Nanoparticles for Brain Drug Delivery

Abstract

The central nervous system, one of the most delicate microenvironments of the body, is protected by the blood-brain barrier (BBB) regulating its homeostasis. BBB is a highly complex structure that tightly regulates the movement of ions of a limited number of small molecules and of an even more restricted number of macromolecules from the blood to the brain, protecting it from injuries and diseases. However, the BBB also significantly precludes the delivery of drugs to the brain, thus, preventing the therapy of a number of neurological disorders. As a consequence, several strategies are currently being sought after to enhance the delivery of drugs across the BBB. Within this review, the recently born strategy of brain drug delivery based on the use of nanoparticles, multifunctional drug delivery systems with size in the order of one-billionth of meters, is described. The review also includes a brief description of the structural and physiological features of the barrier and of the most utilized nanoparticles for medical use. Finally, the potential neurotoxicity of nanoparticles is discussed, and future technological approaches are described. The strong efforts to allow the translation from preclinical to concrete clinical applications are worth the economic investments.

Full text

Hindawi Publishing Corporation
ISRN Biochemistry
Volume , Article ID ,  pages
http://dx.doi.org/.//
Review Article
Nanoparticles for Brain Drug Delivery
Massimo Masserini
DepartmentofHealthSciences,UniversityofMilano-Bicocca,ViaCadore48,20900Monza,Italy
Correspondence should be addressed to Massimo Masserini; massimo.masserini@unimib.it
Received  March ; Accepted  April 
Academic Editors: H. Itoh, H. Pant, and M. Seno
Copyright ©  Massimo Masserini. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
e central nervous system, one of the most delicate microenvironments of the body, is protected by the blood-brain barrier (BBB)
regulating its homeostasis. BBB is a highly complex structure that tightly regulates the movement of ions of a limited number of
small molecules and of an even more restricted number of macromolecules from the blood to the brain, protecting it from injuries
and diseases. However, the BBB also signicantly precludes the delivery of drugs to the brain, thus, preventing the therapy of a
number of neurological disorders. As a consequence, several strategies are currently being sought aer to enhance the delivery
of drugs across the BBB. Within this review, the recently born strategy of brain drug delivery based on the use of nanoparticles,
multifunctional drug delivery systems with size in the order of one-billionth of meters, is described. e review also includes a brief
description of the structural and physiological features of the barrier and of the most utilized nanoparticles for medical use. Finally,
the potential neurotoxicity of nanoparticles is discussed, and future technological approaches are described. e strong eorts to
allow the translation from preclinical to concrete clinical applications are worth the economic investments.
1. Introduction
At the beginning of the third millennium, due to prolonged
ageing, neurological disorders are growing, with a conse-
quent high social impact due to their prevalence and/or high
morbidity and mortality. For the purpose of calculation of
estimates of the global burden of disease, the neurological dis-
orders are included in two categories: neurological disorders
within the neuropsychiatric category and neurological dis-
orders from other categories. Neurological disorders within
the neuropsychiatric category include epilepsy, Alzheimer
and other dementias, Parkinson’s disease, multiple sclerosis,
and migraine. Neurological disorders from other categories
include diseases and injuries which have neurological sequels
such as cerebrovascular disease, neuroinfections, and neuro-
logical injuries.
Neurological disorders are an important cause of mor-
tality and constitute % of total deaths globally. Among
the neurological disorders, Alzheimer and other dementias
are estimated to constitute .% of the total deaths, while
cerebrovasculardiseaseconstituteabout%ofthetotal
deaths in high income countries in  [].
Presently, there are no eective therapies for many of
them. Scientic and technological researches,from molecular
to behavioral levels, have been carried out in many directions
buttheyhavenotyetbeendevelopedinatrulyinterdis-
ciplinary way, and a denitive response is still far to be
prospected.
e immediate consequence of such condition is that
several pathological disorders involving CNS remain untreat-
able. Examples of diseases include neurodegeneration (e.g.,
amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, Hunt-
ington disease, and Prion Disease), genetic deciencies (e.g.,
lysosomal storage diseases, leukodystrophy), and several
types of brain cancer. Even if candidate drugs for therapy of
such diseases may be already available in line of principle,
they cannot be currently utilized because of their insignicant
access to the central nervous system (CNS), due to the
presence of the blood-brain barrier (BBB) []preventingthe
passage from blood to the brain.
2. The Blood Brain Barrier and Drugs
e BBB is a structure formed by a complex system of
endothelial cells, astroglia, pericytes, and perivascular mast
cells [], preventing the passage of most circulating cells and
molecules [,]. e tightness of the BBB is attributed mainly

ISRN Biochemistry
to the vascular layer of brain capillary endothelial cells which
are interconnected side-by-side by tight and adherens junc-
tions. Tight junctions perform two functions: (i) they prevent
thepassageofsmallmoleculesandionsthroughthespace
betweencells,sothattheirpassagemustoccurbyenteringthe
cells (by diusion or active transport). is pathway controls
the type and amount of substances that are allowed to pass (ii)
they prevent the movement of integral membrane proteins
between the apical and basolateral membranes of the cell, so
that each of the cell membrane surfaces preserves its peculiar
functions, for example, receptor-mediated endocytosis at
the apical surface and exocytosis at the basolateral surface.
ree integral proteins are present at the tight junctions:
occludin, claudins, and junctional adhesion molecules. e
former two constitute the backbone of junction strands while
junctional adhesion molecules are important for tracking of
T-lymphocytes, neutrophils, and dendritic cells from the vas-
cular compartment to the brain during immune surveillance
and inammatory responses. Adherens junctions provide
strong mechanical attachments between adjacent cells and
are built from cadherins and catenins. e compact network
of interconnections is conferring to the endothelial layer of
the BBB a transelectrical resistance > Ωcm2,whichis
the highest among all other endothelial districts. e com-
pactness of the endothelial BBB layer precludes the passage
across intercellular junctions (paracellular passage), limiting
the possibility of exchanges between the two compartments
virtually through passages transiting across the cellular body
(transcellular passage).
However, the BBB is not only a mechanical fence but
also a dynamic biological entity, in which active metabolism
and carrier-mediated transports occur. Nutrients, including
glucose, amino acids, and ketone bodies, enter the brain via
specic transporters, whereas receptor-mediated endocyto-
sismediatestheuptakeoflargermolecules,suchasneu-
rotrophins and cytokines [–]. e BBB prevents the brain
uptake of most pharmaceuticals, with the exception of small
hydrophilic compounds with a mass lower than  Da and
highlyhydrophobiccompoundswithamasslowerthan–
 Da that can cross the membrane by passive diusion [].
e list of BBB-permeant drugs includes opiates (e.g., mor-
phine, methadone, and meperidine), anxiolytics (diazepam,
temazepam), SSRIs (paroxetine), and antipsychotics (chlor-
promazine, promethazine) but does not include the majority
of antibiotics and antitumorals.
As above said, the tightness of the BBB is preventing
the pharmacological therapy of a number of neurological
diseases. It should also be mentioned that a further obstacle
for drugs crossing the cerebral capillary endothelium and
entering the brain parenchyma is represented by the pres-
ence of the P-glycoprotein pump in the BBB, allowing the
recognition of molecules necessary for the brain to enter the
brain and the expulsion of other molecules, pharmaceuticals
included.
Given such premises, it is conceivable that dierent ap-
proaches have been tried to allow pharmaceuticals to over-
come the BBB. ese explorative strategies have been ran-
ging from invasive techniques, for example, through osmotic
opening of the BBB [], to chemical modications of drugs
in order to take advantage of physiological carrier-mediated
transports, or exploiting the so-called “Trojan horse” technol-
ogy, coupling BBB-impermeant pharmaceutical to molecules
able to cross the barrier taking advantage of receptor-
mediated transport systems [].
Alternative routes of administration, able to reach the
brain bypassing the BBB (e.g., intranasal), have been actively
investigated, but in the line of principle they are facing
the constraints of the limited surface of adsorption of the
olfactory bulb, which is minimal compared to that of the BBB,
thus, quantitatively reducing the possibility to reach the brain
with relevant amounts of drugs [].
3. Nanotechnology for Brain Drug Delivery
In the recent years, with the advent of nanomedicine, engi-
neered tunable devices with the size in the order of billionth
of meters have been proposed as an intriguing tool potentially
able to solve the unmet problem of enhancing drug transport
across the BBB [,]. Among dierent devices, nanoparti-
cles (NPs) technology is rapidly advancing. NPs are objects
sized between  and  nm []thatworkasawholeunitin
terms of transport and properties.
e types of NPs that are more popular for biomedical
applications are reported in Figure .
e reasons for this expectation are most of all linked to
the possibility of NPs multifunctionalization, coupled to their
ability to carry drug payloads, included BBB-impermeant
drugs. In particular, the rationale of using NPs for brain drug
delivery is that proper surface multifunctionalization may
promote at the same time either their targeting of the BBB
or the enhancement of its crossing. e possibility for BBB-
impermeant drugs to reach the brain, when vehicled by NPs,
is based upon the fact that their crossing of the barrier will
depend completely on the physicochemical and biomimetic
features of the NPs vehicle and will not depend anymore on
the chemical structure of the drug, which is hindered inside
the NPs.
What makes NPs even more attractive for medical appli-
cations is the possibility of conferring on them features
such as high chemical and biological stability, feasibility of
incorporating both hydrophilic and hydrophobic pharma-
ceuticals, and the ability to be administered by a variety
of routes (including oral, inhalational, and parenteral) [].
Moreover, NPs can be functionalized by covalent conjugation
to various ligands (such as antibodies, proteins, or aptamers)
to target specic tissues. e large surface-area-to-volume
ratio of NPs permits multiple copies of a ligand to be attached
and to dramatically increase their binding anity via the
multivalent functionalization [].
When designing NPs for clinical applications, it should
be remembered that their systemic administration generates
important modications. In particular, the nonspecic inter-
action between the shell of NPs and many classes of proteins
circulating in the bloodstream leads to the adsorption of
opsonins on their surface, forming the so-called “corona.”

ISRN Biochemistry 
Liposomes Solid lipid nanoparticle Non-polymeric micelles
Lipoplex
+
++
+
+
+
+
+
+
+
+
+
Dendrimers Polymeric nanoparticle
Polymeric micelle Nanotub es Silica nanoparticle
Quantum dots Gold nanoparticle Magnetic nanoparticle
F : Dierent types of nanoparticles (NPs). Graphical representation of the most commonly used NPs for biomedical applications. NPs
are typically by a size measuring not more than  nm and have signicant potential for delivering drugs across the blood-brain barrier. e
sizeofquantumdotsisusuallylessthannm.
ese proteins substantially change the bare material proper-
ties determining the removal of NPs from circulation by the
reticuloendothelial system, mainly located in spleen and liver.
e most common approaches used for escaping RES are to
formulate the particles with neutral surface charge, to coat
their surface with dierent hydrophilic surfactants, such as
polysorbates and polyethylene glycol (PEG), and to use small
size nanoparticles (e.g., < nm) []. NPs with these features,
called “stealth,” are able to avoid the reticuloendothelial
system, to display long circulation time and stability in blood,

ISRN Biochemistry
and may be functionalized to successfully target and cross
the BBB []. Finally, NPs should be nontoxic either for
cells in the bloodstream or for healthy bystanding cells and
should be biodegradable and biocompatible, noninamma-
tory and nonimmunogenic []. e possibilities of func-
tionalization of MP for brain drug delivery are depicted in
Figure .
ItisalsoimportanttopointoutthatthetailoringofNPsto
enhance drug delivery to the brain does not necessarily imply
their ability to cross the BBB themselves. It is predictable that
NPs could play this role at least in two ways:
(i) by increasing the drug concentration inside, or at
theluminalsurfaceofBBBcells,establishingalocal
high concentration gradient between blood an brain,
higher than that obtainable aer systemic administra-
tion of the free drug. e gradient should then favor
the enhanced passive diusion of the drug. As for
example, this task could be realized by synthesizing
NPs functionalized to target brain capillary endothe-
lial cells. is feature can be followed or not by their
subsequent uptake from targeted cells [];
(ii) by moving themselves into the CNS, together with
their drug cargo. As for example, this task can be
realized enabling NPs targeting of brain capillary
endothelial cells and their subsequent transcellular
passage across the BBB [].
For the completeness of this review, in the next section, the
main features of NPs common ly utilized for medical purposes
and already utilized, or promising candidates for brain drug
delivery, will be described.
4. Nanoparticles for Medical Applications
4.1. Lipid-Based Nanoparticles
4.1.1. Liposomes. Liposomes are the rst generation of
nanoparticulate drug delivery systems []andareconsti-
tuted by one or more vesicular bilayers (lamellae) composed
of amphiphilic lipids, delimiting an internal aqueous com-
partment. Usually, the liposomal lipid bilayer is composed
of biocompatible and biodegradable lipids, present in bio-
logical membranes. Common liposome constituents are sph-
ingomyelin, phosphatidylcholine, and glycerophospholipids.
Cholesterol, an important component of cell membranes,
is frequently included in liposome formulations because it
decreases the bilayer permeability and increases the stability
of the liposome in vivo. Liposomes are classied on the
base of their size and the number of lamellae as follows (i)
small unilamellar vesicles (SUV) with a size up to  nm
and one bilayer, (ii) large unilamellar vesicles (LUV) with a
size > nm and one bilayer, and (iii) multilamellar vesicles
(MLV) that can reach a size of several 𝜇m and made of many
concentric lipid bilayers.
Liposomes have been largely utilized for brain drug
delivery (for a review see []), for the treatment of cerebral
++++++
Targeting
antibody
Fluorescent
probe
Targeting
aptamer
Drug payload
Cationic
molecules
Targeting
peptide
Drug
PEG/spacer
F : Multifunctionalized nanoparticles (NPs). Graphical rep-
resentation of surface-modied NPs with drugs (incorporated
within the core of NPs or conjugated to the surface), targeting
molecules (antibodies, peptides, aptamers, and cationic molecules)
for brain drug delivery, with PEG for stealthiness and with uores-
cent probe as a tracer.
ischemia [], for delivery of opioid peptides [], and brain
tumours [].
Cationic Liposomes. Cationic liposomes containing positively
charged lipids have been developed and initially used as
transfection vehicles, to deliver genetic material (e.g., DNA)
into the cell, avoiding the lysosomal digestion. e most
commonly utilized cationic lipid is ,-dioleoyl--trimethyl-
ammonium-propane (DOTAP), mixed with dioleoyl-phos-
phatidylethanolamine (DOPE). e cholesterol also increases
the levels of transfection and can potentially reduce the
destabilization of the liposomes in the presence of serum [].
e interactions between cationic lipids and nucleic
acids lead to the formation of structures, which are called
“lipoplexes” []. Lipoplexes are typically formed by direct
mixing between cationic liposomes and DNA solutions.
Positively charged liposomes bind to negatively charged
phosphate molecules on the DNA backbone through electro-
static interactions that are embedded and are shielded from
the environment. Generally, complexes are formed with a
slight excess positive charge to permit them to interact with
the negatively charged cell surface. e cationic liposomes
used are typically small before adding to DNA; however,
complexes formed with DNA exhibit diameters that range
from as small as  nm to structures as large as 𝜇m. Unlike
liposomes, cationic liposomes undergo adsorptive-mediated
endocytosis and internalization in endosomes. Upon acid-
ication at pH  to , DOPE fuses and destabilizes the
endosomal membrane, releasing its contents to the cytosol.
erefore, drugs could be vehicled into endothelial cells,
similarly to DNA, enhancingtheir crossing of the barrier and
reaching neurons. As a proof of this p ossibility, transfection of
neuronal SH-SYY cells was achieved with the lipoplexes at a
degree much higher than the degree obtained with the widely
and commonly utilized transfectant Lipofectamine; cationic

ISRN Biochemistry 
liposomes carrying a photoreactive drug resulted in a laser-
stimulated cytotoxic eect on glioblastoma cells and showed
the ability to improve brain drug delivery of paclitaxel
(a mitotic inhibitor used in cancer chemotherapy) in rodents
in vivo [–].
4.1.2. Solid Lipid Nanoparticles. Solid lipid nanoparticles
(SLN) are a stable lipid-based nanocarrier with a solid
hydrophobic lipid core, in which the drug can be dissolved
or dispersed []. ey are made with biocompatible lipids
such as triglycerides, fatty acids, or waxes. ey are generally
of small size (around – nm) allowing them to cross
tight endothelial cells of the BBB and escape from the
reticuloendothelial system (RES) [].
During their fabrication the melted lipid, mixed with
the drug, is commonly dispersed in an aqueous surfactant
by high-pressure homogenization or microemulsication.
e advantages of SLN are their biocompatibility, drug
entrapment eciency comparatively higher than other NPs,
and the ability to provide a continuous release of the drug
for several weeks []. Moreover, the composition of SLN
can be controlled modifying their surface properties to target
molecules to the brain and to limit RES uptake [].
Several reports are available describing an enhanced drug
delivery to the brain mediated by SLN. For instance, SLN
carrying a calcium channel blocker drug, administered i.v.
into rodent, showed that the drug was taken up to a greater
extent by the brain and maintained high drug levels for a
longer time compared to free drug suspension.
Wang et al. have reported the synthesis of 󸀠,-dioctanoyl-
-uoro--deoxyuridine (DO-FUdR) to overcome the lim-
ited access of the drug -uoro-,-deoxyuridine (FUdR) and
its incorporation into SLN. e results indicated that DO-
FUdR-SLN had brain targeting eciency in vivo of about -
fold compared to free FUdR. ese authors report that SLN
can improve the ability of the drug to penetrate through
the BBB and is a promising drug targeting system for the
treatment of central nervous system disorders [,].
4.2. Polymer-Based Nanoparticles
4.2.1. Polymeric Nanoparticles. Polymeric NPs are composed
of a core polymer matrix in which drugs can be embedded
[–], with sizes usually between  and  nm. A range
of materials have been employed for delivery of drugs. In
particular, in recent years some polymers have been designed
primarily for medical applications and have entered the
arena of controlled release of bioactive agents. Many of
these materials are designed to degrade within the body.
Most popular ones are polylactides (PLA), polyglycolides
(PGA). poly(lactide-co-glycolides) (PLGA), polyanhydrides,
polycyanoacrylates, and polycaprolactone. In spite of devel-
opment of various synthetic and semi-synthetic polymers,
alsonaturalpolymerssuchaschitosancanbeutilized.
Also in the case of polymeric NPs, reports are avail-
able describing an enhanced drug delivery to the brain
mediated by these devices. NPs made of PLGA embedding
antituberculosis drugs (rifampicin, isoniazid, pyrazinamide,
and ethambutol) for cerebral drug delivery were administered
to mice, maintaining high drug levels for – days in plasma
and for  days in the brain, much higher as compared
with free drugs []. In Mycobacterium tuberculosis-infected
mice,  doses of the NPs formulation (against  doses of
conventional free drugs) resulted in undetectable bacteria
in the meninges []. In another research [], polybutyl-
cyanoacrylate (PBCA) NPs were successfully utilized for
delivery of functional proteins into neurons and neuronal cell
lines.
4.2.2. Polymeric Micelles. Polymeric micelles are formed
by amphiphilic copolymers whose aggregation in aqueous
media leads to spheroidal structures with a hydrophilic shell
and a hydrophobic core and with a good grade of stability
[]. Stability can be improved by crosslinking between the
shell or the core chains. Additional tunable features of poly-
meric micelles are the possibility to render them responsive
to external stimuli (pH, light, temperature, ultrasound, etc.)
[], triggering a controllable release of entrapped drugs.
One of the most utilized polymer is Pluronic type, block
copolymer based on ethylene oxide and propylene oxide.
e possibility to use these NPs for brain drug deliv-
ery has been described. For instance, chitosan-conjugated
Pluronic nanocarriers with a specic target peptide for
the brain (rabies virus glycoprotein; RVG) following i.v.
injection in mice displayed in vivo brain accumulation either
of a quantum dot uorophore conjugated to the nanocarrier,
or of a protein loaded into the carrier []. Other studies
have shown an increased central analgesic eect of micellar-
vehicled drug [].
4.2.3. Dendrimers. Dendrimers are branched polymers,
reminding the structure of a tree. A dendrimer is typically
symmetric around the core, and when suciently extended it
oen adopts a spheroidal three-dimensional morphology in
water. A central core can be recognized in their structure with
at least two identical chemical functionalities; starting from
these groups, repeated units of other molecules can originate,
having at least one junction of branching. e repetitions of
chains and branching result in a series of radially concentric
layers with increased crowding. e structure is therefore
tightly packed in the periphery and loosely packed in the
core,leavingspaceswhichplayakeyroleinthedrug-
entrapping ability of dendrimers []. Poly(amidoamine),
or PAMAM, is perhaps the most well-known molecule for
synthesis of dendrimers. e core of PAMAM is a diamine
(commonly ethylenediamine), which is reacted with methyl
acrylate and then with another ethylenediamine to make
the generation- PAMAM. Successive reactions create higher
generations. Albertazzi et al. []showedthatfunctionaliza-
tion of PAMAMs dendrimers has a dramatic eect on their
ability to diuse in the CNS tissue in vivo and penetrate living
neurons as shown aer intraparenchymal or intraventricular
injections.
Kannan et al. [] showed that systemically administered
polyamidoamine dendrimers localize in activated microglia
andastrocytesinthebrainofnewbornrabbitswithcerebral

ISRN Biochemistry
palsy, providing opportunities for clinical translation in the
treatment of neuroinammatory disorders in humans.
5. How NPs Can Cross the BBB
Many medicines are not able to reach the brain due to the
lack of drug-specic transport systems through the BBB. e
development of new strategies based on NPs to enhance the
brain drug delivery is of great importance in the therapy and
diagnosis of CNS diseases and it is based on the interactions
between NPs and the BBB and on their intracellular trac
pathways.
5.1. Crossing the BBB without Functionalization. Although
almost all nanomaterials fall into the class of BBB imper-
meable, some exceptions have been reported in recent years.
For instance, gold and silica NPs have been shown to reach
thebrainandaccumulateinneuronsevenintheabsence
of any specic functionalization, with a mechanism that
substantially is still unknown. In the case of silica the results
indicated that NPs administered to rodents via intranasal
instillation entered into the brain and especially deposited
in the striatum [].InthecaseofgoldNPspreciseparticle
distribution in the brain was studied ex vivo by X-ray micro-
tomography, confocal las er and uorescence microscopy [].
e authors found that the particles mainly accumulate in
the hippocampus, thalamus, hypothalamus, and the cerebral
cortex. e same holds true for Titanium dioxide NPs that
were found to cross the mice BBB particularly when smaller
than  nm [].
5.2. Adsorptive-Mediated Transcytosis. e concept of ad-
sorptive-mediated transcytosis through the BBB was orig-
inally suggested by the observation that cationic proteins
can bind the endothelial cell surface but also cross the BBB
[]. e mechanism, applied to NPs, is based on the proper
functionalization of their surface allowing electrostatic inter-
action with the luminal surface of BBB. Given the presence
of negative charges on endothelial cells []thisinteraction
canbepromotedbyconferringapositivechargetotheNPs
surface.
Dierent procedures can be followed to realize this issue.
A rst possibility is to build up NPs made of components
that are bearing a positive charged at physiological pH (.).
is is the case of nanosized vesicles made of bolaamphiphilic
molecules (amphiphilic molecules that have hydrophilic
groups at both ends of an hydrophobic chain) that, following
intravenous administration to mice, showed a marked accu-
mulation of their encapsulated uorescent cargo, whereas
nonencapsulated probe was detected only in peripheral
tissues but not in the brain []. In another investigation Jin
et al. [] used SLN made with lipids extracted from depro-
teinated lipoproteins and enriched with cationic cholesteryl
hydrochloride and phosphatidyl-ethanolamine. e authors,
aer intravenous administration of such cationic NPs for the
delivery of siRNA inhibiting c-Met expression, suppressed
tumor growth without evident signs of systemic toxicity in
an orthotopic xenogra tumor mice model of glioblastoma.
A second possibility is to functionalize the NPs surface
with positively charged biomolecules combining their physic-
ochemical features with biological activity. is is the case
of cell-penetrating peptides (e.g., TAT peptides derived from
HIV, gH derived from Herpes simplex virus type ) and
cationic proteins (e.g., albumin) [] that have been exten-
sively used for NPs decoration, facilitating the BBB passage
of drugs []. As for example, Rao et al. []aeradmin-
istration of ritonavir via TAT-conjugated NPs demonstrated
approximately an -fold higher level of drug in the brain
when compared to that with free drug, with a remarkable
enhancement of drug delivery. e authors suggested that
TAT-conjugated polymeric NPs are rst mobilized in the
brain vasculatures, then transported to the brain parenchyma
where they continue to release the drug.
Polymeric micelles having TAT molecules on the surface
were successfully fabricated also to incorporate antibiotics
andwereabletocrosstheBBB[]. In another study, Xu
et al. [] showed ecacy of TAT-polymeric NPs for the
treatment of C. albicans meningitis in rabbits. Similar results
are reported using another family of cell-penetrating peptides
(SynB peptides RGGRLSYSRRRFSTSTGR) that were able to
increase drug permeability across a cocultured BBB model,
doubling drug transport in vivo in healthy mice [].
Since the eect of cell-penetrating peptides could be
invalidated by the rapid systemic clearance of functionalized
NPs due to their positive charge, in a study dedicated to
investigate this issue, Xia et al. [] utilized penetratin, a
peptide derived from Antennapedia protein (Drosophila),
with relatively lower content of basic amino acids, to func-
tionalize poly(ethylene glycol)-poly(lactic acid) (PLGA) NPs,
uorescently labeled with coumarin-, and detected an
increase of uorescence in the rodent brain with respect to
unfunctionalized NPs.
Xia et al. [] also combined the use of cell-penetrating
peptide to coumarin--loaded NPs for intranasal adminis-
tration. e amount of uorescent probe, detected in the rat
cerebrum and cerebellum, was found to be more than -fold
compared to that of coumarin carried by unfunctionalized
NPs. Brain distribution analysis suggested that NPs aer
intranasal administration could be delivered to the central
nervous system along both the olfactory and trigeminal
nerves pathways.
e ecacy of cationic albumin to functionalize and
promote NPs-carried drug delivery to the brain has been
repeatedlyreported [,]. e property of cationic proteins
to eciently penetrate cells raises the question of the potential
toxicity and immunogenicity of these proteins. e possi-
ble toxic eects include a generalized increase in vascular
permeability since breakdown of BBB permeability and
othervascularbedshasbeenobservedfollowingintravenous
injection of large amounts of cationic proteins but has not
been seen when moderate amounts of these substances were
administered [].
5.3. Receptor-Mediated Transcytosis. One of the most recently
applied strategies for drug delivery across the BBB endo-
thelium using functionalized NPs is the one exploiting

ISRN Biochemistry 
the transcytosis physiological mechanism of transport of
macromolecules, relying on the presence of specic recep-
tors on the luminal surface of cells. Transcytosis is the
process by which extracellular cargo internalized at one
plasma membrane domain (e.g., apical) of a polarized cell is
transported via vesicular intermediates to the controlateral
plasma membrane (e.g., basolateral). As for example, insulin,
transferrin, apolipoproteins, and -macroglobulin are some
of the proteins that reach the brain following the path across
endothelial cells.
e rationale for exploiting this strategy with NPs is that
the existing cellular mechanisms, handling macromolecular
cargoes,maytalsototransportNPsaertheirfunctional-
ization to interact with the same receptors.
Transcytosis in endothelial cells starts with internal-
ization of extracellular cargo into the cell by vesicular
carriers. e cargo is subsequently processed via dierent
pathways to appropriate intracellular organelles and recycled,
degraded, or transcytosed to the contralateral side. Strong
morphological and biochemical evidence suggest that the
two membranes, apical and basolateral, are interconnected
via vesicular intermediates involving multivesicular bodies
or a system of endosomal vacuoles and tubule vesicles.
Phosphoinositide -kinase, an enzyme increasingly demon-
strated to play an important regulatory role in many vesicular
tracking events, also appears to regulate transcytotic trac
between the two endosomal compartments.
Transcytosis in endothelial cells starts with uptake either
through clathrin-coated pits, by caveolae, or caveolae-like
membrane domains [] that will be described here below.
e rst step of internalization through clathrin-
mediated endocytosis is the binding of a ligand to a specic
cell surface receptor. is results in the clustering of the
ligand-receptor complexes in coated pits on the plasma
membrane, which are formed by the assembly of cytosolic
coat proteins; the main assembly units being clathrin, which
form a polygonal lattice in the surface of the membrane; and
adaptor protein complexes, which mediate the assembly of
the clathrin lattice on the membrane. e coated pits then
invaginate and pinch o from the plasma membrane to form
intracellular clathrin-coated vesicles.eclathrincoatthen
depolymerizes, resulting in early endosomes, which fuse
with each other or with other preexisting endosomes to form
late endosomes that further fuse with lysosomes. Vesicular
tracking aer CME is controlled by the action of small
GTPases, the Rab proteins.
e second mechanisms internalization (caveolar) has
been claried following the tracking of some viruses that
usecaveolaetogainentryintothecells.Caveolaeare
small, ask-shaped invaginations of the plasma membrane
with a size of about – nm that are rich in cholesterol
and sphingolipids. Viruses initially associate with the cell
membrane and then become trapped in relatively stationary
caveolae. e subsequent intracellular uptake of caveolae
leads to intracellular organelles that are distinct from classic
endosomes; the presence of the protein caveolin in these
organelles gave rise to the name caveosomes. In contrast to
the dynamic nature of endosomes, caveolae are highly stable
and are only slowly internalized. Another major dierence is
that the caveolar uptake does not lead to a decrease of pH
and to a degradative pathway of their cargo, as in endoso-
mal/lysosomal pathway.
It is clear that the dierent pathways of NPs internal-
ization aect their delivery to dierent intracellular com-
partments and may aect their nal destination. To obtain
insight into the properties of drug delivery vehicles that
direct their intracellular processing in brain endothelial cells,
the intracellular trac of xed-size nanoparticles in an
in vitro BBB model as a function of distinct nanoparticle
surface modications has been investigated. Previous obser-
vations [] that latex particles with a diameter ≥ nm are
internalized by nonphagocytic B cells through caveolae,
whereas particles up to  nm in diameter are eciently
taken up via clathrin-mediated endocytosis, were succes-
sively conrmed with Silica-Hydroxyl NPs on immortalized
human brain capillary endothelial cell cultures []. In the
investigation of Georgieva et al. []nmparticleswere
used uncoated, surface functionalized with cationic polymer
polyethyleneimine (PEI), or with prion protein. e results
suggested that uncoated NPs are internalized through cave-
olae; nanoparticles carrying a net cationic charge, accom-
plishedbyPEI,followedanadsorptiveendocytoticroute;
and particles surface-modied with prion protein followed
receptor-mediated endocytotic route. erefore, the authors
concluded that the NPs intracellular pathway is dictated by
theparticlesurfacecharacteristic,foragivensize.Most
interestingly, they found that the transcytotic potential is
higher for the receptor-mediated and caveolar pathways and
lower for the adsorptive-mediated.
However, the enhancement of brain delivery obtained
with drug-loaded NPs, exploiting receptor- or caveolar-
mediated transcytosis, is currently quantitatively limited in
comparison with the free drug. Consequently also its clinical
relevance is still scarce. If we compare the hydrodynamic
size of most physiological proteins with that of NPs, we are
confronting objects in the order of – nm size with those
that, in most cases, are in the order of tenths of nanome-
ters and commonly reach  nm, and more. erefore, it
is conceivable that the transcytotic trac may become a
bottleneck when transporting NPs. It is also predictable
that smaller size NPs, closer to the size of physiological
molecules, would be facilitated to enter the same intracellu-
lar transport pathways. Following the strategy of receptor-
mediated transport, NPs made of gold, PLGA, chitosan,
PAA, dendrimers, and liposomes have been functionalized,
improving the delivery of drugs such as caspase inhibitors,
endomorphin, tamoxifen, and tramadol and throwing the
basis for treatment of neurological diseases [–].
Exploring and targeting new receptors that could be
utilized for receptor-dependent endocytosis are likely to
provide more ecient systems of brain drug delivery, mainly
because these uptake mechanisms are relatively unaected by
lysosomal degradation.
5.3.1. Lipoprotein Receptors. Apolipoprotein E (apoE) is a -
kDa protein constituent of both very-low-density lipopro-
tein (VLDL) and high-density lipoprotein (HDL), which

ISRN Biochemistry
transports cholesterol and other lipids in the plasma and in
the CNS [,]. e lipoproteins complexes can be taken
up in the brain through the recognition of apoE by specic
receptors at the BBB, which include the low-density lipopro-
tein receptor (LDLR) and the LDLR-related protein (LRP).
Taking into consideration that LRP has been reported to
be highly expressed on endothelial brain microvessels [],
receptor-mediated transcytosis using NPs functionalized to
bind this target has been exploited in several ways.
First, by exploiting the preferential absorption of ApoE
on some types of NPs when in serum. is feature has been
described for PBCA NPs coated with polysorbate , indeed
displaying the ability to cross the BBB in vivo in animal
models [–]andforPEGylatedPHDCANPs,ableto
penetrate into brain endothelial cells in vitro [].
Taking advantage of such opportunity, the formation of
a stable ApoE decoration of NPs surface by covalent binding
has been utilized to functionalize albumin-NPs or liposomes
that showed the capacity of enhancing BBB passage in vivo in
rodent models [–].
It should be questioned whether other apolipoproteins
may be employed for NPs functionalization to confer on
them the ability to enhance brain drug delivery. In order to
answer this question, in an investigation of Kreuter et al. [],
PBCA NPs loaded with dalargin or loperamide were coated
with dierent apo-lipoproteins, AII, B, CII, E, or J, coated
or not with polysorbate , and then injected in mice and
the drug eect on CNS was evaluated. e results showed
that only NPs coated with apolipoprotein B or E were able to
achieve an antinociceptive eect. is eect was signicantly
higher aer polysorbate-precoating and apolipoprotein B or
E-overcoating [].
e region of apoE that is critical for interaction with the
LDL receptor resides between amino acid residues  and
. Several studies with synthetic peptides have investigated
the structural features of the LDLR-binding sequence of
ApoE [,].
Laskowitz et al. [] derived an apoE-mimetic pep-
tide from amino acids –, named COG (LRVR-
LASHLRKLRKRLL), which retains its biological activity in
vitro and in vivo. is capacity is displayed also by a fragment
of  a.a. from ApoE [].
It has also been reported that the tandem dimer (–)2
is recognized by the LDLR, in contrast to the monomeric pep-
tide (–) []. Also a shorter sequence of this peptide, the
tandem dimer (–)2,retainsthisability[]. Probably
as a consequence of these observations, sterically stabilized
liposomes were functionalized only with ApoE tandem dimer
peptide (–)2, but not with the monomer, and were
showntobeecientlytakenupbyratbraincapillary
endothelial cells []. Interestingly, in spite of the theoretical
premises based on receptor-mediated endocytosis, liposomes
tagged with (–)2were nonselectively internalized into
cultured BBB cells, and clathrin- or caveolin-dependent
endocytosis was not demonstrable [].
However, in a successive investigation the ApoE-derived
peptide monomer – was utilized by Re et al. []
in comparison with the dimer, for the functionalization of
liposomes entrapping a radioactive derivative of curcumin.
e investigation showed a higher uptake of the drug by
human endothelial cells and an enhancement of permeability
of the drug across an in vitro model of the BBB made with the
same cells, when using monomer-functionalized liposomes.
Apartfromtheaminoacidsequence,otherfactorsmay
aect the cellular uptake of these peptides []. ese factors
include the type of peptide, its cationic nature, its mode of
exposure to the cell surface, the nature of the cargo, and the
chemical linkage between the peptide and the cargo, other
than the peptide surface density on the cellular uptake of
nanoparticles [,].
Finally, also Angiopep-, a ligand of LRP, possesses a high
brain penetration capability in both in vitro model of the
BBB and in situ brain perfusion in mice []. PEG-PAMAM
dendrimers functionalized using this peptide resulted in
a high accumulation of a delivered gene into brain aer
intravenous administration into mice []; moreover, also
functionalized polymeric NPs and carbon nanotubes (CNT)
have been produced, showing the ability to reach glioma
tumors in mice [,].
5.3.2. Transferrin Receptor (TfR). e transferrin receptor
(TfR) is the most widely studied receptor for BBB targeting.
TfR is a transmembrane glycoprotein, consisting of two
linked -kDa subunits, each one binding a transferrin
molecule. e receptor is highly expressed on immature ery-
throid cells, placental tissue, and rapidly dividing cells, both
normal and malignant []. Furthermore, it is expressed
onhepatocytesandendothelialcellsoftheBBB.eroleof
the receptor is the regulation of cellular uptake of iron via
transferrin, a plasma protein which transports iron in the
circulation. Cellular uptake starts with the binding of trans-
ferrin to the transferrin receptor followed by endocytosis
[].
Iron-bound transferrin has a high anity for the TfR;
therefore, it has been used with success as a ligand for func-
tionalization and targeting of liposomes to cultured brain
endothelial cells [,]. However, it is likely that that
NPs decorated with transferrin would not be performant
in vivo, since transferrin receptors are almost saturated in
physiological conditions, because of the high circulating
levels of endogenous protein []. Nevertheless, successful
brain targeting using transferrin as a targeting ligand has been
accomplished in vivo [,]. Concerning the specicity of
transferrin, a comparative study has been carried out between
transferrin or lactoferrin, a multifunctional protein of the
transferrin family widely represented in various secretory
uids, such as milk []. Lalani et al. []comparedin vivo
distribution in mice of PLGA NPs surface modied with the
two proteins and showed a better performance to reach the
brain in the case of lactoferrin.
In order to avoid the competition with endogenous trans-
ferrin circulating in blood, the use of monoclonal antibodies
(mAbs) directed against transferrin receptors on the BBB has
been suggested because they recognize dierent epitopes on
the receptor [].Forinstance,OX,D,andRmAb
bind to TfR and have also been shown to undergo receptor-
mediated transcytosis.

ISRN Biochemistry 
Amongthese,itisstilldebatedwhichmAbisthebest
performing to be used for the decoration of NPs surface. e
most well known is OX, an antibody directed against the rat
TfR, that has been successfully used in many brain targeting
studies in vivo []andalsoin vitro using BBB cellular
models []. However, Lee et al. [] have shown that the
OX monoclonal antibody is not an eective brain targeting
molecule in mice. Moreover, they demonstrated that other
two monoclonal antibodies, D and RI, directed against
the mouse TfR, had a higher permeability of the mouse
BBB in vivo, with respect to the brain uptake of the OX
antibody, which was negligible. Additionally, they showed
that RI was more selective for the brain than D, because
this antibody was less taken up by the liver and kidney. It
has been shown that RI covalently coupled to human
serum albumin is able to transport loperamide across the
BBB []. More recently, it has been shown that also the
chemical linkage of anti-TfR on the NPs surface could aect
the crossing of BBB in vitro []. e authors compared
liposomes covalently coupled with mAbs (obtained by react-
ing thiolated mAbs to phosphatidyl-ethanolamine maleimide
inserted in the liposome bilayer) with liposomes decorated
with biotinylated mAbs via an avidin bridge. e Authors
found an higher cellular uptake and permeability across an
in vitro BBB model made of immortalized human brain
capillary endothelial cells of the liposomes covalently coupled
with mAbs, suggesting that the covalent ligation could be
preferentially chosen for NPs decoration.
5.4. Retrograde Transport. Transsynaptic retrograde trans-
port could enable some types of nanocarriers to travel from
peripheral nerve terminals to neuronal cell bodies in the CNS
[].StudiesinthisregardhaveshownthatNPsmodied
with PEI and other polyplexes display active retrograde
transport along neurites but are unable to mediate ecient
biological actions upon reaching the neuronal body [].
5.5. BBB Breakdown. BBBbreakdownoccursinneuroin-
ammatory diseases []. NPs can transiently and reversibly
open the tight junctions located at the BBB and other sites,
thus, increasing their paracellular permeability [,].
In particular, blood-brain barrier disruption therapy is an
intensive, eective way of sending medication to brain tumors
[].
Nevertheless,itisknownthattightjunctionscanbe
opened only to a limited extent []; thus, only NPs smaller
thanaboutnmcanusethispathwaytopenetrateintothe
brainthroughtheBBB.
5.6. Exploiting Monocyte/Macrophage Inltration in the CNS.
Monocyte/macrophage inltration in the CNS plays a key
role in neuroinammation, as well as in lesion development
and brain injury in neurological diseases such as multiple
sclerosis (MS) and stroke [–]. Knowledge of active
phases of cell inltration during CNS disorders is important
because anti-inammatory treatments can target cell adhe-
sion molecules and chemokines guiding cellular tracking
[]. e monocyte inltration through the BBB is also
widely believed to play an important role in HIV infection
of the CNS. e blood-brain barrier appears unaltered until
the late stage of HIV encephalitis. HIV ux that moves
toward the brain, thus, relies on hijacking with immune-
activated leukocytes, mainly monocytes/macrophages from
the periphery [].
In this paper, BBB crossing by immune-activated macro-
phages appears to suggest possible strategies for future ther-
apeutic developments employing NPs. is strategy could
be realized at least in two ways: () by embedding NPs into
activated monocytes used as Trojan horses to reach the brain
() by designing NPs mimicking activated monocytes.
5.6.1. Trojan Monocytes for NPs Delivery to the Brain. Nano-
particulate drug delivery systems have been used to avoid
the RES clearance and achieve longer circulation time for
enhanced tissue uptake. However, the enhancing of NPs
phagocytosis by monocytes can be considered as an uncon-
ventional approach to deliver NPs-loaded drugs to the
brain. Aer phagocytosis of NPs, monocytes, just like Trojan
horses, may transport their cargo into the brain. Afergan
et al. [], taking this approach, embedded serotonin, a
BBB impermeable neurological drug, into negatively charged
liposomes and analyzed brain uptake in rats and rabbits.
e performance of liposomal serotonin was signicantly
better, leading to two-fold higher drug concentration in
brain than the free drug. Since treatment of animals by
alendronate resulted with inhibition of monocytes but not of
neutrophils, and with no brain delivery, the authors suggested
that monocytes are the main transporters of liposomes to
the brain. In spite of the fact that the clinical relevance of
this investigation is limited, the Trojan monocyte approach
provides a new possibility of more eective treatment of
brain-associated inammatory disorders, including multiple
sclerosis and Alzheimer’s disease, which are characterized
with increased passage of immune cells across the BBB
[].
In other examples, in order to improve the delivery of
contrast agents to the brain, iron oxide NPs administered
intravenously in vivo have been shown to detect activated
macrophage inltration in multiple sclerosis either in the
CNS of humans [,] or rodent model [–], in stroke
either in human [,] or animal brain ischemia [–
], in human or murine intracranial tumors [,], in
EAE [–], or in an ischemia/reperfusion long-lived rat
model [].
5.6.2. NPs Mimicking Activated Monocytes. e therapeu-
tic ecacy of drug-loaded NPs systemically administered
depends on their ability to evade the immune system, to
cross the biological barriers of the body, and to localize
at target tissues. Parodi et al. [] show that nanoporous
silicon particles can successfully perform all these actions
when they are coated with cellular membranes puried
from leukocytes, avoiding being cleared by the immune sys-
tem. Furthermore, they can communicate with endothelial
cells through receptor-ligand interactions and transport and
release a doxorubicin payload across an inamed endothelial

 ISRN Biochemistry
barrier in vitro. Also the accumulation of coated NPs in
mice inoculated with murine B melanoma was enhanced
compared with that of non coated NPs. Particle coating
using leukocyte membranes led to an approximately twofold
increase in particle density in the tumor.
Since several studies demonstrate increased passage of
monocytesacrosstheBBBinvariouspathologicalconditions,
it is predictable that the research of NPs mimicking immune
cells might be eective in brain-associated disorders and will
receive a stimulus in this direction in the next years.
6. Factors Affecting NPs Brain Drug Delivery
6.1. NPs Diusion inside the Brain Parenchyma. While this
factor is likely not particularly relevant when brain drug
delivery is sought aer, it could be very important when the
passageoftheBBBbyNPsislookedfor,sincetheextracellular
space (ECS) of the brain parenchyma could limit their diu-
sion or even preclude their entrance into the brain. e ability
to achieve brain penetration with larger NPs is expected to
allow more uniform, longer lasting, and eective delivery of
drugswithinthebrainandmayinparticularndpractice
in the treatment of brain tumors, stroke, neuroinammation,
and other brain diseases where the BBB is compromised or
where local delivery strategies are feasible.
TodayitiswellestablishedthattheECSoccupiesavolume
fraction of between % and % in normal adult brain tissue
with a typical value of %, and that this falls to % during
global ischemia [,].
It is less obvious what the true size of the spaces between
cells is. Small molecules such as inulin and sucrose diuse
through the ECS with a decreased diusion coecient with
respect to water, suggesting the existence of limitation to their
movements [].
Possible sources of the limitations to the diusing mol-
ecules are (a) the presence of cellular obstructions; (b) trap-
ping of molecules in dead-space microdomains; (c) a viscous
drag imposed by the macromolecules that compose the extra-
cellular matrix or drag arising from the walls of the channels
when molecules are large, such as in the case of NPs;
(d) transient binding to cell membranes or extracellular
matrix (e) nonspecic interaction with negative charges on
the extracellular matrix when the diusing molecule has
adequate charge density.
A dierent type of model was proposed by orne and
Nicholson [] to explain the higher tortuosity experimen-
tally measured for larger substances such as dextran and
especially large synthetic quantum dot nanocrystals in the in
vivo rat cortex. It was assumed that the quantum dots with a
hydrodynamic diameter of  nm were close to the average
width of the ECS, and depending on whether a planar or
tubular model was adopted, the estimated ECS width was –
 nm.
In counter tendency to orne and Nicholson [],
Nance et al. []reportthatNPsaslargeasnmin
diameter diused within the human and rat brain, only if
they were densely coated with PEG. Using these minimally
adhesive PEG-coated NPs, they estimated that human brain
tissue ECS has some pores larger than  nm and that more
than one-quarter of all pores are ≥ nm.
In addition to ECS in CNS, there is also much interest in
the possibility that perivascular spaces, uid-lled channels
surrounding arteries, arterioles, veins, venules, and possibly
even microvessels oer potential pathways for rapid ow into
and out of brain parenchyma. Measurements of perivascular
space widths in mammals suggest that their typical dimen-
sions may be at least  orders of magnitude greater than
the neocortical ECS width (e.g., arteriole perivascular spaces
have been reported in the range of –𝜇morlarger(in
rodents and humans)).
6.2. Eect of Protein Corona. Dierent physicochemical
properties, not only size, may determine the ability of
nanoparticles to reach the brain. e situation is further
complicated by the formation of a so-called “corona” of
biomolecules on the surfaces of nanoparticles, the composi-
tion of which may vary depending on the route of exposure,
and on the physicochemical surface properties of NPs. e
acquisition of a corona of biomolecules on the surface of
NPs may also determine whether NPs are able to cross from
one compartment to another and whether they are taken up
eectively by cells or not [].
In practical situations in which nanoparticles interact
with living organisms, the nanoparticle surfaces are initially
exposed to a biological uid, such as blood, depending
on the route of administration. For instance, NPs injected
intravenously would be exposed to blood plasma, containing
an excess of proteins and many other complex biomolecules,
which bind competitively with the surface of the nanoparti-
cles.
e current idea is that “bare” nanoparticles do not
exist in vivo, because they are immediately modied by
the adsorption of blood proteins with higher anities for
theparticlesurface,formingamoreorlesstightlybound
layer (the so-called hard corona) and a weakly associated
mobile layer (the so-called so corona). When this issue was
investigated, it has been found that typical coronas contain
a limited number and types of molecules gathered from
the blood, in spite of the fact that biological uids contain
thousandsofproteins[]. Moreover, it should be noted that
thecoronamayalsoplayaroleinotherundesirableeects
of NPs in living systems such as complement activation and
bloodclottingandmaynotnecessarilyplayaroleonlyin
cellular uptake.
It should be pointed out that none of the in vitro exper-
iments conducted to study the process of NPs translocation
to the brain takes into account the surface modication of
NPs inside the blood and how can this aect the crossing
of the BBB. is issue needs much further investigation.
Moreover, NPs once internalized by endothelial BBB cells,
they may also exit towards the brain covered with dierent
biomolecules depending on that they have undergone endo-
cytosis/transcytosis/exocytosis, and, thus, exert additional
eects (e.g., toxicity) on neuron. Also this issue has not yet
been investigated.

ISRN Biochemistry 
6.3. BBB Alterations in Neurological Diseases. e peculiar
physiological features of the BBB aect the extent of drug
permeability and in most cases extremely restrict the amount
of pharmaceuticals taken up by the brain. It should be pointed
out that the anatomy and organization of the BBB are altered
in a number of pathological conditions, and these changes
could have consequences onto the crossing of the barrier by
physiological circulating molecules, drugs, and NPs. It should
be also pointed out that changes are not the same for all the
diseases: the variability is high; thus, it is hard to predict the
eects on any particular drug. In general, the BBB alterations
couldcauseshisinthedosage,ecacy,andsideeectof
commonly used drugs.
For instance, the eect of hypoxia-ischemia on the barrier
has been extensively investigated. Hypoxia-ischemia starts a
series of events, which lead to increased BBB permeability,
possibly due to disruption of tight junctions and mediated
by signaling molecules such as cytokines and nitric oxide
[].Itisexpectedthatthepassageofmacromoleculesacross
the BBB is increased under this condition. It is therefore
signicant that nanostructured erythropoietin may exert a
neuroprotective action against hypoxia-ischemia in animal
models [].
A variety of evidence demonstrates that the BBB is
also compromised in septic encephalopathy, where albumin
[] has been shown to enter brain parenchyma from the
circulation in rodents.
Also a series of neurologic inammatory diseases, includ-
ing HIV-associated dementia and multiple sclerosis, strongly
alter the integrity of the BBB with consequent migration of
leukocytes into the brain [,]. e migration has been
shown to trigger signals leading to loss of tight junctions
molecules and to opening of the BBB [].
Although there is no evidence in humans or animal
modelsforamassivedisruptionoftheBBBinAlzheimer
disease (AD), there is evidence of a decreased glucose and
oxygen use by the AD brain. If the decrease represents a defect
in the ow across the barrier or in response to a decreased
requirement by the CNS is unclear, however, it is consistent
with the AD environment promoting barrier cell secretions
that have detrimental eects on cognition []. What is clear
is that BBB endothelial cells bind and internalize 𝛽-amyloid
peptide (A𝛽),wherethepeptidemostlyremainsadherentto
or internalized by the cells. A𝛽induces chemokine secretion,
monocyte tracking, decreased proliferation, altered per-
meability, and altered nitric oxide synthase activity. Micro-
lesions representing limited protein leakage at capillaries have
been demonstrated in some animal models of AD [].
Interestingly, one of the few drugs available for the treatment
of AD, the NMDA receptor antagonist memantine, protects
against BBB disruption.
6.4. Size. An underestimated issue is whether or not the size
of NPs designed for reaching the brain drug delivery makes
any dierence. Dierent investigations have been carried
in the attempt to clarify this issue. Sarin et al. []aer
having intravenously administered functionalized PAMAM
dendrimers with size less than approximately  nm found
that NPs were able to traverse pores of the blood-brain tumor
barrier of RG- malignant gliomas, while larger ones could
not.
Sonavane et al. [] studied tissue distribution of col-
loidal gold nanoparticles of dierent sizes (, , , and
 nm) aer intravenous administration in mice. e num-
ber of NPs which entered the brain aer  h was inversely
dependent on the size and for  nm gold NPs was -
fold higher than for  nm NPs, while was very low for
 nm NPs. However, the total amount of gold, which is
proportional to total volume of NPs entered, thus, to the
payload of drug in the case of drug-loaded NPs, was similar
for  and  and only % lower for  nm. Oberd¨
orster
et al. [] generated solid ultrane particles of size around
 nm made of graphite that were administered by inhalation
by rodents. e authors concluded that the CNS can be
targeted by airborne NPs of this size via the olfactory nerve
from the olfactory mucosa.
Recent evidence suggests that the transit through the
gastrointestinal tract may strongly inuence the subsequent
access of NPs into the brain. Hillyer and Albrecht []
studied the gastrointestinal uptake and tissue/organ distri-
bution of , , , and  nm diameter metallic colloidal
gold NPs. e gold NPs were administered orally, and gold
concentration in various tissues/organs, brain included, was
determinedaerh.eauthorfoundthatthetotalamount
of gold uptaken in brain was similar for  and  nm particle
size. Also Schleh et al. [] investigated the inuence of size
and surface charge of gold nanoparticles on the absorption
across intestinal barriers and accumulation in brain aer
oral administration. e authors utilizing NPs from . to
 nm found the highest accumulation in secondary organs
for . nm particles, while the highest brain accumulation
was recorded in the case of  nm NPs. ese data suggest
that the route of administration of NPs into the body is of
pharmacological and clinical importance and that NPs may
cross the BBB whichever the initial route of administration.
7. Neurotoxicity Issues
Even if the use of engineered NPs represents one of the main
hopes for innovative pharmacological strategies in neurology
[], it is important to mention that the BBB represents a
mechanism of defense of CNS against potentially neurotoxic
molecules and structures, NPs included. In vivo and clinical
data evaluating the toxic eects of NPs on neural cells are still
scarce, and it is still dicult to extrapolate the results obtained
on in vitro models to the actual situation in vivo [], given
that the application of NPs to the CNS is at a nascent stage.
It should be pointed out that dierent types of NPs,
showing promising features for in vivo applications at the
beginning, were successively discarded aer having demon-
strated their toxicity when utilized in vivo. is is the case, for
instance, of quantum dots and carbon nanotubes that proved
to be toxic in vivo [], and for this reason, their prospective
useinmedicinewillbelikelylimitedtoin vitro diagnostics.
Other cases of NPs-related neurotoxicity have been reported,
and neurological eects were described in mice exposed

 ISRN Biochemistry
to SiO2and MnO NPs []. In vivo experiments showed that
other NPs also have some neurotoxicity by causing transient
microglia activation and induction of TLR- promoter activ-
ity in transgenic mice [].
For these reasons, the benet-risk balance deriving from
theuse of NPs intended for treatment of CNS diseases should
be carefully evaluated for each type of new engineered NPs.
When assessing the possible neurotoxicity of NPs, it should
be pointed out that beyond of the core structure, also surface
functionalization, oen employed for targeted delivery, can
signicantly alter the biological response and induce neuro-
toxicityofotherwisesafeparticles[]. erefore, this aspect
also needs to be taken into consideration. Moreover, it should
also be stressed that toxicity could depend by the opening
of tight junctions of the BBB endothelia, induced by NPs.
For instance, an in vitro study by Olivier et al. []showed
that PBCA NPs induced a permeabilization of a BBB model,
which was presumably attributed to the toxicity of the carrier.
e most reliable data on the safety of NPs towards
CNS have been reported for liposomes and iron oxide NPs.
Liposomes are known from the s and are generally low
toxic because they are composed of naturally occurring lipids
[].Ironoxide-basedNPsarealsobelievedtobescarcely
toxic to the CNS [,].
Another source of neurotoxicity could arise from the
functionalization of NPs with cationized proteins []. Toxic
eects have been detected only when such proteins are
administered to “heterologous” animals. In view of this,
human proteins or recombinant humanized proteins should
be used for cationization and subsequent applications in
humans. Moreover, since the conjugation of proteins to
polyethylene glycol has been shown to decrease their immu-
nogenicity, PEGylation of cationized molecules may be an
alternative to minimize the immunogenic potential of these
molecules.
8. Future Directions
Given the importance of nding new drug delivery systems
fortreatmentofCNSdiseasesitisconceivablethatnew
strategies will be developed in the next future.
Since several studies demonstrate increased passage of
monocytesacrosstheBBBinvariouspathologicalconditions,
the synthesis of NPs mimicking immune cells might be
eective in brain-associated disorders, and it is therefore
predictable that the research will receive a stimulus in this
direction in the next years. It is also hypothesizable that
NPs designed to mimic the molecular interactions occurring
between inamed leukocytes and endothelium should pos-
sess selectivity toward diverse host inammatory responses,
for instance, the incorporation of inammation-sensitive
sensorssuchasintegrinlymphocytefunction-associated
antigen (LFA)- I domain, to mimic activated leukocytes
for the targeting of inamed tumor microenvironments, Or
CCR, since cerebral microglia can recruit an increased
number of activated circulating monocytes into the brain
in response to elevated cerebral monocyte chemoattractant
protein (MCP)-.
Other possibilities are to exploit the absence or at least
the high permeability of some BBB regions. e BBB is
present in all brain regions, with the exception area postrema,
median eminence, neurohypophysis, pineal gland, subfor-
nical organ, and lamina terminalis. e endothelial cells
present in capillaries of these brain areas have fenestrations
that allow diusion of molecules. e most extended area
where, presumably, a more extensive passage of NPs could
be possible is occupied by the leptomeningeal space, which
is the space occupied by Cerebrospinal uid (CSF), including
all spaces continuous with the subarachnoid space, such as
perivascular spaces and ventricles []. e data available
to date suggest that many macromolecules and nanopar-
ticles can be delivered to CNS in biologically signicant
amounts.
Moreover, the perivascular spaces which are in continuity
with leptomeningeal space penetrate into the parenchyma
provide an unexplored avenue for drug transport deep into
thebrainviaCSF.euxoftheinterstitialuidinthe
CNS parenchyma, as well as the macro ux of CSF in the
leptomeningeal space, is believed to be generally opposite to
the desirable direction of CNS-targeted drug delivery. e
nal outcome will depend on the NPs behavior, which has
not been studied yet. It is therefore conceivable that eorts in
this direction will be carried out in the next future, in order
to exploit alternative administration routes.
9. Conclusions
e number of deaths due to neurological or neurodegener-
ative diseases is those of a world war, with connected huge
socioeconomical problems and costs. e treatment of such
diseases is hampered by the presence of BBB, insurmountable
by most available and future potentially eective drugs.
erefore, the discovery and development of novel drug
delivery systems for the treatment of such diseases is a
major challenge for both the academic and pharmaceutical
community. Nanotechnology represents an innovative and
promising approach. Currently, several types of NPs are
available for biomedical use with dierent features and
applications facilitating the delivery of neuroactive molecules
such as drugs, growth factors and genes, and cells to the
brain. NPs oer clinical advantages for drug delivery such
as decreased drug dose, reduced side eects, increased
drug half-life, and the possibility to enhance drug crossing
across the BBB. However, the enhancement of brain delivery
obtained with drug-loaded NPs, although very promising,
is still quantitatively limited in comparison with free drugs.
Consequently, with very few exceptions, NPs are not yet a
viable solution for pharmacology, requiring enhancements of
one order of magnitude or more.
Further investigations are necessary for a better com-
prehension of the mechanisms which manage this dierent
NPs-mediated transport of the drugs to the brain. However,
the strong eorts to allow the translation from preclinical
to concrete clinical applications are worth of the necessary
economic investments.