A Smart Drug Approach Biology Essay
Keywords:BloodBrainBarrier, Nanoparticles, Targeteddrugdelivery
Drug delivery to the brain is a challenge, because the braintissue benefits from a very efficient and effectiveprotective barrier. This mechanism protects the brain from foreign substances, undesired materials as well as the entry of 95% of potentially activetherapeutic moieties. The Blood Brain Barrier (BBB) is the major barrier to the passage of active molecules from the outer compartment to the brain. It islocated at the level of the brain capillaries, where there is a convergence or similarity of different type of cells, like endothelial cells, pericytes, astrocytes and microglia. The brain micro vessel endothelial cells that form the BBB, display important morphological characters such as the presence of tight junction between the cells, the absence of fenestrations or arrangements of inlet or outlet and diminished pinocytic activity, that together help to restrict the passage of compounds from the blood into the extracellular environment of the brain.
This barrier results from the selectivity of the tight junctions between endothelial cells in CNS vessels that blocksthe passage of solutes passing through. At the interface between blood and the brain, endothelial cells are attachedtogether by these tight junctions, which are composed of very smaller subunits, frequently biochemical dimers, that are transmembrane proteins such as occludin, claudins, junctional adhesion molecule (JAM).
The blood brain barrier is composed of high-density cells restricting or blocking passage of substances and solutes from the bloodstream much more than endothelial cells in capillaries elsewhere in the body. Astrocytecellprojections called astrocytic feet (also known as "glia limitans") surround the endothelial cells of the BBB, providing biochemical support to those cells.The connection between these glial and endothelial cells is not very well known, but it may express an influence of the astrocytes on the formation and the maintenance of the Blood Brain Barrier(1).The function of the BBB is dynamically regulated by various cells present at the level of the BBB (2). It is distinct from the quite resembling 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.
CNS drug delivery
To date, delivery of therapeutic molecules into the brain often involves highly invasive techniques (like drilling a hole in the skull). The utter scarcity of techniques for brain-specific delivery of erapeutic molecules using non-invasive approaches has led researchers to increasingly explore the vast potential of nanotechnology toward the diagnosis and treatment of diseases/disorders incurable with present techniques.
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. The function is dynamically regulated by various cells present at the level of BBB (3).
Mechanisms for drug targeting in the brain involve going either "through" or "behind" the BBB. Modalities for drug delivery/Dosage form through the BBB entail its disruption by osmotic means; 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. Methods for drug delivery behind the BBB include intracerebral implantation (such as with needles) and convection-enhanced distribution. Mannitol can be used in bypassing the BBB.It is now well established that the BBB is a unique membranous that tightly segregates the brain from the circulating blood (4, 5).
Peptides are able to cross the blood-brain barrier (BBB) through various mechanisms, opening new diagnostic and therapeutic avenues. Tight epithelium, similar in nature tothis barrier, is also found in other organs (skin, bladder,colon, and lung) (6). However, their BBB transport data are scattered in the literature over different disciplines, using different methodologies reporting different influx or efflux aspects. Therefore, a comprehensive BBB peptide database (Brainpeps) was constructed to collect the BBB data available in the literature. Brainpeps currently contains BBB transport information with positive as well as negative results. The database is a useful tool to prioritize peptide choices for evaluating different BBB responses or studying quantitative structure-property (BBB behavior) relationships of peptides. Because a multitude of methods have been used to assess the BBB behavior of compounds.The choroid plexus may beof importance when considering the transport of peptidedrugs, because it is the major site of cerebrospinal-fluid(CSF) production, and both the CSF and brain ECF freely exchange (7).
The BBB also has an additional enzymatic aspect. Solutescrossing the cell membrane are subsequently exposed todegrading enzymes present in large numbers inside theendothelial cells that contain large densities of mitochondria, metabolically highly active organelles. BBB enzymesalso recognize and rapidly degrade most peptides, including naturally occurring neuropeptides (8, 9).
Nanotechnology may also help in the transfer of drugs across the BBB. Recently, researchers have been trying to build liposomes loaded with nanoparticles to gain access through the BBB. More research is needed to determine which strategies will be most effective and how they can be improved for patients with brain tumors. The potential for using BBB opening to target specific agents to brain tumors has just begun to be explored.
Delivering drugs across the blood–brain barrier is one of the most promising applications of nanotechnology in clinical neuroscience. Nanoparticles could potentially carry out multiple tasks in a predefined sequence, which is very important in the delivery of drugs across the blood–brain barrier.
A significant amount of research in this area has been spent exploring methods of nanoparticle-mediated delivery of antineoplastic drugs to tumors in the central nervous system. For example, radiolabeled polyethylene glycol coated hexadecylcyanoacrylate nanospheres targeted and accumulated in a rat gliosarcoma. However, this method is not yet ready for clinical trials, due to the accumulation of the nanospheres in surrounding healthy tissue.
It should be noted that vascular endothelial cells and associated pericytes are often abnormal in tumors and that the blood–brain barrier may not always be intact in brain tumors. Also, the basement membrane is sometimes incomplete. Other factors, such as astrocytes, may contribute to the resistance of brain tumors to therapy.
Of over 7000 drugs in the comprehensive medicinal chemistry (CMC) database drugs treat the CNS, and these CNS active drugs only treat depression, insomnia and schizophrenia. The average molecular mass of CNS active drug is 357 Dalton. Studies show that only 12% of the drugs were active in the CNS, but only 1% of all the drugs were active in the CNS disease other than effective disorders.
Blood Brain barrier
The blood–brain barrier (BBB) is meant to protect the brain from noxious agents; however, it also significantly hinders the delivery of therapeutics to the brain. Several strategies have been employed to deliver drugs across this barrier and some of these may do structural damage to the BBB by forcibly opening it to allow the uncontrolled passage of drugs. The ideal method for transporting drugs across the BBB should be controlled and should not damage the barrier. Among the various approaches that are available, nano-biotechnology based delivery methods provide the best prospects for achieving this ideal.Three cellular elements of the brain microvasculature compose the BBB-endothelial cells, astrocyte end-feet, and pericytes (PCs). Tight junctions (TJs), present between the cerebral endothelial cells, form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain. Astrocytic end-feet tightly ensheath the vessel wall and appear to be critical for the induction and maintenance of the TJ barrier, but astrocytes are not believed to have a barrier function in the mammalian brain. Dysfunction of the BBB, for example, impairment of the TJ seal, complicates a number of neurologic diseases including stroke and neuro-inflammatory disorders.
The BBB is further reinforced by a high concentration of P-glycoprotein (Pgp), active –drug-efflux-transporter protein in the luminal membranes of the cerebralcapillary endothelium. This efflux transporter activelyremoves a broad range of drug molecules from the endothelial cell cytoplasm before they cross into the brain parenchyma.
The second barrier that a systemically administered drugencounters before entering the CNS is known as theblood-cerebrospinal fluid barrier (BCB). Since the CSFcan exchange molecules with the interstitial fluid of thebrain parenchyma, the passage of blood-borne moleculesinto the CSF is also carefully regulated by the BCB. Physiologically, the BCB is found in the epithelium of the choroids plexus, which are arranged in a manner that limits thepassage of molecules and cells into the CSF. The choroidplexus and the arachnoid membrane act together at thebarriers between the blood and CSF. On the external surface of the brain the ependymal cells fold over onto themselves to form a double layered structure, which liesbetween the dura and pia, this is called the arachnoidmembrane.Within the double layer is the subarachnoidspace, which participates in CSF drainage. Passage of substances from the blood through the arachnoid membraneis prevented by tight junctions (10).
Limitations for drug delivery
Drug may be transported through BBB by active or passive transport.
Passive transport is the movement of molecules through a permeable membrane without expending chemical energy. It plays a key role in a number of biological processes by allowing the body to move nutrients and waste materials in and out of cells without having to use energy to do so. Diffusion, osmosis, and facilitated diffusion are examples of passive transport.
Diffusion reflects the tendency for molecules to spread out, if they have room to do so. In a classic example, oxygen molecules flow from the oxygen-rich environment outside cells to the oxygen-poor environment inside, diffusing across the cell membrane. Diffusion allows molecules to move from areas where their numbers are high to locations where their numbers are low. Cells do not have to expend any energy to move the molecules, as they push through the membrane on their own (11, 12).
In facilitated diffusion, structures in the cell membrane help the process along. Some molecules may not naturally fit through the membrane. They could travel down an ion channel, a structure in the membrane of the cell that allows larger molecules and ions to pass through. They may also connect to carrier proteins. These proteins lock on and act like keys to open the cell membrane and allow a molecule to enter.
Regarding the passive transfer through BBB, the main factor that intervene are ionization of drugs, molecular weight, lipophilicity and protein binding. The transportation through BBB is decreased in case of ionization of acidic compounds. Ionization of basic compounds has got no significant effects. Drug transport via BBB is inversely proportional to its molecular weight. It has been demonstrated that molecular weight may be a limiting factor for values >600 Dalton. Lipophilicity would probably be the most important factors conditioning brain uptake of drugs. Transport of a drug through BBB is generally, directly proportional to its lipophilicity. However, too much high values of lipophilicity can decrease the rate of transport due to trapping of the compound inside the membrane.
Another important factor involved in the drug transport through BBB is protein binding. Logically, due to the size of protein-drug complex and the characteristics of the BBB, only the free fraction of the drug is transported. Different hypotheses have been proposed such as the dissociation of protein drug complex in the microcirculation following the binding of the protein to a specific endothelial receptor.
Active transport is the energy-demanding transfer of a substance across a cell membrane against itsconcentration gradient, i.e., from lower concentration to higher concentration.Special proteins within the cell membrane act as specific protein ‘carriers’.The energy for active transport comes from ATP generated by respiration (in mitochondria).active transport include the uptake of glucose in the intestines in humans and the uptake of mineral ions into root hair cells of plants(13).
Symport uses the downhill movement of one solute species from high to low concentration to move another molecule uphill from low concentration to high concentration (against itselectrochemical gradient).An example is the glucose symporter SGLT1, which co-transportsone glucose (or galactose) molecule into the cell for every two sodium ions it imports into the cell.This symporter is located in the small intestines, trachea, heart, brain, testis, and prostate. It is also located in the S3 segment of the proximaltubule in each nephron in the kidneys (14). Its mechanism is exploited in glucose rehydration therapy and defects in SGLT1 prevent effective reabsorption of glucose, causing familial renal glucosuria (15).
For some drugs, transport through the BBB is higher or lower than that expected from the chemical and physical properties. A facilitated or an active transport can be responsible for a higher rate of transfer. In case of lower rate of transfer, efflux protein may be involved.
Strategies forenhanced CNS drugs.
The diffusion of drugs from the blood into the brain counts primarily upon the ability of the biologically active molecules to cross lipid membranes. Several drugs do not have adequate physicochemical characteristics such as high lipid solubility, low molecular size and positive charge which are essential to succeed traversing BBB. This is the rationale behind developing several strategies to overcome the BBB including invasive and non-invasive techniques.
Disruption of BBB :Despite recent developments for enhanced CNS penetration, the BBB remains a formidable obstacle that compromises successful treatment of many neurological disorders, The second invasive strategy for enhanced CNS drug delivery involves the systemic administration of drugs in the conjunction with transient BBB disruption (BBBD). Theoretically, with the BBB weakened, systematically administered drugs can undergo enhanced extravasation rates in the cerebral endothelium, leading to increased parenchymal drug concentrations. A variety of techniques that transiently disrupt the BBB have been investigated; however, albeit physiologically interesting, many are unacceptably toxic and therefore not clinically useful. These include the infusion of solvents such as dimethyl sulfoxide or ethanol and metals such as aluminum; X-irradiation and the induction of pathological conditions including hypertension, hypercapnia, hypoxia or ischemia. The mechanisms responsible for BBBD with some of these techniques are not well understood. The BBB can also be compromised by the systemic administration of several antineoplastic agents including VP-16, cisplatin, hydroxyl urea, fluorouracil and etoposide.
Direct drug delivery: To overcome the BBB in clinical trial the drugs were administered directly by intraventricular and intracerebral routes, using a plastic reservoir implanted subcutaneously in the scalp and connected to the ventricles within brain by an outlet catheter. Unfortunately there are several problems like insufficient concentration of drug may reach the target site, secreted interstitial fluid flow works against diffusive drug penetration, the high turnover rate of the CSF continuously clears injected drug back into the blood apart from surgical intervention required. In practice, drug injection into the CSF is a suitable strategy for sites close to the ventricles only. For drugs that need to be at elevated levels for long periods for an effective action, continuous or pulsatile infusion may be necessary.
Although, the ease and compliance of non-invasivedelivery methods is often not associated with direct orinvasive delivery of drugs to the brain, it often shows up asthe sole alternative wherein the drugs elicit right physicochemical properties. Generally, only low molecularweight, lipid-soluble molecules and a few peptides andnutrients can cross this barrier to any significant extent,either by passive diffusion or using specific transport mechanisms (16). So, for most drugs it is not possible to achievetherapeutic levels within the brain tissue followingintravenous or oral administration. In addition, highly potentdrugs (e.g., anticancer drugs and neurotrophic factors) thatmay be necessary to be delivered to the CNS, often causeserious toxic side effects when administered systemically.The drug can be administered directly into the brain tissue (17). Many ways are explored for direct intracranial drug delivered by intracerebroventricular, intracerebral or intrathecal administration after creating holes in head orDisrupting the BBB integrity by osmotic blood brain barrierdisruption or biochemical BBB disruption and also byemploying controlled release biodegradable drug deliverysystems which are able to control the release rate of anincorporated drug in a pre-determined manner over periodsof days to months (18- 24).
Non –invasive techniques may be of chemical or biological nature.
Chemical method- The prodrug approach was involved in chemical methods, to improve some deficient physicochemical property, such as membrane permeability or solubility. For example esterification or amidation of hydroxy-, amino- or carboxylic acid containing drugs, may enhance lipid solubility and enter into brain easily. The conversion into active form is realized generally, by an enzymatic cleavage.
A possible choice of CNS prodrug is to link the drugs to a lipid moiety, such as fatty acid, glyceride or a phospholipid. Such prodrug approaches were investigated for an array of acid containing drugs like, levodopa. In order to target the diseased site and to release the active compounds in this environment. Exploiting the pH or an enzymatic process may be theoretically possible, but in the pathological conditions there are some modifications.
The other problems involved with the prodrug are poor selectivity and poor tissue retention of some of these molecules. Increase lipophilicity also tends to increase the rate of oxidative metabolism by cytochrome P450 and other enzymes. The increased lipophilicity may improve diffusion movement across the BBB, causing increased uptake into other tissues and increasing the tissue burden.
Biological method: Biological approaches involve the conjugation of the drug with antibodies which can then be directed towards an antigen residing on or within the target tissues. Antibodies to the transferrin receptors (TfR) like the OX26 antibody, the 8D3 MAb or the R17-217 MAb were able to undergo receptor mediated transcytosis across the mouse BBB via the endogenous TfR. It is difficult to find target tissues unfortunately, bearing specific antigens that will provide a distinct targeting effect. For example, in cancer chemotherapy tumor specific antigens are rare tumor-associated antigens can be present not only within the target tissue but elsewhere in the body.
A basic definition:
Nanotechnology is the engineering of functional systems at the molecular scale. This covers both current work and concepts that are more advanced.
The incidence of neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), multiplesclerosis (MS), and primary brain tumors is increasingspecifically in the ageing population (25). Blood-brain barrier (BBB) presents an impediment for their treatment and diagnosis (26). The BBB is readily permeable onlyto lipophilic molecules or those of a molecular weight below400–600 Da (27).
Nanobiotechnology is attracting increasing interest as a meansof overcoming the issues posed by the BBB in treating CNSdiseases(28). In particular, systems involving nanoparticles (NPs)are providing alternate means for targeted drug delivery to theCNS, as well as for novel therapeutic applications (29). Furthermore, the ability of the NPs to traverse the BBB providesincreased opportunity for the early diagnosis of CNS diseasesbefore BBB inflammation and also the opportunity to monitorand detect further disease progression (30).
In its original sense, 'nanotechnology' refers to the projected ability to construct items from the bottom up, using techniques and tools being developed today to make complete, high performance products.
The use of nanotechnology in medicine offers some exciting possibilities. Some techniques are only imagined, while others are at various stages of testing, or actually being used today. Thanks to progress in polymer chemistry and in polymeric colloids, such systemscan be almost tailor made, and many types of nanoparticulate drug carriers have been proposed (31-39).
Nanotechnology in medicine involves applications ofnanoparticles currently under development, as well as longer range research that involves the use of manufactured nano-robots to make repairs at the cellular level (sometimes referred to as Nano medicine).
The use of nanotechnology in the field of medicine could revolutionize the way we detect and treat damage to the human body and disease in the future, and many techniques only imagined a few years ago are making remarkable progress towards becoming realities.
Nanotechnology in medicine currently being developed involves employing nanoparticles to deliver drugs, heat, light or other substances to specific types of cells (such as cancer cells). Particles are engineered so that they are attracted to diseased cells, which allows direct treatment of those cells. This technique reduces damage to healthy cells in the body and allows for earlier detection of disease.
For example, nanoparticles that deliver chemotherapy drugs directly to cancer cells are under development. Tests are in progress for targeted delivery of chemotherapy drugs and their final approval for their use with cancer patients is pending. One company, CytImmune has published the preliminary results of a Phase 1 Clinical Trial of their first targeted chemotherapy drug and another company, BIND Biosciences, has published preliminary results of a Phase 1 Clinical Trial for their first targeted chemotherapy drug.
Another technique delivers chemotherapy drugs to cancer cells and also applies heat to the cell. Researchers are using gold Nano rods to which DNA strands are attached. The DNA strands act as a scaffold, holding together the Nano rod and the chemotherapy drug.
When Infrared light illuminates the cancer tumor the gold Nano rod absorbs the infrared light, turning it into heat. The heat both releases the chemotherapy drug and helps destroy the cancer cells.
Researchers are also developing a nanoparticle to defeat viruses. The nanoparticle does not actually destroy virus molecules, but delivers an enzyme that prevents the reproduction of virus molecules in the patients’ bloodstream.
Researchers are developing nanoparticles that can delivery drugs across the brain barrier to tackle neurologic disorders. Researchers are developing a method to increase the immune response generated by vaccines by attaching the vaccine molecules to a DNA nanostructure that delivers the vaccine molecules to specific cells that are key in triggering white blood cells to an immune response.
Physico chemical properties of nanoparticles :
The surface morphology of the prepared NPs was determined for by using transmission electron microscopy (TEM). The nano suspension samples were prepared by dispersing a small amount of NPs into distilled water. A drop of nano suspension was placed on a paraffin sheet and carbon coated grid was placed on sample and left for 1 min to allow CS-NPs to adhere on the carbon substrate. The remaining suspension was removed by adsorbing the drop with the corner of a piece of filter paper. Then the grid was placed on a drop of phosphotungstate for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and the sample was air dried. The sample was examined by TEM (Morgagni 268D TEM, Massachusetts, USA).The particle size, particle size distribution, Polydispersity index, and zeta potential were determined by Zetasizer Nano ZS, (Mal-vern Instruments Ltd, Worcestershire, UK). Measurements were performed using Standard laser 4 mW He–Ne, 633 nm, room temperature 25 C at fixed angle of 90. The sample volume used for the analysis was kept constant i.e., 1 ml. The instrument is equipped with appropriate software for analysis of particle size and polydispersityindex.
Determination of the loading capacity, encapsulation efficiency:
Process yield of NPs the EE and LC of NPs were determined by separation of NPs from the aqueous medium containing non-associated RHT by centrifugation at 15,000 rpm at 4 C for 45 min. The amount of free RHT in the supernatant was measured by UV spectrophotometer at 261 nm. The EE and LC of CS-RHT NPs were calculated as per equations given below with all the measurements were performed in triplicate and average.
The process yield was calculated from the weight of dried NPs recovered (W1) and the sum of the initial dry weight of starting materials (W2).
Nanoparticles in drug delivery:
Nanotechnology has provided the possibility of delivering drugs to specific cells using nanoparticles. The overall drug consumption and side-effects may be lowered significantly by depositing the active agent in the morbid region only and in no higher dose than needed. This highly selective approach would reduce costs and human suffering. An example can be found in dendrimers and nanoporous materials. Another example is to use block co-polymers, which form micelles for drug encapsulation. They could hold small drug molecules transporting them to the desired location. Another vision is based on small electromechanical systems; nano electromechanical systems are being investigated for the active release of drugs. Some potentially important applications include cancer treatment with iron nanoparticles or gold shells. A targeted or personalized medicine is intended to reduce the drug consumption and treatment expenses resulting in an overall societal benefit by reducing the costs to the public health system.
Nano medical approaches to drug delivery center on developingnano scale particles or molecules to improve drug bioavailability. Bioavailability refers to the presence of drug molecules where they are needed in the body and where they will do the most good. Drug delivery focuses on maximizing bioavailability both at specific places in the body and over a period of time. This can potentially be achieved by molecular targeting by nano engineered devices It is all about targeting the molecules and delivering drugs with cell precision. More than $65 billion are wasted each year due to poor bioavailability. In vivo imaging is another area where tools and devices are being developed. Using nanoparticle contrast agents, images such as ultrasound and MRI have a favorable distribution and improved contrast. The new methods of nano engineered materials that are being developed might be effective in treating illnesses and diseases such as cancer. What nano scientists will be able to achieve in the future is beyond current imagination. This might be accomplished by self-assembled biocompatible nano devices that will detect, evaluate, treat and report to the clinical doctor automatically.
Drug delivery systems, lipid- or polymer-based nanoparticles, can be designed to improve the pharmacological and therapeutic properties of drugs. The strength of drug delivery systems is their ability to alter the pharmacokinetics and bio distribution of the drug. When designed to avoid the body's defence mechanisms, nanoparticles have beneficial properties that can be used to improve drug delivery. Where larger particles would have been cleared from the body, cells take up these nanoparticles because of their size. Complex drug delivery mechanisms are being developed, including the ability to get drugs through cell membranes and into cell cytoplasm. Efficiency is important because many diseases depend upon processes within the cell and can only be impeded by drugs that make their way into the cell. Triggered response is one way for drug molecules to be used more efficiently. Drugs are placed in the body and only activate on encountering a particular signal. For example, a drug with poor solubility will be replaced by a drug delivery system where both hydrophilic and hydrophobic environments exist, improving the solubility. Also, a drug may cause tissue damage, but with drug delivery, regulated drug release can eliminate the problem. If a drug is cleared too quickly from the body, this could force a patient to use high doses, but with drug delivery systems clearance can be reduced by altering the pharmacokinetics of the drug. Poor bio distribution is a problem that can affect normal tissues through widespread distribution, but the particulates from drug delivery systems lower the volume of distribution and reduce the effect on non-target tissue. Potential nano drugs will work by very specific and well-understood mechanisms; one of the major impacts of nanotechnology and nano science will be in leading development of completely new drugs with more useful behavior and less side effects.
One of the major challenges in drug delivery is to get the drug at the place it is needed in the body thereby avoiding potential side effects to non-diseased organs. This is especially challenging in cancer treatment where the tumor may be localized as distinct metastases in various organs. The non-restricted (cyto)toxicity of chemotherapeutics thus limits the full use of their therapeutic potential. Local drug delivery or drug targeting results in increased local drug concentrations and provides strategies for more specific therapy. Nanoparticles have specific particles as tools to enable these strategies. These include benefits such as their small size which allows penetration of cell membranes, binding and stabilization of proteins, and lysosomal escape after endocytosis.
Liposomes as nanosized phospholipid "fatty" structures have the advantage of being small, flexible and biocompatible thus being able to pass along the smallest arterioles and endothelial fenestrations without causing clotting. Now also other materials, including various (co-)polymers and dendrimers at the nanosize range have become available to alter the distribution of encapsulated or attached drugs.NPs were obtained in accordance with the nanoprecipitation procedure (40).
Future perspectives of nano particles.
Over the past century imaging became a tool that changed theway medicine thinks and practices, mainly for diagnostic purposesand over the past 20 years for therapeutic as well. X-rays gave forthe first time the opportunity to obtain information for body’s interior, with non-invasive methods. As technology evolved in physics,engineering and computer technology, new physical principleswere exploited, resulting to new imaging tools that provided differenttypesofinformation.Startingfromsimpleplanaranatomicalinformation, three-dimensionalfunctionalinformationcanbenowobtained. in vivo information about proteins,genes, molecules, stem cells; thus mechanisms related to biological processes and diseases can be studied, with significant benefits,compared to in vitro and ex vivo studies (41-43).
Localinvasive(directinjection/infusion)deliveryhasbeenassociatedLocalinvasive(directinjection/infusion)deliveryhasbeenassociatedwithriskofinfectionhighneurosurgicalcostandalimiteddeliveryareaespecially in human brain (44). In contrast, global noninvasive strategiestaking advantage of endogenous nutrient transport systems presentingat the BBB can realize widespread transport across the whole brainwithout disruption of the barrier properties (45). Among the variousnoninvasive approaches, receptor-mediated systems seem to be one ofthe most promising ones. By coupling of vectors with specific receptorson the BBB to loading vehicles, it combines the advantages of braintargeting, high incorporation capacity, reduction of side effects, andcircumvention of the multidrug efflux system (46). OX-26 conjugated immune liposome, the most well-known receptor-mediated system, istransportedthroughthetransferrinreceptoronthe brain microvascularendothelium via receptor mediated transcytosis (RMT). It has beenextensivelyinvestigatedtodeliversmallmolecularchemicals (47) aswell as genes (48,49) which manifest the extraordinary capacity of thereceptor-mediated routes. However, the system still has its drawbacks,instability of the liposome and disparity of the effect of the monoclonalantibodiespreparedbydifferentgroupsareofconcern (50-52) .Soit stilldemands new delivery systems to better assist brain drug delivery.
Lactoferrin (Lf), a mammalian cationic iron-binding
Glycoproteinbelongingtothetransferrin(Tf)family,consistsofapolypeptidechainofabout 690 amino acids folded into two globular lobes, each of whichcontains one iron-binding site. Many physiological roles of Lf have beenindicated, such as anti-inflammatory, immune modulatory, anti-microbial, and anti-carcinogenic activity (53). Lf receptor (LfR) has beendemonstratedexistingontheBBB in differentspecies and involved in Lftransport across the BBB in vitro and in vivo (54-56). Recently, it was compared that the brain uptake of Lf with that of Tf and OX-26, and theresultsshowedthattheuptakeofLfwasmuchhigherthantheothertwo (57). These intriguing results inspired us to construct a brain deliverysystem employing the brain targeting ability of Lf.The objective of this paper is to develop a novel brain deliverysystem Lf conjugated nanoparticle (Lf–NP) and to explore thepotential of Lf to be a brain delivery vector. Poly (ethylene glycol) poly (lactide) (PEG–PLA) nanoparticles were chosen to conjugate withLf for its main advantages over liposomes which include the lownumber of excipients used in their formulations, the simple procedures for preparation, a high physical stability, and the possibility ofsustained drug release that may be suitable in the treatment of chronic diseases (58). What's more, both the PEG and the PLA arematerials approved by FDA thus will endow good safety to our system (59,60). To investigate the brain delivery property of the system, alipophilic fluorescent dye, coumarin-6, was incorporated into it as ananoparticle probe. Lf–NP loaded with coumarin-6 was quantitativelyand qualitatively evaluated using an uptake experiment by the bEnd.3cell line, a mouse brain endothelia cell line in vitro and its localizationin brain tissue sections was visualized by fluorescence microscopein vivo compared with common pegylated nanoparticles (NP). ThebEnd.3 cytotoxicity of Lf–NP was also investigated. The blood andbrain concentrations of the probe associated to Lf-NP were detectedwith a high-performance liquid chromatography (HPLC)-fluorescencedetection method, compared with those of coumarin-6 loaded NP.
Preparation and characterization of NP and Lf-NP.
Preparation of NP:
MePEG–PLA and maleimide–PEG–PLA block copolymers weresynthesized and characterized as described previously (61). PEG-PLA nanoparticles were prepared using the double emulsion andsolvent evaporation method with minor modification (62). Coumarin-6 loadednanoparticles were prepared with the same procedure except that0.1% or 0.003%(w/v) of coumarin-6 was added to the dichloromethanesolution before primaryemulsification and the obtained nanoparticleswere subjected to a 1.5×20 cm sepharose CL-4B column and elutedwith 0.05 M HEPES buffer pH 7.0 containing0.15 M NaCl to removetheunentrapped coumarin-6.
Preparation of Lf–NP:
Lf was thiolated by reaction for 60 min with a 40:1 molar excess of2-iminothiolane in 0.15 M sodium borate buffer, pH 8.0 supplementedwith 0.1 mM EDTA, as described previously (63). The product was then applied to Desalting columnand eluted with 0.01 M HEPES pH 7.0 containing 0.15 MNaCl. The protein fractions were collected and introduced thiol groupswere determined spectrophotometrically (λ=412 nm) with Ellman'sreagent (64).
Lf–NP was prepared by incubating the purified thiolated Lf with theNP at room temperature for 9 h. The products were then subjected to a1.5×20 cm sepharose CL-4B column and eluted with 0.01 M phosphatebufferedsaline(PBS)bufferpH7.4 toremovetheunconjugatedproteins.The anoparticle concentration was determined by turbidimetry usingUV2401 spectrophotometer at 350 nm. The averagenumberofLfmoleculesconjugatedpernanoparticlewasdeterminedbyaLactoferrinELISAKitandcalculatedbydividingthenumberofLfbythecalculated average number of nanoparticles using the methods described by Oliver et al. (65).
In vitro release of coumarin-6 from NP and Lf–NP:
In vitro release experiments of coumarin-6 from the nanoparticleswere performed at 37 °C in pH 4 and pH 7.4 PBS and DMEM mediumcontaining 10% FBS to evaluate if the fluorescence probe remainedassociated withthe particlesduring a 24hincubationperiod.pH4 andpH 7.4 represented the pH in the endolysosomal compartment andphysiologic pH respectively. Coumarin-6-loaded NP and Lf–NP wereincubated at a nanoparticle concentration of 7 μg/ml with a shaking at100 rpm under a predetermined sink condition. Periodic sampleswere subject to centrifugation at 21,000 g for 45 min and the supernatant was further diluted with methanol and analyzed for thereleased coumarin-6 by HPLC assay(66).
Brain distribution of coumarin-6-labeled NP and Lf–NP:
According to qualitative studies Coumarin-6-labeled Lf–NP and NP were injected into the caudalveins (dose 60 mg/kg) of mice, respectively. After 1 h, the animals wereanaesthetized and frozen brain sections of 20 μm thicknesses wereprepared after a series process (refer to S4 for details) and stained with1 μg/ml DAPI for 10 min at room temperature. After PBS washing, thesections were mounted in Dako fluorescent mounting medium andexamined under the fluorescence microscope(66).
According to quantitative studies Fifty-four mice were randomly divided into two groups, receivingcoumarin-6 loaded Lf–NP and NP nanoparticles, respectively. Theanimals were injected in tail vein with a dose of 60 mg/kgfluorescence-labeled nanoparticles. At 0.083, 0.25, 0.5, 1, 2, 4, 8, 12and 24 h following i.v. injection, the blood samples were collected andthe micewere sacrificed. The brain tissues wereharvestedfollowed byquick washing with cold saline and then subjected to n-hexaneextraction for HPLC analysis with coumarin-7 as internal standard (66).
Although the number of papers related to NPs continuouslyincreases, at the moment most of the published studies do notuse in vivo small animal imaging. One reason for this could be theresourcesnecessarytoobtainandmaintainimagingfacilities.AtthemomentevenmoreconventionalstudiesthatdonotuseNPs,donotproceed to in vivo imaging. However, as stated in this article, lowcost prototypes can provide accurate dynamic scintigraphic data (67) and in some cases even tomographic data. As they becomemore and more popular and economically affordable, it is expectedthat in the next years a number of studies that have been carriedout using radioisotopes and post-mortem biodistributions will be repeated in vivo(68).