2. 478 13 Role of Biotechnology in Drug Delivery to the Nervous System
Historical Evolution of Drug Delivery for CNS Disorders
Landmarks in the historical evolution of drug delivery technology to the brain are
shown 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 to
administer 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 injected
into 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 fluid
have 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 attributed
to Lewandowski in 1900. Despite the BBB, intracerebral distribution of various
substances was observed. “Barrière hémato-encéphalique” was defined as a cerebral
blood vessel compartment in which choroid plexus was semipermeable, facilitating
the flow of substances from the blood into the CSF (Stern and Gautier 1921). BBB
permeability 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 first
injections into the cerebral circulation were of contrast materials for cerebral angiog-
raphy (Moniz 1927). The injection of a therapeutic substance (diazepam) into the
carotid 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 for
placing instruments at selected targets in the depths of the brain for the treatment
of movement disorders (Spiegel et al. 1947). This approach was used some years
later to perform chemopallidectomy by injection of a mixture of procaine and alco-
hol into the globus pallidus (Cooper 1954). The techniques of creating lesions in
basal ganglia have been refined, but these principles of localization and injection
continue to be used for the introduction of novel therapeutic agents into the brain
for the treatment of movement disorders. The first implantable pump for intrathecal
and intraventricular injection of morphine for the treatment of cancer pain was
described in 1978 (Lazorthes et al. 1991). During the past 25 years, further progress
has taken place with the development of intra-arterial chemotherapy, direct injec-
tions of therapeutic substances into intracranial lesions, and strategies to overcome
the BBB. Further advances have taken place with the development of cell and gene
therapies as well as nanobiotechnology.
The Neurovascular Unit
A neurovascular unit, consisting of endothelial cells, neurons, and glia, regulates the
BBB. The fact that endothelial cells of brain capillaries differ greatly from those in
the periphery confers on the BBB its discriminatory characteristics. Brain endothelial
cells display tight junctions, absence of intercellular clefts and fenestrations, minor
6. 482 13 Role of Biotechnology in Drug Delivery to the Nervous System
Receptor-Mediated Peptide and Protein Transcytosis
The transport of peptides and proteins across cellular barriers- transcytosis- has
been documented in a number of systems. Examples include the transport of IgG
across the intestinal epithelium and human placenta, the transport of insulin and
insulin-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 bidirectional
transport 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 to
maintain their function and brain may substitute its iron through transcytosis of
iron-loaded transferrin across the brain microvasculature. Other biologically active
proteins such as insulin and immunoglobulin G are actively transcytosed through
BBB endothelia. The presence of receptors involved in the transcytosis of ligands
from the blood to the brain offers opportunities for developing new approaches to
the delivery of therapeutic compounds across the BBB.
Molecular Biology of the BBB
The molecular composition of the BBB has been studied by immunocytochemistry,
and the results of these studies show that the BBB exhibits a specific collection of
structural and metabolic properties that are also found in the tight-transporting epi-
thelia. These conclusions are substantiated by the use of antibodies that recognize
proteins of nonBBB origin and BBB-specific proteins. BBB-specific immunoprobes
have a potential application for investigating the pathomechanisms that lead to the
breakdown of BBB. Different patterns of BBB disintegration are anticipated under
different pathological conditions, e.g., inflammatory reactions versus tumors.
Genes that are selectively expressed at the BBB have been cloned. These include
GLUT-1 (glucose transporter) and GGTP (gamma-glutamyl transpeptidase). The
BBB GLUT-1 transporter maintains the availability in the brain of glucose and the
regulation of the protein is mediated at the levels of the gene transcription, mRNA
translation and stability, and posttranscriptional processes. GLUT-1 plays a role in
the 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 for
intactness of the tight junctions with resulting leaky BBB and vasogenic cerebral
edema (Zheng et al. 2010). This finding suggests that research into modulation of
GLUT-1 expression may lead to therapeutic strategies for preventing vasogenic
cerebral 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
7. 483Passage of Substances Across the Blood–Brain Barrier
clinical relevance for the development of new therapeutic strategies for neurologic
disorders.
Genomics of BBB
Genomic and proteomic analyses have been used to study the BBB and how it
relates to the pathogenesis of major neurologic diseases. Shear stress associated
with blood flow in arteries has variable effects on endothelial cells, which are
modulated 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 of
developing 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 in
BBB research. Genomic methods include gene microarray technologies, serial
analysis of gene expression (SAGE) and suppression subtractive hybridization
(SSH). Gene microarray technologies are useful for generating semiquantitative
data 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 all
expressed 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 SAGE
has been reported (Enerson and Drewes 2006). This resulted in identification of 864
genes, including several known for their abundant expression at the BBB, such as
the transferrin receptor. Sorting enriched genes based on function revealed groups
that encode transporters (11%), receptors (5%), proteins involved in vesicle
trafficking (4%), structural proteins (10%), and components of signal transduction
pathways (17%). This genomic repertoire emphasizes the unique cellular pheno-
type existing within the brain and further implicates the BBB as a mediator between
the brain and periphery. These results may provide a useful resource and reference
point from which to determine the effects of different physiological, developmental,
and disease processes on BBB gene expression. Currently, some of the research
priorities include examination of the genes and proteins that are uniquely expressed
by the intact BBB and mechanisms by which brain cells regulate endothelial cell
gene expression.
Proteomics of BBB
Proteomics analyses are currently being used to examine BBB function in healthy
and diseased brain to better characterize this dynamic interface. Because the levels
of mRNA and protein in cells do not always correlate, proteomic methods have been
developed to examine proteins, the real actors in many cell functions. A widely used
8. 484 13 Role of Biotechnology in Drug Delivery to the Nervous System
technique for creating differential proteomic profiles is 2D polyacrylamide gel elec-
trophoresis (2D PAGE). This technique separates proteins according to charge and
mass allowing for the resolution of up to 10,000 individual protein spots on a single
gel. Mass spectrometry (MS) is often used in conjunction with this method to iden-
tify the resolved proteins. Another proteomic technique employed in BBB research
uses isotope coded affinity tags (ICAT). Labeling of protein samples with discrete
isotope tags allows for a semiquantitative comparison of protein expression using
MS. Finally, similar to genomic arrays, proteomic arrays can be used to evaluate
differential protein expression. One type of protein array used in BBB research is the
antibody array. In this technology, antibodies are immobilized at high concentrations
on a substrate to capture antigens from protein mixtures such as cell lysates.
It is becoming increasingly evident that proteomic approaches have the potential to
clarify 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 against
these targets. It has been estimated that approximately 3% of the proteins encoded by
the human genome function as molecular transporters. It is also likely that a significant
portion 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 expression
profiling 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 cells
were profiled using microarray and two-dimensional gel electrophoresis. It was dis-
covered that cell adhesion, apoptosis, and chemotaxis genes were differentially
expressed, and these findings were corroborated by proteomic analysis.
Damage to BBB Manifested as Increased Permeability
BBB is damaged in several neurological disorders such as stroke, TBI, brain tumors,
infections, multiple sclerosis, and neurodegenerative disorders. The impairment of
permeability of BBB is variable and cannot be used to improve drug delivery for
treatment 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 BBB
In animal studies, BBB permeability can be quantitatively evaluated by measuring
the concentration of non-permeable radioactive materials, traceable macromolecules
or dyes in the brain. However, this approach is not applicable in humans due to its
invasiveness and the potential dangers. Therefore, in most human studies, BBB per-
9. 485Passage of Substances Across the Blood–Brain Barrier
meability is usually qualitatively evaluated using brain imaging techniques. Two
main 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 of
damage, and (2) functional imaging is used to study the transport of substances
across the BBB − both intact and damaged. Structural imaging employs contrast
agents with CT scanning and is relatively insensitive. MRI with the contrast agent
gadolinium is more sensitive. Functional imaging is done with PET and can quantify
cerebral uptake of therapeutic agents, such as cytotoxic agents and MAbs. SPECT
is less versatile than PET, but can provide semiquantitative measurement of BBB
leakage of albumin or red blood cells. Quantitative approaches are available to mea-
sure BBB permeability using differentiated images and statistical analyses of CT or
MRI images following the administration of standard contrast agents. This enables
quantification of the spatial characteristics of BBB-disruption and behavior of con-
trast agents with time under different neurological conditions. This method may
enable assessment of the functional implications of the BBB integrity in various
CNS diseases as well as matching the required drug dose with BBB penetrability in
specific patients. PET can also be used to image regional P-glycoprotein activity and
inhibition at the human BBB as it affects the distribution of drugs in the various
brain regions protected by the barrier (Eyal et al. 2010).
Biomarkers of Disruption of BBB
There 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 believed
to be a precursor to hemorrhagic transformation (HT) and poor outcome in acute
stroke patients. A novel MRI biomarker has been used to characterize early BBB
disruption 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 as
defined by this imaging biomarker may be a promising target for adjunctive therapy
to reduce the complications associated with thrombolytic therapy, broaden the
therapeutic window, and improve clinical outcome in acute stroke.
The astrocytic protein S-100beta is a potentially useful peripheral biomarker of
BBB permeability. Other biomarkers of BBB have been recently discovered by
proteomic approaches. These proteins are virtually absent in normal blood, appear
in serum from patients with cerebral lesions, and can be easily detected.
Peripheral assessment of BBB opening can be achieved by detection in blood of
brain-specific proteins that extravasate when these endothelial junctions are
breached. A proteomic approach was used to discover clusters of CNS-specific
proteins with extravasation into serum that correlates with BBB openings. Protein
profiles from blood samples obtained from patients undergoing BBB disruption
with intra-arterial hyperosmotic mannitol are compared with pre-BBB opening
serum. A low molecular weight protein (14 kDa) was identified by MS as transthy-
11. 487Passage of Substances Across the Blood–Brain Barrier
to the exposure of the whole brain to the therapeutic agent. Genetic and other
defects leading to brain changes in Down’s syndrome, Alzheimer’s disease, amyo-
trophic lateral sclerosis, Huntington’s disease, Gaucher disease, hypertension, and
other disorders are rapidly being identified. Several effective therapeutic agents are
available but their use is limited pending improvement of drug delivery across the
BBB. In silico methods are available to predict BBB penetration by drugs
(Lanevskij et al. 2010).
2B-Trans™ Technology
Systems directed at endogenous receptor-mediated uptake mechanisms have been
shown to be effective in animal models including primates. Various systems use the
low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-
related protein 2 receptor (also known as megalin and glycoprotein 330) or the
diphtheria 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 effective
transport system with a specific carrier protein that has an excellent proven safety
profile in human (Gaillard and de Boer 2008). Advantages of 2B-Trans™ receptor
are as follows:
It uses receptor-mediated endocytosis, an effective and safe transport mecha-•
nism, for delivery of large proteins and liposomes containing drugs and genes
across 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 targeting
to-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 BBB
A significant number of lipid soluble molecules, among them many useful thera-
peutic drugs have lower brain permeability than would be predicted from a
determination of their lipid solubility. These molecules are substrates for the ABC
efflux transporters, which are present in the BBB and BCSB, and the activity of
these transporters very efficiently removes the drug from the CNS, thus limiting
brain uptake. P-glycoprotein (Pgp) was the first of these ABC transporters to be
described, followed by the multidrug resistance-associated proteins and more
recently breast cancer resistance protein. All are expressed in the BBB and BCSFB
and 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
12. 488 13 Role of Biotechnology in Drug Delivery to the Nervous System
influence of these ABC transporters and for the design of effective drugs that are
not substrates and the development of inhibitors for the ABC transporters becomes
a high imperative for the pharmaceutical industry (Begley 2004).
Another potentially promising approach to enhancing the delivery of otherwise
non-permeating drugs to the brain is the use of excipients, which are able to interact
with ABC transporters and modify function. Certain Pluronic block co-polymers
appear to target pgp by two mechanisms: depletion of cellular ATP and altering the
physicochemical properties of the membrane lipid phase. ABC transporters may be
of therapeutic benefit in situations where acute dosing is indicated. However, it is
uncertain whether chronic administration of blocking agents is feasible given the
protective role of these transporters in the BBB and other organs. Several strategies
designed to bypass pgp at the BBB without direct inhibition have been described and
tested including a system that uses antibody-coupled immunoliposomes to transport
pgp substrates. The strategy is to move the encapsulated drug through the lumenal
plasma membrane of capillary endothelial cells avoiding direct interaction with pgp.
Immunoliposomes, which are coupled to an antitransferrin receptor antibody, have
been 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. Similar
results 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 side
effects of non-CNS drugs. From the molecular biology and pharmacology of the
proteins involved, it might be possible to identify specific probes to distinguish
transporter 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 drug
delivery to the CNS.
Carrier-Mediated Drug Delivery Across the BBB
Of the various carrier systems, those for glucose and neutral amino acids have
high enough transport capacity to hold promise of significant drug delivery to the
brain. Glucose transporter has the limitation that only molecules closely resem-
bling D-glucose are transported. Neutral amino acids are less specific. Entry via
this carrier may explain the central effects of the muscle relaxant baclofen.
Transport systems for peptides may prove to be effective targets for peptide drugs
required to control natural peptide hormones.
Drugs used to treat neurologic disorders appear to cross the BBB more easily
when an ascorbic acid molecule is attached. Ascorbic acid works like a shuttle and,
theoretically, could transport any compound into the brain. The ascorbic acid
SVCT2 transporter, which is believed to play a major role in regulating the transport
of ascorbic acid into the brain, provides a targeted delivery to the brain. Potential
applications include drugs to treat neurodegenerative diseases, e.g. AD and PD.
13. 489Passage of Substances Across the Blood–Brain Barrier
A feasible method to achieve carrier-mediated drug transport into the rat brain
was shown to be via the specific, large neutral amino acid transporter (LAT1) by
conjugating a model compound to L-tyrosine (Gynther et al. 2008). A hydrophilic
drug, 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 determined
with an in situ rat brain perfusion technique and found to be concentration-depen-
dent. Moreover, a specific LAT1 inhibitor significantly decreased the brain uptake
of the prodrug.
The iron binding protein p97 (melanotransferrin) is closely related to Tf and
lactoferrin and as a result of alternative splicing, it exists in both a soluble form and
a cell surface GPI-linked form. However, in normal brain it appears to discretely
localize on the surface of endothelial cells and transiting through brain capillary
endothelium. Studies on the structure and function of p97 suggest it might be an
ideal carrier for transport of drug conjugates into the brain. A study provides the
initial proof of concept for p97 as a carrier capable of shuttling therapeutic levels
of 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). This
novel delivery platform may be useful in various clinical settings for therapeutic
intervention 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 the
BBB. Glutathione is found in high levels in the brain and cerebral vasculature.
Glutathione has favorable antioxidant-like properties and plays a central role in
detoxification of intracellular metabolites. Glutathione transporters are conserved
across all mammalian species, including humans. Glutathione is considered to be
safe to administer to humans for a prolonged period of time. Glutathione is mar-
keted as functional food ingredient and antioxidant, and applied as supportive
therapy in cancer and HIV treatments and as excipient in parenteral formulations.
Glutathione coated on the surface of nanosized liposomes was shown to be well
tolerated and to effectively deliver several classes of drugs to the brain in a range
of experimental studies reproducibly performed by independent laboratories. This
is the basis of G-Technology® (to-BBB), which uses pegylated liposomes coated
with glutathione, an endogenous tripepeptide transporter expressed on the BBB, to
facilitate delivery of drugs to the brain (Gaillard 2011). Applied to anticancer drugs,
it improves targeted delivery to the brain tumors after systemic administration and
reduces adverse effects. Glutathione pegylated liposomal doxorubicin (2B3-101) is
in phase I/II clinical trials in patients with brain cancer. G-Technology has also been
applied for delivery of methylprednisolone, which is used for several diseases with
a neuroinflammatory component. A product, 2B3-201(to-BBB), is in preclinical
studies for potential applications in multiple sclerosis, acute spinal cord injury and
lupus erythematosus involving the CNS (Gaillard et al. 2012).
14. 490 13 Role of Biotechnology in Drug Delivery to the Nervous System
Glycosylation Independent Lysosomal Targeting
Enzyme replacement therapies use a novel, proprietary technology, known as
Glycosylation Independent Lysosomal Targeting (GILT), which improves the
delivery of lysosomal enzymes to clinically significant tissues (LeBowitz 2005).
A target directing molecule (the tag) is embedded within the therapeutic enzyme to
promote internalization into cells. The internalization process is accomplished by
the binding of the tag to a receptor found on the surface of the target cell therefore
facilitating endocytosis. GILT technology might be used to deliver drugs across the
BBB. This would be an important application because several lysosomal storage
diseases have profound neurological components and conventionally glycosylated
drugs are unable to address this problem.
Inhibition of P-glycoprotein to Enhance Drug Delivery Across the BBB
P-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 number
of prescribed drugs, contributes to the poor success rate of CNS drug candidates, and
patient-to-patient variability in response to CNS pharmacotherapy. It pumps out
some 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 in
the 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 several
signaling pathways have been mapped (Miller et al. 2008). Three pathways that are
triggered by elements of the brain’s innate immune response are: (1) by glutamate;
(2) by xenobiotic-nuclear receptor (pregnane X receptor) interactions; and (3) by
elevated Ab levels. Signaling is complex, with several pathways sharing common
signaling elements − TNFR- 1, endothelin B receptor, PKC, and NOS − suggesting
a regulatory network. Several pathways include autocrine/paracrine elements,
involving release of the proinflammatory cytokine, TNF-a, and the polypeptide
hormone, endothelin-1. Several steps in signaling are potential therapeutic targets
that 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 specifically
reduce P-gp function and thus selectively increase brain permeability of P-gp
substrates. A crosslinked dimer of galantamine, Gal-2, inhibits P-gp mediated
efflux mechanism at the BBB by competing for the substrate binding sites (Namanja
et al. 2009). Several specific inhibitors of P-gp as efflux transporters are in clinical
development.
LipoBridge™ Technology
LipoBridge™ (Genzyme Pharmaceuticals) temporarily and reversibly opens tight
junctions to facilitate transport of drugs across the BBB and into the CNS.
15. 491Passage of Substances Across the Blood–Brain Barrier
LipoBridge 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 a
simple mixture of Lipobridge™ and model compounds or pharmaceutical actives
can deliver these actives into one or both hemispheres of the brain allowing for
increased concentration in a selected hemisphere. It can be administered orally as
well as intravenously. LipoBridge has been used to administer anticancer drugs for
brain cancer in animals. Safety clinical studies in humans are in progress.
Modification of the Drug to Enhance Its Lipid Solubility
There is a good correlation between the lipid solubility of a drug and the BBB
penetration in vivo. The lipophilic pathway also provides a large surface area for
drug delivery. It is approximately 12 m2
in an average human brain. Therefore,
addition of hydrophobic groups to molecules increases their ability to penetrate the
BBB. Addition of methyl groups in a series of barbiturates improves lipophilicity
and brain penetration, leading to increased hypnotic action. It is also possible to
generate a lipophilic prodrug that is broken down to release the more active drug
within the brain. An example of this is heroin, which enters the brain readily due to
its lipophilicity but, after entry, hydrolyses to morphine, which is less lipophilic and
less likely to diffuse back across the BBB, leading to prolongation of duration of
its 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 delivery
of antiviral agents. An example of this is improvement of the BBB penetration of
GABA (gamma amino butyric acid), an anticonvulsant agent with poor CNS pen-
etration, by use of lipophilic esters.
Although increasing lipophilicity generally increases penetration across the
BBB, 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 and
rate of bioconversion are required.
Lipid-binding carriers may reduce the binding of neurotrophic factors to serum
lipids and increase transport across the BBB. Liposomes have been considered, but
their size is too large to cross the BBB.
Monoclonal Antibody Fusion Proteins
These involve conjugation of a drug to a transport vector. These have diagnostic and
therapeutic applications for the treatment of brain tumors. Nontransportable
specific antigen-binding monoclonal antibodies such as IgG3 have been attached to
a transport vector such as insulin-like growth factor. The bifunctional molecule can
cross the BBB through interaction with the receptor for insulin-like growth factor.
16. 492 13 Role of Biotechnology in Drug Delivery to the Nervous System
Transferrin is a specific receptor for molecules that are not synthesized in the
brain but play an essential biological role. This transfer mechanism can be exploited
in an approach in which antiferritin receptor antibodies are covalently linked to
NGF, resulting in a substantial transfer of biologically active nerve growth factor
across 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 an
endogenous BBB receptor-mediated transcytosis system, such as the transferrin
receptor. The MAb carries any drug attached to it across the BBB. However, the
number of small molecules that can be conjugated to monoclonal antibodies vectors
is limited. The carrying capacity of the vector can be greatly expanded by attaching
liposomes to the vector.
A new generation of multifunctional fusion proteins are being engineered at
ArmaGen Technologies to cross the BBB following intravenous administration and
to produce a therapeutic effect on brain disorders (Boado 2008). These fusion pro-
teins are comprised of both a transport and a therapeutic domain. The transport
domain 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 access
to the brain via receptor-mediated transcytosis without interfering with the normal
transport of insulin. Both human-chimeric and fully humanized versions of the anti-
human HIRMAb have already been produced. The therapeutic domain of these
fusion proteins consists of the peptide or protein of interest fused to the carboxyl
terminus of the CH3 region of the heavy chain of the anti-human HIRMAb. A
variety of HIRMAb fusion proteins were engineered aiming at the development of
therapeutics 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, and
HIRMAb-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 biotherapeutic
family discussed in the present review, designed to overcome the brain drug deliv-
ery hurdle, are positioned to become a new generation of neuropharmaceutical
drugs for the treatment of human CNS disorders.
Neuroimmunophilins
Neuroimmunophilin ligands are small molecules that in can repair and regenerate
damaged nerves without affecting normal, healthy nerves. Neuroimmunophilin ligands
may 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 of
allograft rejection following organ transplantation. Immunophilins can regulate neu-
ronal survival and nerve regeneration although the molecular mechanisms are poorly
understood. Neuroimmunophilin can be administered orally and can cross the BBB.
17. 493Passage of Substances Across the Blood–Brain Barrier
Peptide-Mediated Transport Across the BBB
Under 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 deliver
successfully various cargoes across the cell membrane into the cytoplasm or
nucleus. Some of these vectors have also shown the ability to deliver hydrophilic
molecules across the BBB.
Another approach involves forming a chimeric peptide by coupling an otherwise
nontransportable drug to a BBB transporter vector by a disulfide bond. The chime-
ric peptide is then endocytosed by the capillary endothelial cells and transported to
the 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 or
penicillin 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 BBB
Lipid-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, other
small molecules lacking these molecular properties, antisense drugs, and peptide-
based pharmaceuticals ordinarily undergo negligible transport through the BBB in
pharmacologically significant amounts. Some small-molecule neuroprotective
agents have failed in human trials due to poor transport of these agents across the
BBB. Strategies that enable drug transport through the BBB arise from knowledge
of the molecular and cellular biology of BBB transport processes. As biology-driven
drug discovery progresses, more large molecules are being discovered as potential
therapeutics. Some of the strategies for samll molecules transport may be used for
transporting larger molecules such as gene medicine and recombinant proteins.
Trojan Horse Approach
Attaching an active drug molecule to a vector that accesses a specific catalyzed
transporter mechanism creates a Trojan horse-like deception that tricks the BBB
into welcoming the drug through its gates. Transport vectors, such as endogenous
peptides, modified proteins, or peptidomimetic MAbs are one way of tricking the
brain into allowing these molecules to pass. Intravenously administered molecules,
attached to Trojan horses for CNS effect in experimental animals, are shown in
Table 13.4.
Biopharmaceuticals, including recombinant proteins, MAb therapeutics, and
antisense or siRNAs, cannot be developed as drugs for the brain, because these
large molecules do not cross the BBB. Biopharmaceuticals must be re-engineered
20. 496 13 Role of Biotechnology in Drug Delivery to the Nervous System
The high level of expression of transferrin receptors (TfR) on the surface of
endothelial 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 neuroprotective
drug for stroke that does not readily cross the BBB because of its strong polar
nature. Low concentrations of citicoline encapsulated in transferrin-coupled lipo-
somes could offer therapeutic benefit in treating stroke compared to administration
of 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 of
either fusion partner (Prince et al. 2004). Studies indicate that radio-labeled
NeuroTrans™ may be transcytosed across the BBB and, that fusions between
NeuroTrans™ and therapeutic proteins may be manufactured economically (Pan
et al. 2004). Scanning electron microscopy imaging is being used to determine
whether 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 of
NeuroTrans™ within various brain compartments.
Use of Nanobiotechnology for Therapeutic Delivery
Across the BBB
Among 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 are
reviewed elsewhere (Jain 2012). Some strategies use multifunctional NPs. An
important application of nanobiotechnology is delivery of therapy for brain tumors
across the BBB as well as combination of diagnostics with therapeutics. Despite
some current limitations, future prospects for NP-based therapeutic delivery to the
brain are excellent.
Delivery of Cell Therapy to the Brain
Cell therapy is described in Chap. 12. Although cells may deliver therapeutics
themselves, there is also a need for drug delivery systems for cell therapies. Various
methods of delivery of cells for therapeutic purposes are listed in Table 13.5.
22. 498 13 Role of Biotechnology in Drug Delivery to the Nervous System
2005). 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. The
preferential cell adhesion of the cells to be grafted on the microcarriers permits
their preparation or transformation in vitro without the use of enzymes of animal
origin. The cell adhesion molecules as well as the growth factors may induce the
survival and differentiation of stem cells towards a determined phenotype. The
microspheres are spontaneously degraded, without toxicity and without interfering
with the activity or integration of the grafted cells, in a few weeks or months after
implantation, depending on the composition of the polymer. PAM may serve as a
support for cell culture and may be used as cell carriers presenting a controlled
delivery of active protein. They can thus support the survival and differentiation of
the transported cells as well as their microenvironment. They reduce the host
immune reaction and favor the tissue integration of the grafted cells.
To develop this tool, nerve growth factor (NGF)-releasing PAM, conveying
PC12 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 parameters
such as the size of the microcarriers, the conditions enabling the coating of the
microparticles and the subsequent adhesion of cells were thus studied to produce
optimized PAM. NGF-releasing PAM coated with fibronectin plus polylysine and
transporting PC12 cells were evaluated in an animal model of PD. After transplan-
tation, the PAM induced the differentiation, reduced cell death and proliferation of
the PC12 cells and the animals presented an ameliorated behavior.
PAM may be used in any type of cell therapy: (1) tissue reconstruction by
implanting 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 gene
transfer; 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 Specific
Tissues via Circulation
Minimally invasive delivery of a large quantity of viable cells to a tissue of interest
with high engraftment efficiency is a challenge in cell therapy. Low and inefficient
homing of systemically delivered MSCs, e.g., is considered to be a major limita-
tion of existing MSC-based therapeutic approaches, caused predominantly by
inadequate expression of cell surface adhesion receptors. The surface of MSCs
was modified without genetic manipulation with a nanometer-scale polymer con-
struct containing sialyl Lewisx (sLex) that is found on the surface of leukocytes
and mediates cell rolling within inflamed tissue (Sarkar et al. 2011). The sLex
engineered MSCs exhibited a robust rolling response on inflamed endothelium
23. 499Delivery of Cell Therapy to the Brain
in vivo and homed to inflamed tissue with higher efficiency compared with native
MSCs. This is a simple method to potentially target any cell type to specific tissues
via the circulation.
Devices for Delivery of Cell Therapy
A 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 cell
therapy (Gimi et al. 2005). Because of their metallic nature, the location of cubic
containers in the body could easily be tracked by MRI. The microcontainers could
someday incorporate electronic components that would allow the cubes to act as
biosensors to release medication on demand in response to a remote-controlled
radio frequency signal. Biohybrid implants represent a new class of medical
device in which living cells, supported by hydrogel matrix and surrounded by a
semipermeable membrane, produce and deliver therapeutic reagents to specific
sites within a host.
Cell Encapsulation
Modern encapsulation techniques involve surrounding the cells with selectively
permeable membranes. The pores of the membranes should be small enough to
block entry of immune mediators but large enough to allow inward diffusion of
oxygen and nutrients required for the survival of cells and for outward diffusion of
active molecules produced by the cells. Encapsulation avoids some of the compli-
cations of free cell transplants including local reaction at the site of transplantation
and 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•
manipulation
Pore size should be adequate to allow the passage of oxygen and nutrients for•
the cells
It 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 interactions
between solvents and cells during the formation of thermoplastic-based microcap-
sules. Biodegradable materials can be synthetic or natural and they are degraded
in vivo both enzymatically as well as nonenzymatically. Their by-products are usu-
ally nontoxic and excreted via physiological pathways. Examples of natural biode-
24. 500 13 Role of Biotechnology in Drug Delivery to the Nervous System
gradable materials are human serum albumin and collagen. Because of their cost
and the possibility of contamination, several synthetic biodegradable polymers
have been developed. Polyelectrolyte microcapsules are fragile, both physically and
chemically. Great care is required in handling during transplantation and in vivo
failure 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 that
can 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 regulate
permeability, providing controlled delivery of desired substances.
Encapsulated Cell Biodelivery
The encapsulated cell (EC)-biodelivery (NsGene) is a general biodelivery system
of cell-derived substances to the CNS that provides a controlled, site-specific and
safe delivery of a variety of therapeutic substances. For CNS indications one or
multiple EC-biodelivery devices can be implanted in defined regions of the brain to
deliver any proteins or neurotransmitters across the BBB. EC-biodelivery system
consists of a catheter-like device that in the active portion contains a genetically
modified 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 cells
and the host tissue. The encapsulated cells provide for long-term (>12 months)
secretion of a therapeutic factor from the implanted device. This offers great safety
advantages over direct cell/gene therapy approaches and technical and functional
advantages over pump technologies. The device is compatible with stereotactic
neurosurgical techniques and instrumentation adapters to common stereotactic
frames have been made. EC-biodelivery devices are suitable for intraparenchymal,
intracerebroventricular, or intrathecal placement.
Therapeutic Applications of Encapsulated Cells
in Neurological Disorders
Table 13.6 lists therapeutic applications of encapsulated cells in neurological
disorders.
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.
26. 502 13 Role of Biotechnology in Drug Delivery to the Nervous System
approach is to encapsulate genetically engineered cells and implant them into the
body, e.g., beta-endorphin secreting cells for pain treatment and neurotrophic factor
secreting cells as trophic factors for neurodegenerative diseases. There are some
problems related to implantation including the following:
Safety problems related to the introduction of genetically engineered material•
into the body
Although the cells are protected from rejection by leucocytes and antibodies,•
there is potential rejection by complements and cytokines.
Ferrofluid Microcapsules for Tracking with MRI
Implanting recombinant cells encapsulated in alginate microcapsules to express
therapeutic proteins has been proven effective in treating several mouse models of
human neurological disorders. In anticipation of clinical application, magnetized
ferrofluid alginate microcapsules have been synthesized, which can be tracked
in vivo by MRI (Shen et al. 2005). Ferrofluid-enhanced alginate microcapsules are
comparable to classic alginate microcapsules in permeability and biocompatibility.
Their visibility and stability to MRI monitoring permits qualitative and quantitative
tracking of the implanted microcapsules without invasive surgery. These properties
are important advantages for the application of immunoisolation devices in human
cell/gene therapy.
Delivery of Gene Therapy to the Brain
Gene therapy, a sophisticated method of delivery of therapeutics, is described in
Chap. 13. Various methods of delivery of DNA and genes for therapeutic purposes
that are relevant to neurological disorders are listed in Table 13.7.
Clinical Applications of Biotechnology for CNS Drug Delivery
Several techniques are used for drug delivery in CNS disorders (Jain 2012). This
chapter will emphasize the role of biotechnology. Of the various approaches
described, the following have been used clinically.
28. 504 13 Role of Biotechnology in Drug Delivery to the Nervous System
the entry of anticancer agents into brain tumors in phase III trials. Although this
approach increases the efficacy of the cytotoxic drugs, it also increases their neuro-
toxicity by increasing the permeability of the BBB of the normal brain. This
approach has also been used to facilitate the delivery of adenoviral vectors for gene
therapy of brain tumors and for the administration of bifunctional fusion proteins
of tumor-specific MAbs for the treatment of brain tumors. Opening of the BBB
facilitates the entry of superparamagnetic iron oxide conjugates used as adjuncts to
MRI for diagnosis of brain metastases.
Intraarterial Administration of Therapeutic
Substances for CNS Disorders
Acrylic and thrombosis inducing material for occluding arteriovenous malforma-
tions are administered intraarterilly in neuroradiology and neurosurgery. A number
of drugs have also been administered by this route. This route is used for direct
injection of gene vectors into the arterial circulation of the brain. The approved
method 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 lesions
have been demonstrated by angiography prior to thrombolysis.
The BBB hinders the penetration of anti-HIV drugs into the brain for treatment
of AIDS encephalopathy, promoting viral replication, the development of drug
resistance, and, ultimately, subtherapeutic concentrations of drugs reaching the
brain, leading to therapeutic failure. The specificity and efficiency of anti-HIV drug
delivery can be enhanced by using nanocarriers with specific brain-targeting, cell-
penetrating ligands (Wong et al. 2010).
Drug Delivery to the Brain in PD
GDNF is potentially useful in the treatment of PD (PD), but penetration into brain
tissue from either the blood or the CSF is limited. GDNF was delivered directly into
the 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 infusion
for 6 months via a catheter in patients with advanced PD (Slevin et al. 2005).
Drug Delivery to the Brain in AD
Several 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
29. 505Clinical Applications of Biotechnology for CNS Drug Delivery
bloodstream, improving tolerability and allowing a higher proportion of patients to
receive 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 polysorbate
80 (Wilson et al. 2008).
Drug Delivery in Epilepsy
Special methods of drug delivery would improve the control of seizures, reduce
toxic effects, and increase compliance in patients with epilepsy, such as by use of
long-acting formulations and subcutaneous implants. Overexpression of
P-glycoprotein and other efflux transporters in the cerebrovascular endothelium, in
the region of the epileptic focus, may also lead to drug resistance in epilepsy. This
hypothesis is supported by the findings of elevated expression of efflux transporters
in epileptic foci in patients with drug-resistant epilepsy, induction of expression by
seizures in animal models, and experimental evidence that some commonly used
antiepileptic 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 Glioblastoma
Multiforme
Several innovative therapies inclusing biological are being investigated for treat-
ment of glioblastoma multiforme. Methods relevant to biotechnology are shown in
Table 13.8.
Gene therapy for GBM is described in Chap. 12. Examples of other strategies
are as follows:
Biodegradable polymer implants containing anticancer drugs. Polymer-based
drug delivery to the brain has special applications for the delivery of anticancer
agents 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 cm
of the primary lesion. Strategies to prevent local recurrence include implantation of
delivery devices containing chemotherapeutic agents. Biodegradable polymer
impregnated with carmustine (Gliadel), an approved product, is implanted into the
tumor cavity after surgery improves the survival of patients.
32. 508 13 Role of Biotechnology in Drug Delivery to the Nervous System
cardiotoxicity and also the testicular toxicity of this drug. The drug transport across
the BBB by nanoparticles is due to a receptor-mediated interaction with the brain
capillary endothelial cells, which is facilitated by certain plasma apolipoproteins
adsorbed by nanoparticles in the blood.
A polymeric nanobioconjugate drug based on biodegradable, nontoxic, and
nonimmunogenic polymalic acid as a universal delivery nanoplatform is used for
design of a nanomedicine for intravenous treatment of brain tumors (Ding et al.
2010). The polymeric drug passes through the BTB and tumor cell membrane using
tandem monoclonal antibodies targeting the BTB and tumor cells. The next step for
polymeric drug action is inhibition of tumor angiogenesis by specifically blocking
the 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 trileucine
endosome escape unit results in significantly increased antisense oligonucleotide
delivery to tumor cells, inhibition of laminin-411 synthesis, specific accumulation
in brain tumors, and suppression of intracranial glioma growth compared with pH-
independent leucine ester. The availability of a systemically active polymeric drug
delivery system that crosses BTB, targets tumor cells, and inhibits tumor growth is
a promising strategy of glioma treatment.
In vivo application of nanoparticle-based platforms in brain tumors is limited by
insufficient accumulation and retention within tumors due to limited specificity for
the target, and an inability to traverse the BBB. A nanoprobe has been designed that
can cross the BBB and specifically target brain tumors in a genetically engineered
mouse model, by using in vivo magnetic resonance and biophotonic imaging, as
well as histologic and biodistribution analyses (Veiseh et al. 2009). The nanoprobe
is made of an iron oxide nanoparticle coated with biocompatible PEG-grafted chi-
tosan copolymer, to which a tumor-targeting agent, chlorotoxin (a small peptide
isolated from scorpion venom), and a near-IR fluorophore are conjugated. The
particle was about 33 nm in diameter when wet, i.e. about a third the size of similar
particles used in other parts of the body. The nanoprobe shows an innocuous toxic-
ity profile and sustained retention in tumors. The nanoparticles remained in mouse
tumors for up to 5 days and did not show any evidence of damaging the BBB. With
the versatile affinity of the targeting ligand and the flexible conjugation chemistry
for alternative diagnostic and therapeutic agents, this nanoparticle platform can be
potentially used for the diagnosis and treatment of a variety of brain tumors. The
fluorescent nanoparticles improved the contrast between the tumor tissue and the
normal tissue in both MRI and optical imaging, which are used during surgery to
see the tumor boundary more precisely. Precise imaging of brain tumor margins is
important because patient survival for brain tumors is directly related to the amount
of tumor that can be resected.
Nano-imaging could also help with early detection of brain tumors. Current
imaging techniques have a maximum resolution of 1 mm. Nanoparticles could
improve the resolution by a factor of 10 or more, allowing detection of smaller
tumors and earlier treatment. Future research will evaluate this nanoparticle’s
potential for treating tumors. It has already been shown that chlorotoxin combined
33. 509References
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that ability could extend to medulloblastoma, the most common malignant solid
tumor to affect children.
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