body PET scan of an adult female.

A positron emission tomography, or PET scan, is a scan that shows areas of high activity in the body. The way it works is that a patient is first given a radioactive substance (called a tracer) via an injection in the hand or arm. The tracer then circulates through the body and attaches to a specific substance that the organ or tissue produces during metabolism, such as glucose. As a result, positrons are created, and those positrons are scanned by the PET camera. After they are scanned, a computer produces either a 2D or 3D image of the activity occurring within the organ or tissue. The idea for the PET scan was originally proposed by William Sweet in the 1950's, but the first full-body PET scanner wasn't actually developed until 1974 by Michael Phelp.

Similarly, the single-photon emission computed tomography scan, or SPECT scan, also works by scanning a tracer within the patient. The difference, however, is that the SPECT directly scans the gamma rays from where the tracer attaches rather than the positrons that the PET scans. As a result, the images that the SPECT scan creates are not as clear as the images produced by a PET scan, but it's typically a cheaper procedure to undertake. SPECT was developed by David Kuhl in the 1950's. Kuhl also helped set the foundation that would lead to the PET scan.

Magnetoencephalography

MEG device with patient.

Magnetoencephalography (MEG) is a technique that looks for regions of activity in the brain by detecting large groups of electrically charged ions moving through cells. It was originally developed by physicist David Cohen in the early 1970's as a noninvasive procedure. In order to be noninvasive, the MEG was designed like a giant helmet that the patient would put their head inside of and, once turned on, would read the electromagnetic pulses coming from their brain. Later on, in 1972, Cohen invented the SQUID (superconducting quantum interference device), which gave the MEG the ability to detect extremely small changes in ions and magnetic fields in the brain.   

Xenon CT Scanning

Godfrey Hounsfield, inventor of first CT scanner

Xenon computed tomography is a modern scanning technique that reveals the flow of blood to the areas of the brain. The scan tests for consistent and sufficient blood flow to all areas of the brain by having patients breathe in xenon gas, a contrast agent, to show the areas of high and low blood flow. Although many trial scans and tests were ran during the development process of computed tomography, British biomedical engineer Godfrey Hounsfield is the founder of the technique and invented the first CT scanner in 1967, which he won a Nobel Prize for in 1979. However, the adoption of the scanners in the United States didn't occur until six years later in 1973. Regardless, the CT scanner was already gaining a notable reputation and popularity beforehand.

Magnetic resonance imaging

Shortly after the initial development of CT, magnetic resonance imaging (MRI or MR scanning) was developed. Rather than using ionizing or X-radiation, MRI uses the variation in signals produced by protons in the body when the head is placed in a strong magnetic field. Associated with early application of the basic technique to the human body are the names of Jackson (in 1968), Damadian (in 1972), and Abe and Paul Lauterbur (in 1973). Lauterbur and Sir Peter Mansfield were awarded the 2003 Nobel Prize in Physiology or Medicine for their discoveries concerning MRI. At first, structural imaging benefited more than functional imaging from the introduction of MRI. During the 1980s a veritable explosion of technical refinements and diagnostic MR applications took place, enabling even neurological tyros to diagnose brain pathology that would have been elusive or incapable of demonstration in a living person only a decade or two earlier.

Blood–brain barrier

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Blood%E2%80%93brain_barrier

 

Blood–brain barrier
Solute permeability at the BBB vs. choroid plexus

The blood–brain barrier (BBB) is a highly selective semipermeable border of endothelial cells that regulates the transfer of solutes and chemicals between the circulatory system and the central nervous system, thus protecting the brain from harmful or unwanted substances in the blood. The blood–brain barrier is formed by endothelial cells of the capillary wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules such as glucose and amino acids that are crucial to neural function.

The blood–brain barrier restricts the passage of pathogens, the diffusion of solutes in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O₂, CO₂, hormones) and small non-polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins. The barrier also restricts the passage of peripheral immune factors, like signaling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to peripheral immune events.

Specialized brain structures participating in sensory and secretory integration within brain neural circuits—the circumventricular organs and choroid plexus—have in contrast highly permeable capillaries.

Structure

Part of a network of capillaries supplying brain cells

The astrocytes type 1 surrounding capillaries in the brain

Sketch showing constitution of blood vessels inside the brain

The BBB results from the selectivity of the tight junctions between the endothelial cells of brain capillaries, restricting the passage of solutes. At the interface between blood and the brain, endothelial cells are adjoined continuously by these tight junctions, which are composed of smaller subunits of transmembrane proteins, such as occludin, claudins (such as Claudin-5), junctional adhesion molecule (such as JAM-A). Each of these tight junction proteins is stabilized to the endothelial cell membrane by another protein complex that includes scaffolding proteins such as tight junction protein 1 (ZO1) and associated proteins.

The BBB is composed of endothelial cells restricting passage of substances from the blood more selectively than endothelial cells of capillaries elsewhere in the body. Astrocyte cell projections called astrocytic feet (also known as "glia limitans") surround the endothelial cells of the BBB, providing biochemical support to those cells. The BBB is distinct from the quite similar blood-cerebrospinal fluid barrier, which is a function of the choroidal cells of the choroid plexus, and from the blood-retinal barrier, which can be considered a part of the whole realm of such barriers.

Not all vessels in the human brain exhibit BBB properties. Some examples of this include the circumventricular organs, the roof of the third and fourth ventricles, capillaries in the pineal gland on the roof of the diencephalon and the pineal gland. The pineal gland secretes the hormone melatonin "directly into the systemic circulation", thus melatonin is not affected by the blood–brain barrier.

Development

The BBB appears to be functional by the time of birth. P-glycoprotein, a transporter, exists already in the embryonal endothelium.

Measurement of brain uptake of various blood-borne solutes showed that newborn endothelial cells were functionally similar to those in adults, indicating that a selective BBB is operative at birth.

In mice, Claudin-5 loss during development is lethal and results in size-selective loosening of the BBB.

Function

See also: Neuroimmune system

The blood–brain barrier acts effectively to protect brain tissue from circulating pathogens and other potentially toxic substances. Accordingly, blood-borne infections of the brain are rare. Infections of the brain that do occur are often difficult to treat. Antibodies are too large to cross the blood–brain barrier, and only certain antibiotics are able to pass. In some cases, a drug has to be administered directly into the cerebrospinal fluid where it can enter the brain by crossing the blood-cerebrospinal fluid barrier.

Circumventricular organs

Main article: Circumventricular organs

Circumventricular organs (CVOs) are individual structures located adjacent to the fourth ventricle or third ventricle in the brain, and are characterized by dense capillary beds with permeable endothelial cells unlike those of the blood–brain barrier. Included among CVOs having highly permeable capillaries are the area postrema, subfornical organ, vascular organ of the lamina terminalis, median eminence, pineal gland, and three lobes of the pituitary gland.

Permeable capillaries of the sensory CVOs (area postrema, subfornical organ, vascular organ of the lamina terminalis) enable rapid detection of circulating signals in systemic blood, while those of the secretory CVOs (median eminence, pineal gland, pituitary lobes) facilitate transport of brain-derived signals into the circulating blood. Consequently, the CVO permeable capillaries are the point of bidirectional blood–brain communication for neuroendocrine function.

Specialized permeable zones

The border zones between brain tissue "behind" the blood–brain barrier and zones "open" to blood signals in certain CVOs contain specialized hybrid capillaries that are leakier than typical brain capillaries, but not as permeable as CVO capillaries. Such zones exist at the border of the area postrema—nucleus tractus solitarii (NTS), and median eminence—hypothalamic arcuate nucleus. These zones appear to function as rapid transit regions for brain structures involved in diverse neural circuits—like the NTS and arcuate nucleus—to receive blood signals which are then transmitted into neural output. The permeable capillary zone shared between the median eminence and hypothalamic arcuate nucleus is augmented by wide pericapillary spaces, facilitating bidirectional flow of solutes between the two structures, and indicating that the median eminence is not only a secretory organ, but may also be a sensory organ.

Therapeutic research

As a drug target

The blood–brain barrier is formed by the brain capillary endothelium and excludes from the brain 100% of large-molecule neurotherapeutics and more than 98% of all small-molecule drugs. Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of most brain disorders. In its neuroprotective role, the blood–brain barrier functions to hinder the delivery of many potentially important diagnostic and therapeutic agents to the brain. Therapeutic molecules and antibodies that might otherwise be effective in diagnosis and therapy do not cross the BBB in adequate amounts to be clinically effective. The BBB represents an obstacle to some drugs reaching the brain, thus to overcome this barrier some peptides able to naturally cross the BBB have been widely investigated as a drug delivery system.

Mechanisms for drug targeting in the brain involve going either "through" or "behind" the BBB. Modalities for drug delivery to the brain in unit doses through the BBB entail its disruption by osmotic means, or biochemically by the use of vasoactive substances, such as bradykinin, or even by localized exposure to high-intensity focused ultrasound (HIFU).

Other methods used to get through the BBB may entail the use of endogenous transport systems, including carrier-mediated transporters, such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and the blocking of active efflux transporters such as p-glycoprotein. Some studies have shown that vectors targeting BBB transporters, such as the transferrin receptor, have been found to remain entrapped in brain endothelial cells of capillaries, instead of being ferried across the BBB into the targeted area.

Nanoparticles

Main article: Nanoparticles for drug delivery to the brain

Nanotechnology is under preliminary research for its potential to facilitate the transfer of drugs across the BBB. Capillary endothelial cells and associated pericytes may be abnormal in tumors and the blood–brain barrier may not always be intact in brain tumors. Other factors, such as astrocytes, may contribute to the resistance of brain tumors to therapy using nanoparticles. Fat soluble molecules less than 400 daltons in mass can freely diffuse past the BBB through lipid mediated passive diffusion.

Damage in injury and disease

The blood–brain barrier may become damaged in select neurological diseases, as indicated by neuroimaging studies of Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy, ischemic stroke, and brain trauma, and in systemic diseases, such as liver failure. Effects such as impaired glucose transport and endothelial degeneration may lead to metabolic dysfunction within the brain, and an increased permeability of the BBB to proinflammatory factors, potentially allowing antibiotics and phagocytes to move across the BBB.

Prediction

There have been many attempts to correlate the experimental blood–brain barrier permeability with physicochemical properties. In 1988, the first QSAR study of brain–blood distribution conducted reported the in vivo values in rats for a large number of H2 receptor histamine agonists.

The first papers modelling blood-brain barrier permeability identified three properties, i.e., molecular volume, lipophilicity, and hydrogen bonding potential, as contributing to solute transport through the blood-brain barrier. A 2022 dataset selected different classification models based on molecular fingerprints, MACCS166 keys and molecular descriptors.

History

A 1898 study observed that low-concentration "bile salts" failed to affect behavior when injected into the blood of animals. Thus, in theory, the salts failed to enter the brain.

Two years later, Max Lewandowsky may have been the first to coin the term "blood–brain barrier" in 1900, referring to the hypothesized semipermeable membrane. There is some debate over the creation of the term blood–brain barrier as it is often attributed to Lewandowsky, but it does not appear in his papers. The creator of the term may have been Lina Stern. Stern was a Russian scientist who published her work in Russian and French. Due to the language barrier between her publications and English-speaking scientists, this could have made her work a lesser-known origin of the term.

All the while, bacteriologist Paul Ehrlich was studying staining, a procedure that is used in many microscopy studies to make fine biological structures visible using chemical dyes. As Ehrlich injected some of these dyes (notably the aniline dyes that were then widely used), the dye stained all of the organs of some kinds of animals except for their brains. At that time, Ehrlich attributed this lack of staining to the brain simply not picking up as much of the dye.

However, in a later experiment in 1913, Edwin Goldmann (one of Ehrlich's students) injected the dye directly into the cerebrospinal fluid of animal brains. He found then the brains did become dyed, but the rest of the body did not, demonstrating the existence of a compartmentalization between the two. At that time, it was thought that the blood vessels themselves were responsible for the barrier, since no obvious membrane could be found.

Drug delivery to the brain

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Drug_delivery_to_the_brain

Drug delivery to the brain is the process of passing therapeutically active molecules across the blood–brain barrier into the brain. This is a complex process that must take into account the complex anatomy of the brain as well as the restrictions imposed by the special junctions of the blood–brain barrier.

Anatomy

The blood–brain barrier is formed by special tight junctions between endothelial cells lining brain blood vessels. Blood vessels of all tissues contain this monolayer of endothelial cells, however only brain endothelial cells have tight junctions preventing passive diffusion of most substances into the brain tissue. The structure of these tight junctions was first determined in the 1960s by Tom Reese, Morris Kranovsky, and Milton Brightman. Furthermore, astrocytic "end feet", the terminal regions of the astrocytic processes, surround the outside of brain capillary endothelial cells". The astrocytes are glial cells restricted to the brain and spinal cord and help maintain blood-brain barrier properties in brain endothelial cells.

Physiology

The main function of the blood–brain barrier is to protect the brain and keep it isolated from harmful toxins that are potentially in the blood stream. It accomplishes this because of its structure, as is usual in the body that structure defines its function. The tight junctions between the endothelial cells prevent large molecules as well as many ions from passing between the junction spaces. This forces molecules to go through the endothelial cells in order to enter the brain tissue, meaning that they must pass through the cell membranes of the endothelial cells. Because of this, the only molecules that are easily able to transverse the blood–brain barrier are ones that are very lipid-soluble. These are not the only molecules that can transverse the blood–brain barrier; glucose, oxygen and carbon dioxide are not lipid-soluble but are actively transported across the barrier, to support normal cellular function of the brain. The fact that molecules have to fully transverse the endothelial cells makes them a perfect barricade to unspecified particles from entering the brain, working to protect the brain at all costs. Also, because most molecules are transported across the barrier, it does a very effective job of maintaining homeostasis for the most vital organ of the human body.

Drug delivery to the blood–brain barrier

Because of the difficulty for drugs to pass through the blood–brain barrier, a study was conducted to determine the factors that influence a compound’s ability to transverse the blood–brain barrier. In this study, they examined several different factors to investigate diffusion across the blood–brain barrier. They used lipophilicity, Gibbs Adsorption Isotherm, a Co CMC Plot, and the surface area of the drug to water and air. They began by looking at compounds whose blood–brain permeability was known and labeled them either CNS+ or CNS- for compounds that easily transverse the barrier and those that did not. They then set out to analyze the above factors to determine what is necessary to transverse the blood–brain barrier. What they found was a little surprising; lipophilicity is not the leading characteristic for a drug to pass through the barrier. This is surprising because one would think that the most effective way to make a drug move through a lipophilic barrier is to increase its lipophilicity, it turns out that it is a complex function of all of these characteristics that makes a drug able to pass through the blood–brain barrier. The study found that barrier permittivity is "based on the measurement of the surface activity and as such takes into account the molecular properties of both hydrophobic and charged residues of the molecule of interest." They found that there is not a simple answer to what compounds transverse the blood–brain barrier and what does not. Rather, it is based on the complex analysis of the surface activity of the molecule as well as relative size.

Problems faced in drug delivery

Other problems persist besides just simply getting through the blood–brain barrier. The first of these is that a lot of times, even if a compound transverses the barrier, it does not do it in a way that the drug is in a therapeutically relevant concentration. This can have many causes, the most simple being that the way the drug was produced only allows a small amount to pass through the barrier. Another cause of this would be the binding to other proteins in the body rendering the drug ineffective to either be therapeutically active or able to pass through the barrier with the adhered protein. Another problem that must be accounted for is the presence of enzymes in the brain tissue that could render the drug inactive. The drug may be able to pass through the membrane fine, but will be deconstructed once it is inside the brain tissue rendering it useless. All of these are problems that must be addressed and accounted for in trying to deliver effective drug solutions to the brain tissue.

Possible solutions

Exosomes to deliver treatments across the blood–brain barrier

A group from the University of Oxford led by Prof. Matthew Wood claims that exosomes can cross the blood–brain barrier and deliver siRNAs, antisense oligonucleotides, chemotherapeutic agents and proteins specifically to neurons after inject them systemically (in blood). Because these exosomes are able to cross the blood–brain barrier, this protocol could solve the issue of poor delivery of medications to the central nervous system and cure Alzheimer's, Parkinson's Disease and brain cancer, among other diseases. The laboratory has been recently awarded a major new €30 million project leading experts from 14 academic institutions, two biotechnology companies and seven pharmaceutical companies to translate the concept to the clinic.

Pro-drugs

This is the process of disguising medically active molecules with lipophilic molecules that allow it to better sneak through the blood–brain barrier. Drugs can be disguised using more lipophilic elements or structures. This form of the drug will be inactive because of the lipophilic molecules but then would be activated, by either enzyme degradation or some other mechanism for removal of the lipophilic disguise to release the drug into its active form. There are still some major drawbacks to these pro-drugs. The first of which is that the pro-drug may be able to pass through the barrier and then also re-pass through the barrier without ever releasing the drug in its active form. The second is the sheer size of these types of molecules makes it still difficult to pass through the blood–brain barrier.

Peptide masking

Similar to the idea of pro-drugs, another way of masking the drugs chemical composition is by masking a peptide’s characteristics by combining with other molecular groups that are more likely to pass through the blood–brain barrier. An example of this is using a cholesteryl molecule instead of cholesterol that serves to conceal the water soluble characteristics of the drug. This type of masking as well as aiding in traversing the blood–brain barrier. It also can work to mask the drug peptide from peptide-degrading enzymes in the brain Also a "targetor" molecule could be attached to the drug that helps it pass through the barrier and then once inside the brain, is degraded in such a way that the drug cannot pass back through the brain. Once the drug cannot pass back through the barrier the drug can be concentrated and made effective for therapeutic use. However drawbacks to this exist as well. Once the drug is in the brain there is a point where it needs to be degraded to prevent overdose to the brain tissue. Also if the drug cannot pass back through the blood–brain barrier, it compounds the issues of dosage and intense monitoring would be required. For this to be effective there must be a mechanism for the removal of the active form of the drug from the brain tissue.

Receptor-mediated permabilitizers

These are drug compounds that increase the permeability of the blood–brain barrier. By decreasing the restrictiveness of the barrier, it is much easier to get a molecule to pass through it. These drugs increase the permeability of the blood–brain barrier temporarily by increasing the osmotic pressure in the blood which loosens the tight junctions between the endothelial cells. By loosening the tight junctions normal injection of drugs through an [IV] can take place and be effective to enter the brain. This must be done in a very controlled environment because of the risk associated with these drugs. Firstly, the brain can be flooded with molecules that are floating through the blood stream that are usually blocked by the barrier. Secondly, when the tight junctions loosen, the homeostasis of the brain can also be thrown off which can result in seizures and the compromised function of the brain.

Nanoparticles

The most promising drug delivery system is using nanoparticle delivery systems, these are systems where the drug is bound to a nanoparticle capable of traversing the blood–brain barrier. The most promising compound for the nanoparticles is Human Serum Albumin (HSA). The main benefits of this is that particles made of HSA are well tolerated without serious side effects as well as the albumin functional groups can be utilized for surface modification that allows for specific cell uptake. These nanoparticles have been shown to transverse the blood–brain barrier carrying host drugs. To enhance the effectiveness of nanoparticles, scientists are attempting to coat the nanoparticles to make them more effective to cross the blood–brain barrier. Studies have shown that "the overcoating of the [nanoparticles] with polysorbate 80 yielded doxorubicin concentrations in the brain of up to 6 μg/g after i.v. injection of 5 mg/kg" as compared to no detectable increase in an injection of the drug alone or the uncoated nanoparticle. This is very new science and technology so the real effectiveness of this process has not been fully understood. However young the research is, the results are promising pointing to nanotechnology as the way forward in treating a variety of brain diseases.

Loaded microbubble-enhanced focused ultrasound

Microbubbles are small "bubbles" of mono-lipids that are able to pass through the blood–brain barrier. They form a lipophilic bubble that can easily move through the barrier. One barrier to this however is that these microbubbles are rather large, which prevents their diffusion into the brain. This is counteracted by a focused ultrasound. The ultrasound increases the permeability of the blood–brain barrier by causing interference in the tight junctions in localized areas. This combined with the microbubbles allows for a very specific area of diffusion for the microbubbles, because they can only diffuse where the ultrasound is disrupting the barrier. The hypothesis and usefulness of these is the possibility of loading a microbubble with an active drug to diffuse through the barrier and target a specific area. There are several important factors in making this a viable solution for drug delivery. The first is that the loaded microbubble must not be substantially greater than the unloaded bubble. This ensures that the diffusion will be similar and the ultrasound disruption will be enough to induce diffusion. A second factor that must be determined is the stability of the loaded micro-bubble. This means is the drug fully retained in the bubble or is there leakage. Lastly, it must be determined how the drug is to be released from the microbubble once it passes through the blood–brain barrier. Studies have shown the effectiveness of this method for getting drugs to specific sites in the brain in animal models.