478 13 Role of Biotechnology in Drug Delivery to the Nervous SystemHistorical Evolution of Drug Delivery for CNS DisordersLandmarks in the historical evolution of drug delivery technology to the brain areshown in Table 13.1. They are related to the history of the blood–brain barrier (BBB).Although the cerebral ventricles were tapped for hydrocephalus in ancient times,the ﬁrst perforation of subarachnoid space by lumbar puncture was made in 1885 toadminister cocaine for anesthesia (Corning 1885). Paul Ehrlich described the con-cept of the blood–brain barrier in the same year when he observed that dyes injectedinto the vascular system were rapidly taken up by all the organs except the brain(Ehrlich 1885). Later, research showed that dyes injected into the cerebrospinal ﬂuidhave free access to the neural tissues but do not enter the blood supply of the brain.Coining of the term “blood–brain barrier” to describe this phenomenon is attributedto Lewandowski in 1900. Despite the BBB, intracerebral distribution of varioussubstances was observed. “Barrière hémato-encéphalique” was deﬁned as a cerebralblood vessel compartment in which choroid plexus was semipermeable, facilitatingthe ﬂow of substances from the blood into the CSF (Stern and Gautier 1921). BBBpermeability to hexoses, amino acids, amines, and neurotransmitters was demon-strated 50 years later by radiolabeled substances (Oldendorf 1971).Broman ﬁrst observed the transient opening or disruption of the BBB after intraca-rotid arterial administration of hypertonic solutions in 1941 (Broman 1941). The ﬁrstinjections into the cerebral circulation were of contrast materials for cerebral angiog-raphy (Moniz 1927). The injection of a therapeutic substance (diazepam) into thecarotid arteries was not reported until almost a half-century later (Doppman 1973).The advent of stereotactic surgery more than 50 years ago opened the way forplacing instruments at selected targets in the depths of the brain for the treatmentof movement disorders (Spiegel et al. 1947). This approach was used some yearslater to perform chemopallidectomy by injection of a mixture of procaine and alco-hol into the globus pallidus (Cooper 1954). The techniques of creating lesions inbasal ganglia have been reﬁned, but these principles of localization and injectioncontinue to be used for the introduction of novel therapeutic agents into the brainfor the treatment of movement disorders. The ﬁrst implantable pump for intrathecaland intraventricular injection of morphine for the treatment of cancer pain wasdescribed in 1978 (Lazorthes et al. 1991). During the past 25 years, further progresshas taken place with the development of intra-arterial chemotherapy, direct injec-tions of therapeutic substances into intracranial lesions, and strategies to overcomethe BBB. Further advances have taken place with the development of cell and genetherapies as well as nanobiotechnology.The Neurovascular UnitA neurovascular unit, consisting of endothelial cells, neurons, and glia, regulates theBBB. The fact that endothelial cells of brain capillaries differ greatly from those inthe periphery confers on the BBB its discriminatory characteristics. Brain endothelialcells display tight junctions, absence of intercellular clefts and fenestrations, minor
482 13 Role of Biotechnology in Drug Delivery to the Nervous SystemReceptor-Mediated Peptide and Protein TranscytosisThe transport of peptides and proteins across cellular barriers- transcytosis- hasbeen documented in a number of systems. Examples include the transport of IgGacross the intestinal epithelium and human placenta, the transport of insulin andinsulin-like growth factors across the aortic endothelium, and the transport of epi-dermal growth factor across the kidney epithelium. It is not surprising that transcy-tosis occurs across the BBB. In addition to the unidirectional and bidirectionaltransport of small molecules, other macromolecules are able to enter the brain tis-sue from the blood by a receptor-mediated process. An example of this is the trans-port of transferrin across the BBB. Brain cells require a constant supply of iron tomaintain their function and brain may substitute its iron through transcytosis ofiron-loaded transferrin across the brain microvasculature. Other biologically activeproteins such as insulin and immunoglobulin G are actively transcytosed throughBBB endothelia. The presence of receptors involved in the transcytosis of ligandsfrom the blood to the brain offers opportunities for developing new approaches tothe delivery of therapeutic compounds across the BBB.Molecular Biology of the BBBThe molecular composition of the BBB has been studied by immunocytochemistry,and the results of these studies show that the BBB exhibits a speciﬁc collection ofstructural and metabolic properties that are also found in the tight-transporting epi-thelia. These conclusions are substantiated by the use of antibodies that recognizeproteins of nonBBB origin and BBB-speciﬁc proteins. BBB-speciﬁc immunoprobeshave a potential application for investigating the pathomechanisms that lead to thebreakdown of BBB. Different patterns of BBB disintegration are anticipated underdifferent pathological conditions, e.g., inﬂammatory reactions versus tumors.Genes that are selectively expressed at the BBB have been cloned. These includeGLUT-1 (glucose transporter) and GGTP (gamma-glutamyl transpeptidase). TheBBB GLUT-1 transporter maintains the availability in the brain of glucose and theregulation of the protein is mediated at the levels of the gene transcription, mRNAtranslation and stability, and posttranscriptional processes. GLUT-1 plays a role inthe development of the cerebral endothelial cells with BBB properties in vivo.Knockdown of GLUT-1 in an animal model was shown to produce loss of the cere-bral endothelial cells and downregulation of the junctional proteins important forintactness of the tight junctions with resulting leaky BBB and vasogenic cerebraledema (Zheng et al. 2010). This ﬁnding suggests that research into modulation ofGLUT-1 expression may lead to therapeutic strategies for preventing vasogeniccerebral edema.Molecular mechanisms of the “tight junctions” of the BBB are just being unrav-eled. In addition to occludens, other molecules (such as claudins) may be respon-sible for the integrity of the tight junctions. A molecular analysis of the BBB has
483Passage of Substances Across the Blood–Brain Barrierclinical relevance for the development of new therapeutic strategies for neurologicdisorders.Genomics of BBBGenomic and proteomic analyses have been used to study the BBB and how itrelates to the pathogenesis of major neurologic diseases. Shear stress associatedwith blood ﬂow in arteries has variable effects on endothelial cells, which aremodulated by induction or suppression of genes regulating endothelial physiology,e.g., formation of inter-endothelial tight junction and expression of speciﬁc carrier-mediated transporters (Cucullo et al. 2011). These ﬁndings can form the basis ofdeveloping innovative therapeutic strategies to improve the management of BBB-related diseases.Many aspects of how the BBB functions at the molecular level remain unre-solved; therefore, a variety of genomic and proteomic techniques have been used inBBB research. Genomic methods include gene microarray technologies, serialanalysis of gene expression (SAGE) and suppression subtractive hybridization(SSH). Gene microarray technologies are useful for generating semiquantitativedata regarding gene expression across an entire genome. SAGE generates informa-tion about the entire gene expression proﬁle by parsing mRNAs into short nucle-otide fragments called tags, which allow for the quantitative cataloging of allexpressed genes in cells or tissues. Finally, SSH is a PCR-based method for identi-fying differentially regulated (up- or down-regulated) and tissue-speciﬁc (not detect-able in compared samples) gene transcripts.A comprehensive gene expression proﬁle of rat brain microvessels using SAGEhas been reported (Enerson and Drewes 2006). This resulted in identiﬁcation of 864genes, including several known for their abundant expression at the BBB, such asthe transferrin receptor. Sorting enriched genes based on function revealed groupsthat encode transporters (11%), receptors (5%), proteins involved in vesicletrafﬁcking (4%), structural proteins (10%), and components of signal transductionpathways (17%). This genomic repertoire emphasizes the unique cellular pheno-type existing within the brain and further implicates the BBB as a mediator betweenthe brain and periphery. These results may provide a useful resource and referencepoint from which to determine the effects of different physiological, developmental,and disease processes on BBB gene expression. Currently, some of the researchpriorities include examination of the genes and proteins that are uniquely expressedby the intact BBB and mechanisms by which brain cells regulate endothelial cellgene expression.Proteomics of BBBProteomics analyses are currently being used to examine BBB function in healthyand diseased brain to better characterize this dynamic interface. Because the levelsof mRNA and protein in cells do not always correlate, proteomic methods have beendeveloped to examine proteins, the real actors in many cell functions. A widely used
484 13 Role of Biotechnology in Drug Delivery to the Nervous Systemtechnique for creating differential proteomic proﬁles is 2D polyacrylamide gel elec-trophoresis (2D PAGE). This technique separates proteins according to charge andmass allowing for the resolution of up to 10,000 individual protein spots on a singlegel. Mass spectrometry (MS) is often used in conjunction with this method to iden-tify the resolved proteins. Another proteomic technique employed in BBB researchuses isotope coded afﬁnity tags (ICAT). Labeling of protein samples with discreteisotope tags allows for a semiquantitative comparison of protein expression usingMS. Finally, similar to genomic arrays, proteomic arrays can be used to evaluatedifferential protein expression. One type of protein array used in BBB research is theantibody array. In this technology, antibodies are immobilized at high concentrationson a substrate to capture antigens from protein mixtures such as cell lysates.It is becoming increasingly evident that proteomic approaches have the potential toclarify the unique attributes of a healthy BBB, to identify therapeutic targets in dis-eased brain, and to identify novel conduits for noninvasive delivery of drugs againstthese targets. It has been estimated that approximately 3% of the proteins encoded bythe human genome function as molecular transporters. It is also likely that a signiﬁcantportion of the genes encoding proteins with unknown functional roles are also molecu-lar transporters. These can be discovered through genomic/proteomic approaches.Membrane proteins are somewhat difﬁcult to study with conventional proteomic tech-niques. It is now possible to perform differential membrane protein expressionproﬁling of the BBB as a complement to differential gene expression proﬁling.Genomics and proteomics approaches have also been applied to analyze other func-tions of the BBB including immunological response, bacterial invasion, and trans-porter expression in epilepsy. The effects of TNFa on human cerebral endothelial cellswere proﬁled using microarray and two-dimensional gel electrophoresis. It was dis-covered that cell adhesion, apoptosis, and chemotaxis genes were differentiallyexpressed, and these ﬁndings were corroborated by proteomic analysis.Damage to BBB Manifested as Increased PermeabilityBBB is damaged in several neurological disorders such as stroke, TBI, brain tumors,infections, multiple sclerosis, and neurodegenerative disorders. The impairment ofpermeability of BBB is variable and cannot be used to improve drug delivery fortreatment of these disorders. BBB is also damaged due to neurotoxicity of drugs.Methods used for opening the BBB usually produce only transient increase of per-meability but excessive force used may damage the BBB.Brain Imaging for Testing Permeability of the BBBIn animal studies, BBB permeability can be quantitatively evaluated by measuringthe concentration of non-permeable radioactive materials, traceable macromoleculesor dyes in the brain. However, this approach is not applicable in humans due to itsinvasiveness and the potential dangers. Therefore, in most human studies, BBB per-
485Passage of Substances Across the Blood–Brain Barriermeability is usually qualitatively evaluated using brain imaging techniques. Twomain approaches are used for studying the integrity of human BBB in vivo: (1)structural imaging employs contrast agents that only penetrate the BBB at sites ofdamage, and (2) functional imaging is used to study the transport of substancesacross the BBB − both intact and damaged. Structural imaging employs contrastagents with CT scanning and is relatively insensitive. MRI with the contrast agentgadolinium is more sensitive. Functional imaging is done with PET and can quantifycerebral uptake of therapeutic agents, such as cytotoxic agents and MAbs. SPECTis less versatile than PET, but can provide semiquantitative measurement of BBBleakage of albumin or red blood cells. Quantitative approaches are available to mea-sure BBB permeability using differentiated images and statistical analyses of CT orMRI images following the administration of standard contrast agents. This enablesquantiﬁcation of the spatial characteristics of BBB-disruption and behavior of con-trast agents with time under different neurological conditions. This method mayenable assessment of the functional implications of the BBB integrity in variousCNS diseases as well as matching the required drug dose with BBB penetrability inspeciﬁc patients. PET can also be used to image regional P-glycoprotein activity andinhibition at the human BBB as it affects the distribution of drugs in the variousbrain regions protected by the barrier (Eyal et al. 2010).Biomarkers of Disruption of BBBThere is a need for biomarkers to detect early changes in BBB in various condi-tions. Loss of integrity of the BBB resulting from ischemia/reperfusion is believedto be a precursor to hemorrhagic transformation (HT) and poor outcome in acutestroke patients. A novel MRI biomarker has been used to characterize early BBBdisruption in human focal brain ischemia and its association with reperfusion, HT,and poor outcome (Latour et al. 2004). Reperfusion was found to be the most pow-erful independent predictor of early BBB disruption and thus of HT and is impor-tant for the decision for acute thrombolytic therapy. Early BBB disruption asdeﬁned by this imaging biomarker may be a promising target for adjunctive therapyto reduce the complications associated with thrombolytic therapy, broaden thetherapeutic window, and improve clinical outcome in acute stroke.The astrocytic protein S-100beta is a potentially useful peripheral biomarker ofBBB permeability. Other biomarkers of BBB have been recently discovered byproteomic approaches. These proteins are virtually absent in normal blood, appearin serum from patients with cerebral lesions, and can be easily detected.Peripheral assessment of BBB opening can be achieved by detection in blood ofbrain-speciﬁc proteins that extravasate when these endothelial junctions arebreached. A proteomic approach was used to discover clusters of CNS-speciﬁcproteins with extravasation into serum that correlates with BBB openings. Proteinproﬁles from blood samples obtained from patients undergoing BBB disruptionwith intra-arterial hyperosmotic mannitol are compared with pre-BBB openingserum. A low molecular weight protein (14 kDa) was identiﬁed by MS as transthy-
487Passage of Substances Across the Blood–Brain Barrierto the exposure of the whole brain to the therapeutic agent. Genetic and otherdefects leading to brain changes in Down’s syndrome, Alzheimer’s disease, amyo-trophic lateral sclerosis, Huntington’s disease, Gaucher disease, hypertension, andother disorders are rapidly being identiﬁed. Several effective therapeutic agents areavailable but their use is limited pending improvement of drug delivery across theBBB. In silico methods are available to predict BBB penetration by drugs(Lanevskij et al. 2010).2B-Trans™ TechnologySystems directed at endogenous receptor-mediated uptake mechanisms have beenshown to be effective in animal models including primates. Various systems use thelow-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or thediphtheria toxin receptor, which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.The 2B-Trans™ technology (to-BBB) uses a well characterized and effectivetransport system with a speciﬁc carrier protein that has an excellent proven safetyproﬁle in human (Gaillard and de Boer 2008). Advantages of 2B-Trans™ receptorare as follows:It uses receptor-mediated endocytosis, an effective and safe transport mecha-•nism, for delivery of large proteins and liposomes containing drugs and genesacross the BBB.Because the receptor has no endogenous ligands, there is no competition from•endogenous ligands, or blockade of transport to the brain of essential nutrients.It is constitutively expressed on the BBB, neurons and glial cells•The receptor expression is highly ampliﬁed in disease conditions and thus•allows for site-speciﬁc disease targetingto-BBB holds a patent claim on the use of all known ligands/carrier proteins for•the receptor to deliver drugs to the brain.ABC Afﬂux Transporters and Penetration of the BBBA signiﬁcant number of lipid soluble molecules, among them many useful thera-peutic drugs have lower brain permeability than would be predicted from adetermination of their lipid solubility. These molecules are substrates for the ABCefﬂux transporters, which are present in the BBB and BCSB, and the activity ofthese transporters very efﬁciently removes the drug from the CNS, thus limitingbrain uptake. P-glycoprotein (Pgp) was the ﬁrst of these ABC transporters to bedescribed, followed by the multidrug resistance-associated proteins and morerecently breast cancer resistance protein. All are expressed in the BBB and BCSFBand combine to reduce the brain penetration of many drugs. This phenomenon of“multidrug resistance” is a major hurdle when it comes to the delivery of therapeu-tics to the brain. Therefore, the development of strategies for bypassing the
488 13 Role of Biotechnology in Drug Delivery to the Nervous Systeminﬂuence of these ABC transporters and for the design of effective drugs that arenot substrates and the development of inhibitors for the ABC transporters becomesa high imperative for the pharmaceutical industry (Begley 2004).Another potentially promising approach to enhancing the delivery of otherwisenon-permeating drugs to the brain is the use of excipients, which are able to interactwith ABC transporters and modify function. Certain Pluronic block co-polymersappear to target pgp by two mechanisms: depletion of cellular ATP and altering thephysicochemical properties of the membrane lipid phase. ABC transporters may beof therapeutic beneﬁt in situations where acute dosing is indicated. However, it isuncertain whether chronic administration of blocking agents is feasible given theprotective role of these transporters in the BBB and other organs. Several strategiesdesigned to bypass pgp at the BBB without direct inhibition have been described andtested including a system that uses antibody-coupled immunoliposomes to transportpgp substrates. The strategy is to move the encapsulated drug through the lumenalplasma membrane of capillary endothelial cells avoiding direct interaction with pgp.Immunoliposomes, which are coupled to an antitransferrin receptor antibody, havebeen shown in vivo and in vitro to be internalized at the BBB by means of receptor-mediated endocytosis and to deliver p-gp substrates efﬁciently to the brain. Similarresults have been obtained with liposomes coupled to cationized albumin.The discovery of active carrier proteins and cytotic mechanisms and their con-tribution to drug permeation across the barrier would promote the successful devel-opment of efﬁcient CNS drugs and to an understanding of unwanted CNS sideeffects of non-CNS drugs. From the molecular biology and pharmacology of theproteins involved, it might be possible to identify speciﬁc probes to distinguishtransporter subtypes as well as tools to transiently modify barriers to drug absorp-tion through competition. An understanding of the mechanisms by which expres-sion and function of drug transporters is regulated would help in improving drugdelivery to the CNS.Carrier-Mediated Drug Delivery Across the BBBOf the various carrier systems, those for glucose and neutral amino acids havehigh enough transport capacity to hold promise of signiﬁcant drug delivery to thebrain. Glucose transporter has the limitation that only molecules closely resem-bling D-glucose are transported. Neutral amino acids are less speciﬁc. Entry viathis carrier may explain the central effects of the muscle relaxant baclofen.Transport systems for peptides may prove to be effective targets for peptide drugsrequired to control natural peptide hormones.Drugs used to treat neurologic disorders appear to cross the BBB more easilywhen an ascorbic acid molecule is attached. Ascorbic acid works like a shuttle and,theoretically, could transport any compound into the brain. The ascorbic acidSVCT2 transporter, which is believed to play a major role in regulating the transportof ascorbic acid into the brain, provides a targeted delivery to the brain. Potentialapplications include drugs to treat neurodegenerative diseases, e.g. AD and PD.
489Passage of Substances Across the Blood–Brain BarrierA feasible method to achieve carrier-mediated drug transport into the rat brainwas shown to be via the speciﬁc, large neutral amino acid transporter (LAT1) byconjugating a model compound to L-tyrosine (Gynther et al. 2008). A hydrophilicdrug, ketoprofen, that is not a substrate for LAT1 was chosen as a model compound.The mechanism and the kinetics of the brain uptake of the prodrug were determinedwith an in situ rat brain perfusion technique and found to be concentration-depen-dent. Moreover, a speciﬁc LAT1 inhibitor signiﬁcantly decreased the brain uptakeof the prodrug.The iron binding protein p97 (melanotransferrin) is closely related to Tf andlactoferrin and as a result of alternative splicing, it exists in both a soluble form anda cell surface GPI-linked form. However, in normal brain it appears to discretelylocalize on the surface of endothelial cells and transiting through brain capillaryendothelium. Studies on the structure and function of p97 suggest it might be anideal carrier for transport of drug conjugates into the brain. A study provides theinitial proof of concept for p97 as a carrier capable of shuttling therapeutic levelsof drugs from the blood to the brain for the treatment of neurological disorders,including classes of resident and metastatic brain tumors (Karkan et al. 2008). Thisnovel delivery platform may be useful in various clinical settings for therapeuticintervention in acute and chronic neurological diseases and is in commercial devel-opment for CNS drug delivery.G-Technology®Glutathione, an endogenous tripepeptide transporter, is highly expressed on theBBB. Glutathione is found in high levels in the brain and cerebral vasculature.Glutathione has favorable antioxidant-like properties and plays a central role indetoxiﬁcation of intracellular metabolites. Glutathione transporters are conservedacross all mammalian species, including humans. Glutathione is considered to besafe to administer to humans for a prolonged period of time. Glutathione is mar-keted as functional food ingredient and antioxidant, and applied as supportivetherapy in cancer and HIV treatments and as excipient in parenteral formulations.Glutathione coated on the surface of nanosized liposomes was shown to be welltolerated and to effectively deliver several classes of drugs to the brain in a rangeof experimental studies reproducibly performed by independent laboratories. Thisis the basis of G-Technology® (to-BBB), which uses pegylated liposomes coatedwith glutathione, an endogenous tripepeptide transporter expressed on the BBB, tofacilitate delivery of drugs to the brain (Gaillard 2011). Applied to anticancer drugs,it improves targeted delivery to the brain tumors after systemic administration andreduces adverse effects. Glutathione pegylated liposomal doxorubicin (2B3-101) isin phase I/II clinical trials in patients with brain cancer. G-Technology has also beenapplied for delivery of methylprednisolone, which is used for several diseases witha neuroinﬂammatory component. A product, 2B3-201(to-BBB), is in preclinicalstudies for potential applications in multiple sclerosis, acute spinal cord injury andlupus erythematosus involving the CNS (Gaillard et al. 2012).
490 13 Role of Biotechnology in Drug Delivery to the Nervous SystemGlycosylation Independent Lysosomal TargetingEnzyme replacement therapies use a novel, proprietary technology, known asGlycosylation Independent Lysosomal Targeting (GILT), which improves thedelivery of lysosomal enzymes to clinically signiﬁcant tissues (LeBowitz 2005).A target directing molecule (the tag) is embedded within the therapeutic enzyme topromote internalization into cells. The internalization process is accomplished bythe binding of the tag to a receptor found on the surface of the target cell thereforefacilitating endocytosis. GILT technology might be used to deliver drugs across theBBB. This would be an important application because several lysosomal storagediseases have profound neurological components and conventionally glycosylateddrugs are unable to address this problem.Inhibition of P-glycoprotein to Enhance Drug Delivery Across the BBBP-glycoprotein (P-gp) drug efﬂux transporter is present at high densities in the lumi-nal membranes of brain endothelium. It limits entry into the CNS for a large numberof prescribed drugs, contributes to the poor success rate of CNS drug candidates, andpatient-to-patient variability in response to CNS pharmacotherapy. It pumps outsome cytotoxic agents used to treat brain tumors and excludes them from the brain.Recent studies focused on understanding the mechanisms by which P-gp activity inthe BBB can be modulated to improve drug delivery into the brain.Using intact brain capillaries from rats and mice, scientists have identiﬁed mul-tiple extracellular/intracellular signals that regulate this transporter and severalsignaling pathways have been mapped (Miller et al. 2008). Three pathways that aretriggered by elements of the brain’s innate immune response are: (1) by glutamate;(2) by xenobiotic-nuclear receptor (pregnane X receptor) interactions; and (3) byelevated Ab levels. Signaling is complex, with several pathways sharing commonsignaling elements − TNFR- 1, endothelin B receptor, PKC, and NOS − suggestinga regulatory network. Several pathways include autocrine/paracrine elements,involving release of the proinﬂammatory cytokine, TNF-a, and the polypeptidehormone, endothelin-1. Several steps in signaling are potential therapeutic targetsthat could be used to modulate P-gp activity in the clinic. Strategies for P-gp modu-lation include (1) direct inhibition by speciﬁc competitors, (2) functional modula-tion, and (3) transcriptional modulation. Each has the potential to speciﬁcallyreduce P-gp function and thus selectively increase brain permeability of P-gpsubstrates. A crosslinked dimer of galantamine, Gal-2, inhibits P-gp mediatedefﬂux mechanism at the BBB by competing for the substrate binding sites (Namanjaet al. 2009). Several speciﬁc inhibitors of P-gp as efﬂux transporters are in clinicaldevelopment.LipoBridge™ TechnologyLipoBridge™ (Genzyme Pharmaceuticals) temporarily and reversibly opens tightjunctions to facilitate transport of drugs across the BBB and into the CNS.
491Passage of Substances Across the Blood–Brain BarrierLipoBridge itself forms a clear suspension of nanoparticles in water and can solu-bilize or stabilize some drugs, is non-immunogenic and is excreted unmetabolized.It has been demonstrated in several laboratories that intracarotid injections of asimple mixture of Lipobridge™ and model compounds or pharmaceutical activescan deliver these actives into one or both hemispheres of the brain allowing forincreased concentration in a selected hemisphere. It can be administered orally aswell as intravenously. LipoBridge has been used to administer anticancer drugs forbrain cancer in animals. Safety clinical studies in humans are in progress.Modiﬁcation of the Drug to Enhance Its Lipid SolubilityThere is a good correlation between the lipid solubility of a drug and the BBBpenetration in vivo. The lipophilic pathway also provides a large surface area fordrug delivery. It is approximately 12 m2in an average human brain. Therefore,addition of hydrophobic groups to molecules increases their ability to penetrate theBBB. Addition of methyl groups in a series of barbiturates improves lipophilicityand brain penetration, leading to increased hypnotic action. It is also possible togenerate a lipophilic prodrug that is broken down to release the more active drugwithin the brain. An example of this is heroin, which enters the brain readily due toits lipophilicity but, after entry, hydrolyses to morphine, which is less lipophilic andless likely to diffuse back across the BBB, leading to prolongation of duration ofits action on the brain. Ester formation is another approach for increasing the lipo-philicity of polar molecules exhibiting poor CNS penetration. A number of investi-gators have explored the lipophilic-ester concept for improving the CNS deliveryof antiviral agents. An example of this is improvement of the BBB penetration ofGABA (gamma amino butyric acid), an anticonvulsant agent with poor CNS pen-etration, by use of lipophilic esters.Although increasing lipophilicity generally increases penetration across theBBB, it may also result in reduction of biological action due to drug-receptor inter-action, drug metabolism, or binding to plasma proteins as in the case of barbitu-rates. Therefore, optimization rather than maximization of both lipophilicity andrate of bioconversion are required.Lipid-binding carriers may reduce the binding of neurotrophic factors to serumlipids and increase transport across the BBB. Liposomes have been considered, buttheir size is too large to cross the BBB.Monoclonal Antibody Fusion ProteinsThese involve conjugation of a drug to a transport vector. These have diagnostic andtherapeutic applications for the treatment of brain tumors. Nontransportablespeciﬁc antigen-binding monoclonal antibodies such as IgG3 have been attached toa transport vector such as insulin-like growth factor. The bifunctional molecule cancross the BBB through interaction with the receptor for insulin-like growth factor.
492 13 Role of Biotechnology in Drug Delivery to the Nervous SystemTransferrin is a speciﬁc receptor for molecules that are not synthesized in thebrain but play an essential biological role. This transfer mechanism can be exploitedin an approach in which antiferritin receptor antibodies are covalently linked toNGF, resulting in a substantial transfer of biologically active nerve growth factoracross the BBB into the CNS. NGF can be transported across the BBB by conjugat-ing with OX-2, an antibody directed against the transferrin receptor.One suitable transport vector is a peptidomimetic MAb that is transported by anendogenous BBB receptor-mediated transcytosis system, such as the transferrinreceptor. The MAb carries any drug attached to it across the BBB. However, thenumber of small molecules that can be conjugated to monoclonal antibodies vectorsis limited. The carrying capacity of the vector can be greatly expanded by attachingliposomes to the vector.A new generation of multifunctional fusion proteins are being engineered atArmaGen Technologies to cross the BBB following intravenous administration andto produce a therapeutic effect on brain disorders (Boado 2008). These fusion pro-teins are comprised of both a transport and a therapeutic domain. The transportdomain is a MAb directed to an exofacial epitope of the BBB human insulin recep-tor (HIR), which uses the BBB endogenous insulin transport system to gain accessto the brain via receptor-mediated transcytosis without interfering with the normaltransport of insulin. Both human-chimeric and fully humanized versions of the anti-human HIRMAb have already been produced. The therapeutic domain of thesefusion proteins consists of the peptide or protein of interest fused to the carboxylterminus of the CH3 region of the heavy chain of the anti-human HIRMAb. Avariety of HIRMAb fusion proteins were engineered aiming at the development oftherapeutics for stroke and PD, as in the case of HIRMAb-BDNF and HIRMAb-GDNF, respectively, HIRMAb-IDUA for the treatment of Hurler’s disease,HIRMAb-Ab single chain antibody for passive immunotherapy of AD, andHIRMAb-avidin as delivery system for biotinylated drugs, like siRNAs. The mul-tifunctionality of these fusion proteins has been validated in preclinical work,including brain update in primates. Pending further development into pharmaco-logical and toxicological studies, and clinical trials, members of the biotherapeuticfamily discussed in the present review, designed to overcome the brain drug deliv-ery hurdle, are positioned to become a new generation of neuropharmaceuticaldrugs for the treatment of human CNS disorders.NeuroimmunophilinsNeuroimmunophilin ligands are small molecules that in can repair and regeneratedamaged nerves without affecting normal, healthy nerves. Neuroimmunophilin ligandsmay have application in the treatment of a broad range of diseases, including PD,spinal cord injury, brain trauma, and peripheral nerve injuries. The immunosuppres-sants tacrolismus (FK-506) and cyclosporin are in clinical use for the treatment ofallograft rejection following organ transplantation. Immunophilins can regulate neu-ronal survival and nerve regeneration although the molecular mechanisms are poorlyunderstood. Neuroimmunophilin can be administered orally and can cross the BBB.
493Passage of Substances Across the Blood–Brain BarrierPeptide-Mediated Transport Across the BBBUnder normal conditions, vesicular transport that involves receptor-mediated endocy-tosis is responsible for only a small amount of molecular trafﬁcking across the BBB,but it may be suitable for the delivery of agents that are too large to use other routes.A number of different peptide families with the ability to cross the cell mem-branes have been identiﬁed. Certain of these families enter the cells by a receptor-independent mechanism, are short (10–27 amino acid residues), and can deliversuccessfully various cargoes across the cell membrane into the cytoplasm ornucleus. Some of these vectors have also shown the ability to deliver hydrophilicmolecules across the BBB.Another approach involves forming a chimeric peptide by coupling an otherwisenontransportable drug to a BBB transporter vector by a disulﬁde bond. The chime-ric peptide is then endocytosed by the capillary endothelial cells and transported tothe brain where it can be cleaved by disulﬁde reductase to release the pharmaco-logically active compound. BBB peptide receptor systems include those for insulin,insulin-like growth factor, transferrin, and leptin. Conjugation of doxorubicin orpenicillin to peptide vectors signiﬁcantly enhances their brain uptake. Peptide-mediated strategies can improve the availability and efficacy of CNS drugs.Transport of Small Molecules Across the BBBLipid-soluble small molecules with a molecular mass less than 400 Da are trans-ported readily through the BBB in vivo via lipid-mediated transport. However, othersmall molecules lacking these molecular properties, antisense drugs, and peptide-based pharmaceuticals ordinarily undergo negligible transport through the BBB inpharmacologically signiﬁcant amounts. Some small-molecule neuroprotectiveagents have failed in human trials due to poor transport of these agents across theBBB. Strategies that enable drug transport through the BBB arise from knowledgeof the molecular and cellular biology of BBB transport processes. As biology-drivendrug discovery progresses, more large molecules are being discovered as potentialtherapeutics. Some of the strategies for samll molecules transport may be used fortransporting larger molecules such as gene medicine and recombinant proteins.Trojan Horse ApproachAttaching an active drug molecule to a vector that accesses a speciﬁc catalyzedtransporter mechanism creates a Trojan horse-like deception that tricks the BBBinto welcoming the drug through its gates. Transport vectors, such as endogenouspeptides, modiﬁed proteins, or peptidomimetic MAbs are one way of tricking thebrain into allowing these molecules to pass. Intravenously administered molecules,attached to Trojan horses for CNS effect in experimental animals, are shown inTable 13.4.Biopharmaceuticals, including recombinant proteins, MAb therapeutics, andantisense or siRNAs, cannot be developed as drugs for the brain, because theselarge molecules do not cross the BBB. Biopharmaceuticals must be re-engineered
496 13 Role of Biotechnology in Drug Delivery to the Nervous SystemThe high level of expression of transferrin receptors (TfR) on the surface ofendothelial cells of the BBB have been widely utilized to deliver drugs to the brain.This approach has been explored for the delivery of citicoline, a neuroprotectivedrug for stroke that does not readily cross the BBB because of its strong polarnature. Low concentrations of citicoline encapsulated in transferrin-coupled lipo-somes could offer therapeutic beneﬁt in treating stroke compared to administrationof free citicoline (Suresh Reddy et al. 2006). This is likely due to the entry of citi-coline into cells via TfR-mediated endocytosis.NeuroTrans™, a proprietary technology based on receptor-associated protein(RAP) is being developed for the delivery of therapeutics across the BBB. In pre-clinical studies, NeuroTrans™ has been conjugated to a variety of protein drugs,including enzymes and growth factors, without interfering with the function ofeither fusion partner (Prince et al. 2004). Studies indicate that radio-labeledNeuroTrans™ may be transcytosed across the BBB and, that fusions betweenNeuroTrans™ and therapeutic proteins may be manufactured economically (Panet al. 2004). Scanning electron microscopy imaging is being used to determinewhether the NeuroTrans™ peptide is able to enter the brain tissue through the pro-cess of transcytosis. This will also help the assessment of the time frame of trans-port, the extent or amount of NeuroTrans™ transported, and the biodistribution ofNeuroTrans™ within various brain compartments.Use of Nanobiotechnology for Therapeutic DeliveryAcross the BBBAmong the various approaches that are available, nanobiotechnology-based deliv-ery methods provide the best prospects for achieving this ideal. This topic is dis-cussed in more detail in the chapter on “Nanomedicine”. Various nanoparticles(NPs) used for drug delivery to the brain and their known mechanism of action arereviewed elsewhere (Jain 2012). Some strategies use multifunctional NPs. Animportant application of nanobiotechnology is delivery of therapy for brain tumorsacross the BBB as well as combination of diagnostics with therapeutics. Despitesome current limitations, future prospects for NP-based therapeutic delivery to thebrain are excellent.Delivery of Cell Therapy to the BrainCell therapy is described in Chap. 12. Although cells may deliver therapeuticsthemselves, there is also a need for drug delivery systems for cell therapies. Variousmethods of delivery of cells for therapeutic purposes are listed in Table 13.5.
498 13 Role of Biotechnology in Drug Delivery to the Nervous System2005). These biodegradable spherical microparticles are made with poly(D,L-lactic-co-glycolic acid) (PLGA) and coated with adhesion molecules. Their diam-eter may vary, depending on the type of transported cell, from 10 to 500 mm. Thepreferential cell adhesion of the cells to be grafted on the microcarriers permitstheir preparation or transformation in vitro without the use of enzymes of animalorigin. The cell adhesion molecules as well as the growth factors may induce thesurvival and differentiation of stem cells towards a determined phenotype. Themicrospheres are spontaneously degraded, without toxicity and without interferingwith the activity or integration of the grafted cells, in a few weeks or months afterimplantation, depending on the composition of the polymer. PAM may serve as asupport for cell culture and may be used as cell carriers presenting a controlleddelivery of active protein. They can thus support the survival and differentiation ofthe transported cells as well as their microenvironment. They reduce the hostimmune reaction and favor the tissue integration of the grafted cells.To develop this tool, nerve growth factor (NGF)-releasing PAM, conveyingPC12 cells, were produced and characterized. These cells have the ability to dif-ferentiate into sympathetic-like neurons after adhering to a substrate, in the pres-ence of NGF, and can then release large amounts of dopamine. Certain parameterssuch as the size of the microcarriers, the conditions enabling the coating of themicroparticles and the subsequent adhesion of cells were thus studied to produceoptimized PAM. NGF-releasing PAM coated with ﬁbronectin plus polylysine andtransporting PC12 cells were evaluated in an animal model of PD. After transplan-tation, the PAM induced the differentiation, reduced cell death and proliferation ofthe PC12 cells and the animals presented an ameliorated behavior.PAM may be used in any type of cell therapy: (1) tissue reconstruction byimplanting embryonic cells, cell lines, genetically modiﬁed cells or stem cells;(2) for the grafting of cells for nd central nervous system (3) cell therapy for genetransfer; and (4) anticancer vaccination as PAM may present tumor cells or frag-ments of these cells to immunocompetent cells while delivering immunostimulat-ing cytokines.Targeted Delivery of Engineered Cells to SpeciﬁcTissues via CirculationMinimally invasive delivery of a large quantity of viable cells to a tissue of interestwith high engraftment efﬁciency is a challenge in cell therapy. Low and inefﬁcienthoming of systemically delivered MSCs, e.g., is considered to be a major limita-tion of existing MSC-based therapeutic approaches, caused predominantly byinadequate expression of cell surface adhesion receptors. The surface of MSCswas modiﬁed without genetic manipulation with a nanometer-scale polymer con-struct containing sialyl Lewisx (sLex) that is found on the surface of leukocytesand mediates cell rolling within inﬂamed tissue (Sarkar et al. 2011). The sLexengineered MSCs exhibited a robust rolling response on inﬂamed endothelium
499Delivery of Cell Therapy to the Brainin vivo and homed to inﬂamed tissue with higher efﬁciency compared with nativeMSCs. This is a simple method to potentially target any cell type to speciﬁc tissuesvia the circulation.Devices for Delivery of Cell TherapyA self-assembling cube-shaped perforated container, no larger than a dust speck,has been devised to could serve as a delivery system for medications and celltherapy (Gimi et al. 2005). Because of their metallic nature, the location of cubiccontainers in the body could easily be tracked by MRI. The microcontainers couldsomeday incorporate electronic components that would allow the cubes to act asbiosensors to release medication on demand in response to a remote-controlledradio frequency signal. Biohybrid implants represent a new class of medicaldevice in which living cells, supported by hydrogel matrix and surrounded by asemipermeable membrane, produce and deliver therapeutic reagents to speciﬁcsites within a host.Cell EncapsulationModern encapsulation techniques involve surrounding the cells with selectivelypermeable membranes. The pores of the membranes should be small enough toblock entry of immune mediators but large enough to allow inward diffusion ofoxygen and nutrients required for the survival of cells and for outward diffusion ofactive molecules produced by the cells. Encapsulation avoids some of the compli-cations of free cell transplants including local reaction at the site of transplantationand tumor formation. The required characteristics of a membrane device are:The material should be biologically inert and nontoxic to the tissues•It should be nonimmunogenic•It should be sturdy enough to withstand a considerable amount of•manipulationPore size should be adequate to allow the passage of oxygen and nutrients for•the cellsIt should be possible to vary pore size according to need•Availability of an economical large-scale process for production•It is difﬁcult to encapsulate living cells using polymers because of toxic interactionsbetween solvents and cells during the formation of thermoplastic-based microcap-sules. Biodegradable materials can be synthetic or natural and they are degradedin vivo both enzymatically as well as nonenzymatically. Their by-products are usu-ally nontoxic and excreted via physiological pathways. Examples of natural biode-
500 13 Role of Biotechnology in Drug Delivery to the Nervous Systemgradable materials are human serum albumin and collagen. Because of their costand the possibility of contamination, several synthetic biodegradable polymershave been developed. Polyelectrolyte microcapsules are fragile, both physically andchemically. Great care is required in handling during transplantation and in vivofailure may occur 3 months post-transplantation. Thermoplastic-based microcap-sules are mechanically more durable.Another method of encapsulating cells is use of permeable alginate shells thatcan maintain structural integrity in vivo for extended periods of time. It is prefera-ble to most of the currently used techniques for capsule generation that yield micro-spheres prone to wall degradation. Shell properties may be modiﬁed to regulatepermeability, providing controlled delivery of desired substances.Encapsulated Cell BiodeliveryThe encapsulated cell (EC)-biodelivery (NsGene) is a general biodelivery systemof cell-derived substances to the CNS that provides a controlled, site-speciﬁc andsafe delivery of a variety of therapeutic substances. For CNS indications one ormultiple EC-biodelivery devices can be implanted in deﬁned regions of the brain todeliver any proteins or neurotransmitters across the BBB. EC-biodelivery systemconsists of a catheter-like device that in the active portion contains a geneticallymodiﬁed human cell line enclosed behind a semi-permeable hollow ﬁber mem-brane. The membrane allows for the inﬂux of nutrients and outﬂow of the therapeu-tic factor(s) but does not allow for the direct contact between the therapeutic cellsand the host tissue. The encapsulated cells provide for long-term (>12 months)secretion of a therapeutic factor from the implanted device. This offers great safetyadvantages over direct cell/gene therapy approaches and technical and functionaladvantages over pump technologies. The device is compatible with stereotacticneurosurgical techniques and instrumentation adapters to common stereotacticframes have been made. EC-biodelivery devices are suitable for intraparenchymal,intracerebroventricular, or intrathecal placement.Therapeutic Applications of Encapsulated Cellsin Neurological DisordersTable 13.6 lists therapeutic applications of encapsulated cells in neurologicaldisorders.Advantages of encapsulated technology include the following:Continuous delivery of therapeutic proteins and peptides in the treatment of•chronic diseases. Reduction of side effects associated with systemic administra-tion of proteins and peptides, which can cause peaks in serum protein levels.
502 13 Role of Biotechnology in Drug Delivery to the Nervous Systemapproach is to encapsulate genetically engineered cells and implant them into thebody, e.g., beta-endorphin secreting cells for pain treatment and neurotrophic factorsecreting cells as trophic factors for neurodegenerative diseases. There are someproblems related to implantation including the following:Safety problems related to the introduction of genetically engineered material•into the bodyAlthough the cells are protected from rejection by leucocytes and antibodies,•there is potential rejection by complements and cytokines.Ferroﬂuid Microcapsules for Tracking with MRIImplanting recombinant cells encapsulated in alginate microcapsules to expresstherapeutic proteins has been proven effective in treating several mouse models ofhuman neurological disorders. In anticipation of clinical application, magnetizedferroﬂuid alginate microcapsules have been synthesized, which can be trackedin vivo by MRI (Shen et al. 2005). Ferroﬂuid-enhanced alginate microcapsules arecomparable to classic alginate microcapsules in permeability and biocompatibility.Their visibility and stability to MRI monitoring permits qualitative and quantitativetracking of the implanted microcapsules without invasive surgery. These propertiesare important advantages for the application of immunoisolation devices in humancell/gene therapy.Delivery of Gene Therapy to the BrainGene therapy, a sophisticated method of delivery of therapeutics, is described inChap. 13. Various methods of delivery of DNA and genes for therapeutic purposesthat are relevant to neurological disorders are listed in Table 13.7.Clinical Applications of Biotechnology for CNS Drug DeliverySeveral techniques are used for drug delivery in CNS disorders (Jain 2012). Thischapter will emphasize the role of biotechnology. Of the various approachesdescribed, the following have been used clinically.
504 13 Role of Biotechnology in Drug Delivery to the Nervous Systemthe entry of anticancer agents into brain tumors in phase III trials. Although thisapproach increases the efﬁcacy of the cytotoxic drugs, it also increases their neuro-toxicity by increasing the permeability of the BBB of the normal brain. Thisapproach has also been used to facilitate the delivery of adenoviral vectors for genetherapy of brain tumors and for the administration of bifunctional fusion proteinsof tumor-speciﬁc MAbs for the treatment of brain tumors. Opening of the BBBfacilitates the entry of superparamagnetic iron oxide conjugates used as adjuncts toMRI for diagnosis of brain metastases.Intraarterial Administration of TherapeuticSubstances for CNS DisordersAcrylic and thrombosis inducing material for occluding arteriovenous malforma-tions are administered intraarterilly in neuroradiology and neurosurgery. A numberof drugs have also been administered by this route. This route is used for directinjection of gene vectors into the arterial circulation of the brain. The approvedmethod of administration of thrombolytic agents such as recombinant tissue plas-minogen activator, a biotechnology product, is intravenous but considerable experi-ence exists with the use of intraarterial thrombolysis in patients in whom the lesionshave been demonstrated by angiography prior to thrombolysis.The BBB hinders the penetration of anti-HIV drugs into the brain for treatmentof AIDS encephalopathy, promoting viral replication, the development of drugresistance, and, ultimately, subtherapeutic concentrations of drugs reaching thebrain, leading to therapeutic failure. The speciﬁcity and efﬁciency of anti-HIV drugdelivery can be enhanced by using nanocarriers with speciﬁc brain-targeting, cell-penetrating ligands (Wong et al. 2010).Drug Delivery to the Brain in PDGDNF is potentially useful in the treatment of PD (PD), but penetration into braintissue from either the blood or the CSF is limited. GDNF was delivered directly intothe putamen of a patient with AD in a phase 1 safety trial (Gill et al. 2003). The treat-ment was effective with no serious complications. An open-label study has demon-strated the safety and potential efﬁcacy of unilateral intraputaminal GDNF infusionfor 6 months via a catheter in patients with advanced PD (Slevin et al. 2005).Drug Delivery to the Brain in ADSeveral routes of drug delivery other than oral have been explored for the manage-ment of AD (AD). Transdermal rivastigmine maintains steady drug levels in the
505Clinical Applications of Biotechnology for CNS Drug Deliverybloodstream, improving tolerability and allowing a higher proportion of patients toreceive therapeutic doses compared to the capsule form of the medication. A num-ber of studies are exploring the nasal route of drug delivery for AD.Biological therapies for the treatment of AD that exploit mechanisms of penetra-tion of the BBB include peptides, vaccines, antibodies, and antisense oligonucle-otides (Banks 2008).An experimental study demonstrated that the brain concentration of intrave-nously injected rivastigmine can be enhanced over 3.82-fold by binding to poly(n-butylcyanoacrylate) nanoparticles coated with 1% nonionic surfactant polysorbate80 (Wilson et al. 2008).Drug Delivery in EpilepsySpecial methods of drug delivery would improve the control of seizures, reducetoxic effects, and increase compliance in patients with epilepsy, such as by use oflong-acting formulations and subcutaneous implants. Overexpression ofP-glycoprotein and other efﬂux transporters in the cerebrovascular endothelium, inthe region of the epileptic focus, may also lead to drug resistance in epilepsy. Thishypothesis is supported by the ﬁndings of elevated expression of efﬂux transportersin epileptic foci in patients with drug-resistant epilepsy, induction of expression byseizures in animal models, and experimental evidence that some commonly usedantiepileptic drugs are substrates. Further studies to delineate the exact role, if any,of P-glycoprotein and other efﬂux transporters in drug-resistant epilepsy are war-ranted (Kwan and Brodie 2005).Innovative Methods of Drug Delivery for GlioblastomaMultiformeSeveral innovative therapies inclusing biological are being investigated for treat-ment of glioblastoma multiforme. Methods relevant to biotechnology are shown inTable 13.8.Gene therapy for GBM is described in Chap. 12. Examples of other strategiesare as follows:Biodegradable polymer implants containing anticancer drugs. Polymer-baseddrug delivery to the brain has special applications for the delivery of anticanceragents to malignant brain tumors. One example is the use of carmustine implants.One of the problems with surgical excision of GBM is local recurrence within 2 cmof the primary lesion. Strategies to prevent local recurrence include implantation ofdelivery devices containing chemotherapeutic agents. Biodegradable polymerimpregnated with carmustine (Gliadel), an approved product, is implanted into thetumor cavity after surgery improves the survival of patients.
508 13 Role of Biotechnology in Drug Delivery to the Nervous Systemcardiotoxicity and also the testicular toxicity of this drug. The drug transport acrossthe BBB by nanoparticles is due to a receptor-mediated interaction with the braincapillary endothelial cells, which is facilitated by certain plasma apolipoproteinsadsorbed by nanoparticles in the blood.A polymeric nanobioconjugate drug based on biodegradable, nontoxic, andnonimmunogenic polymalic acid as a universal delivery nanoplatform is used fordesign of a nanomedicine for intravenous treatment of brain tumors (Ding et al.2010). The polymeric drug passes through the BTB and tumor cell membrane usingtandem monoclonal antibodies targeting the BTB and tumor cells. The next step forpolymeric drug action is inhibition of tumor angiogenesis by speciﬁcally blockingthe synthesis of a tumor neovascular trimer protein, laminin-411, by attached anti-sense oligonucleotides, which are released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Introduction of a trileucineendosome escape unit results in signiﬁcantly increased antisense oligonucleotidedelivery to tumor cells, inhibition of laminin-411 synthesis, speciﬁc accumulationin brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drugdelivery system that crosses BTB, targets tumor cells, and inhibits tumor growth isa promising strategy of glioma treatment.In vivo application of nanoparticle-based platforms in brain tumors is limited byinsufﬁcient accumulation and retention within tumors due to limited speciﬁcity forthe target, and an inability to traverse the BBB. A nanoprobe has been designed thatcan cross the BBB and speciﬁcally target brain tumors in a genetically engineeredmouse model, by using in vivo magnetic resonance and biophotonic imaging, aswell as histologic and biodistribution analyses (Veiseh et al. 2009). The nanoprobeis made of an iron oxide nanoparticle coated with biocompatible PEG-grafted chi-tosan copolymer, to which a tumor-targeting agent, chlorotoxin (a small peptideisolated from scorpion venom), and a near-IR ﬂuorophore are conjugated. Theparticle was about 33 nm in diameter when wet, i.e. about a third the size of similarparticles used in other parts of the body. The nanoprobe shows an innocuous toxic-ity proﬁle and sustained retention in tumors. The nanoparticles remained in mousetumors for up to 5 days and did not show any evidence of damaging the BBB. Withthe versatile afﬁnity of the targeting ligand and the ﬂexible conjugation chemistryfor alternative diagnostic and therapeutic agents, this nanoparticle platform can bepotentially used for the diagnosis and treatment of a variety of brain tumors. Theﬂuorescent nanoparticles improved the contrast between the tumor tissue and thenormal tissue in both MRI and optical imaging, which are used during surgery tosee the tumor boundary more precisely. Precise imaging of brain tumor margins isimportant because patient survival for brain tumors is directly related to the amountof tumor that can be resected.Nano-imaging could also help with early detection of brain tumors. Currentimaging techniques have a maximum resolution of 1 mm. Nanoparticles couldimprove the resolution by a factor of 10 or more, allowing detection of smallertumors and earlier treatment. Future research will evaluate this nanoparticle’spotential for treating tumors. It has already been shown that chlorotoxin combined
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