Advanced Drug Delivery Reviews 62 (2010) 503–517

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Nanotechnology applications for improved delivery of antiretroviral drugs to

the brain☆
Ho Lun Wong a, Niladri Chattopadhyay b, Xiao Yu Wu b, Reina Bendayan b,⁎
a
School of Pharmacy, Temple University, 3307 North Broad Street, Philadelphia, Pennsylvania, 19140, USA
b
Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 2S2

a r t i c l e i n f o a b s t r a c t

Article history: Human immunodeficiency virus (HIV) can gain access to the central nervous system during the early course
Received 22 June 2009 of primary infection. Once in the brain compartment the virus actively replicates to form an independent
Accepted 14 September 2009 viral reservoir, resulting in debilitating neurological complications, latent infection and drug resistance.
Available online 13 November 2009
Current antiretroviral drugs (ARVs) often fail to effectively reduce the HIV viral load in the brain. This, in part,
is due to the poor transport of many ARVs, in particular protease inhibitors, across the blood-brain barrier
Keywords:
Human immunodeficiency virus
(BBB) and blood-cerebrospinal fluid barrier (BCSBF). Studies have shown that nanocarriers including
Brain delivery polymeric nanoparticles, liposomes, solid lipid nanoparticles (SLN) and micelles can increase the local drug
Antiretroviral concentration gradients, facilitate drug transport into the brain via endocytotic pathways and inhibit the
Nanotechnology ATP-binding cassette (ABC) transporters expressed at the barrier sites. By delivering ARVs with nanocarriers,
Blood-brain barrier significant increase in the drug bioavailability to the brain is expected to be achieved. Recent studies show
ATP-binding cassette membrane transporters that the specificity and efficiency of ARVs delivery can be further enhanced by using nanocarriers with
specific brain targeting, cell penetrating ligands or ABC-transporters inhibitors. Future research should focus
on achieving brain delivery of ARVs in a safe, efficient, and yet cost-effective manner.
© 2009 Elsevier B.V. All rights reserved.

Contents

1. HIV infection and CNS illnesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504

1.1. Human immunodeficiency virus (HIV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
1.2. HIV epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
1.3. Complications associated with HIV infection of the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
1.3.1. Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
1.3.2. Clinical manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
1.3.3. Current treatment and its limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
2. Barriers to antiretroviral (ARV) penetration into the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
2.1. BBB and BCSFB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
2.2. ABC transporters at the BBB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
2.2.1. ABC transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
2.2.2. Role of ABC-transporters in ARVs delivery to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
2.3. Strategies to improve ARVs penetration across the BBB and BCFSB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
2.3.1. Inhibition of ABC transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
2.3.2. Hyper-osmotic opening of the BBB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
2.3.3. Pharmacological disruption of BBB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

Abbreviations: ABC transporter, ATP-binding cassette membrane transporter; AIDS, acquired immunodeficiency syndrome; apoE, apolipoprotein E; ARVs, antiretroviral drugs;
BBB, blood-brain barrier; BCSFB, blood-cerebro spinal fluid barrier; CD4, cluster of differentiation 4; CNS, central nervous system; CSF, cerebrospinal fluid; HAART, highly active
antiretroviral therapy; HAD, human immunodeficiency virus-associated dementia; hCMEC/D3, human brain microvessel endothelial cell line; HIV, human immunodeficiency virus;
HIVE, human immunodeficiency virus encephalitis; LDL, low-density lipoprotein; MCMD, minor cognitive/motor disorder; MMA-SPM, methylmethacrylate-sulfopropylmetha-
crylate; MRP, multidrug resistance-associated proteins; NNRT, non-nucleoside reverse transcriptase inhibitors; NRTI, nucleoside reverse transcriptase inhibitor; PBCA, poly(butyl
cyanoacryalate); PEG, polyethylene glycol; PIs, HIV protease inhibitors; PIL, PEGylated immunoliposomes; P-gp, P-glycoprotein; PLA, polylactide; PLGA, poly(D,L-lactide-co-
glycolide); SLN, solid lipid nanoparticles; Tat, transcriptional activator; Vpr, viral protein R.
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Nanotechnology Solutions for Infectious Diseases in Developing Nations”.
⁎ Corresponding author. Tel.: +1 416 978 6979.
E-mail address: r.bendayan@utoronto.ca (R. Bendayan).

0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.11.020
504 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517

2.3.4. Drug modification approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

2.3.5. Focused ultrasound and microbubble approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
2.3.6. Nanotechnology for ARVs delivery to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3. Nanotechnology to improve ARVs delivery to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.1. General principles of brain delivery using nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.1.1. Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.1.2. Overview of nanocarrier-mediated drug delivery across the BBB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.2. Current use of nanocarriers for brain delivery of ARVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.2.1. Polymer or dendrimer-based nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3.2.2. Lipid-based nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.2.3. Micelle-based nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
4. Recent trends to optimize nanotechnology use for ARV brain delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.1. Optimization of nanocarrier properties to improve passive brain targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.2. Development of specific brain targeting strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.3. Cell penetrating peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4.4. Other nanotechnology-based strategies to improve ARVs brain delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4.4.1. Use of macrophages for BBB passage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4.4.2. Alternative route for nanocarriers administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4.4.3. Advanced delivery of ABC-transporter blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
5. Future perspectives and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
5.1. Improve understanding of the barrier structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.2. Tailor-made nano-formulations for brain delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.3. Use of better experimental models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.4. Refine targets and endpoints of ARV delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

1. HIV infection and CNS illnesses
1.1. Human immunodeficiency virus (HIV)
Human immunodeficiency virus (HIV) is a lentivirus from the
Retroviridae family responsible for the acquired immunodeficiency
syndrome (AIDS). At present, there are two known types of HIV, HIV-1
and HIV-2, with HIV-1 being much more virulent, transmittable and
prevalent, and the cause of the majority of HIV infections in the world
[1]. HIV infection results in compromised immune defense by causing
extensive destruction of T-helper cells, macrophages, dendritic cells
and other cellular components associated with cell-mediated immu-
nity [2,3]. As a result, HIV-infected patients are substantially more
vulnerable to opportunistic infections. The abnormal immune
responses triggered by HIV infection can also result in other
complications such as neurological illnesses.
1.2. HIV epidemiology
According to the 2007 update by the Joint United Nations Program
on HIV/AIDS and World Health Organization [4], every day over 6800
individuals become newly infected and over 5700 patients die from
AIDS. It is estimated that 33.2 million persons worldwide are infected
with HIV-1, and the developing nations continue to be its primary
victims. Although signs of a decline in the cases of new infection have
been observed due to better prevention efforts, the sub-Saharan
African region remains as the epicenter of the pandemic. An estimated
22.5 million people in these countries, equivalent to 5% prevalence,
are living with HIV-1 infection. The prevalence is also alarmingly high
in the Caribbean Islands (1.0%), Latin America (0.5%), Eastern Europe
and Central Asia (0.9%). In East Asia, 92,000 adults and children were
found newly infected with HIV-1 in 2007, representing almost a 20%
increase from 2001. Even though the numbers of new HIV-1 infections
have been relatively stable in the developed nations, this disease is
still an unresolved health issue. In North America only, 1.3 million
people are living with HIV-1, equivalent to 0.6% prevalence. These
data indicate that the current treatment of HIV-1 still needs significant
improvement.
1.3. Complications associated with HIV infection of the central nervous
system
1.3.1. Pathophysiology
HIV and other lentiviruses are unique from other viruses due to their
ability to infect and replicate in non dividing cells including those of the
monocyte/macrophage lineage. In particular, HIV targets the cluster of
differentiation 4 positive (CD4+) T lymphocytes and cells of the
monocyte-macrophage lineage [5]. CD4− negative cells may also be
targeted, but these viral strains are highly sensitive to neutralization by
host antibodies and are present only at sites where circulating antibody
levels are low (e.g. in brain) [6,7]. Once the virus fuses with the host cell,
DNA is produced from its RNA genome via the enzyme reverse
transcriptase, and then DNA is incorporated into the host's genome by
an integrase enzyme and replicates as a part of the host DNA [1,8].
HIV is known to invade the central nervous system (CNS) early in the
course of the infection and primarily targets brain mononuclear
macrophages, perivascular macrophages and microglia. [7,9]. The virus
can enter the CNS compartment from the systemic circulation via two
routes: i) through the blood-cerebro spinal fluid barrier (BCSFB) at the
choroid plexus as cell-free viral particles [10], and/or ii) through the
blood-brain barrier (BBB) in form of infected monocytes [11]. The second
route is known as the “Trojan horse approach”. In brief, monocytes
infected by HIV-1 are able to cross the BBB between the capillary
endothelial cells in a complex process regulated by the secretion of
chemokines (e.g. MIP-1a/b, MCP-1, RANTES) from glial cells [12]. The
brain macrophages and microglial cells, upon infection are responsible
for further production of HIV-1 virus, and can also release viral proteins
such as glycoprotein 120 (gp120), Tat (transcriptional activator) and Vpr
(viral protein R) [13–16]. These viral proteins have been shown to be
neurotoxic in vitro and trigger various harmful events such as activation
of apoptotic pathways, cell-cycle arrest of neuronal cells and stimulation
of the production of reactive oxidative species, glutamate, cytokines and
other inflammatory factors from uninfected astrocytes [17–19], which
further accelerate the neurodegeneration process. Additionally, gp120
and Tat can render the BBB leakier which further promotes the
permeability of HIV-infected monocytes [20–22]. Other CNS cell types
can also be infected by HIV. Low-grade production of provirus has been
 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
detected in some cell populations such as astrocytes, and in vitro HIV
susceptibility in oligodendrocytes and microvascular endothelial cells
has been observed [23]. Non-CD4 entry pathway(s) may play a role as
these cells do not express CD4 on their surface [24].
1.3.2. Clinical manifestations
CNS infection by HIV leads to various forms of neurological
complications. It has been estimated from AIDS patients brain tissue
obtained at autopsy that the prevalence of neuropathologic abnormal-
ities can be as high as over 80% [25]. Approximately 10 to 20% treated
patients demonstrate some forms of overt illnesses [26]. Minor
cognitive/motor disorder (MCMD), which presents with symptoms
such as cognitive and motor slowing, poor concentration and impaired
memory, often occurs during early HIV infection [27]. During the late-
stage of the infection, a more severe form of neurological complications
collectively termed HIV-associated dementia (HAD) or AIDS dementia
complex may develop, usually in patients who have had a CD4 count
nadir of b200 cells/mm3 [16,28]. Patients with HAD may present with
diverse symptoms ranging from confusion, behavioral abnormalities,
motor dysfunctions, to psychosis and seizure. Without proper treat-
ment, the mental conditions of HAD patients can further deteriorate. In
5% to 8% of patients, a syndrome known as AIDS mania develops in
addition to HAD [29]. Pediatric HIV patients are particularly vulnerable
to HAD. About 50% of untreated children infected with HIV-1 was
estimated to have HAD, and the symptoms are often more severe, with
many of them showing compromised intellectual development [11].
Overall, the prognosis of advanced HAD patients is poor, and even
though less severe, MCMD has been identified as a significant
independent risk factor for AIDS mortality [27].
1.3.3. Current treatment and its limitations
Zidovudine was the first antiretroviral compound commercially
available for the treatment of AIDS [30]. Since its launch in 1987,
intensive research efforts have led to the discovery of several classes
of ARVs including: i) nucleoside reverse transcriptase inhibitors
(NRTIs), ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs),
iii) protease inhibitors (PIs), iv) integrase inhibitors, and v) entry
inhibitors [31]. With better understanding of the detailed mechan-
isms of HIV replication, single agent ARV therapy has been generally
replaced by combination therapy. The primary rationale for using
multiple agents is to disrupt HIV replication at multiple points in the
lifecycle. Each of these “cocktail” regimens often comprises two
nucleoside analogues and a PI to achieve potential synergistic effect,
sometimes with a secondary PI at low dose (typically ritonavir)
included to “boost” up the bioavailability of the primary PI. Because of
their higher clinical efficacy in lowering the mortality and morbidity
in HIV patients, these therapeutic combinations are referred as the
highly active ARV therapy (HAART). Table 1 lists the major ARV
components of HAART recommended by the US Department of Health
and Human Services and International AIDS Society in 2008 [31,32].
Table 1
Recommended ARVs in highly active antiretroviral therapy (HAART).
Class of antiretroviral Component
NRTI/NtRTI Tenofovir + emtricitabine
Abacavir + lamivudine
Zidovudine + lamivudine
Stavudine + lamivudine
NNRTI Efavirenz
PI⁎ Lopinavir
Atazanavir
Fosamprenavir
Darunavir
Saquinavir
NRTI: nucleoside reverse transcriptase inhibitors; NtRTI: nucleotide reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitors; PI: protease inhibitors.
⁎ All PIs require “ritonavir-boosting”, i.e. co-administration with low dose of ritonavir, to increase the plasma concentration and area-under-the-curve of the ARVs. (data obtained
from Hammer et al, 2008 [32]).
505
It has been reported that HAART is, in general, less effective for the
treatment of CNS complications than other AIDS-related illnesses [33].
In the short term, HAART remains fairly effective against the more
severe CNS illnesses such as HAD. Although, the incidence of HAD has
been reduced from over 30% of the AIDS population to around 10% in
post-HAART era [34,35], this is accompanied by a significant increase in
the prevalence of HAD since patients now live longer. Relapses of HAD in
HAART-treated patients are also common [26,35]. The therapeutic value
of HAART for other HIV-related CNS complications is lower. Clinical data
indicate that since the advent of HAART, there has been a steady increase
in the MCMD incidence [34]. It has been reported that the rate of MCMD
among adults with symptomatic HIV-1 disease is over 30%, and the
prevalence of MCMD did not significantly change when the pre-HAART
and post-HAART cohorts of HIV-1 infected homosexual males were
compared. Overall, the data suggest a lack of protection against MCMD
provided by HAART [26]. Studies also showed that low-grade
inflammation frequently persists in patients receiving HAART, suggest-
ing the occurrence of ongoing immune activation in the CNS [36,37].
This limited efficacy is in part attributable to the inefficiency of the
current HAART regimens to eradicate the HIV-1 in the CNS. This
phenomenon bears a number of therapeutic and pathologic implications.
Some studies show that in HIV-1 infection, the severity of neurological
complications is positively correlated with the viral load in the
cerebrospinal fluid (CSF), and this CSF viral load has been shown fairly
independent from the plasma viral load [38,39]. Therefore, ARVs will
have little therapeutic value for HIV-related CNS complications unless
these drugs can efficiently reduce the viral load in the CNS compartment.
Failure to eliminate such a “viral reservoir” in the CNS, wherein
replicating virus accumulate and survive with more stable kinetic
properties than in the main pool of virus in the peripheral compartment
[40], could be responsible for the latent reinfection. In addition, the
presence of CNS viral reservoir may also promote the development of
drug resistance. Wong et al have shown the presence of different ARV
resistance mutations in viruses from the brain as compared with the
peripheral sites of infection [39]. Unfortunately, using the current HAART
regimens, most recent studies estimate that it will take up to 7.7 years of
uninterrupted therapy to eliminate this viral reservoir early in the course
of HIV infection [41]. Such long-term use of HAART is highly undesirable.
The risk of side effects, including peripheral neuropathy, liver dysfunc-
tions, and metabolic complications will significantly increase, and other
issues such as poor patient compliance, high drug cost, and drug-drug
interactions are more likely to arise [42,43].
2. Barriers to antiretroviral (ARV) penetration into the brain
2.1. BBB and BCSFB
To effectively treat HIV-associated CNS complications, it is highly
critical to improve the efficiency of CNS penetration by ARVs. A
number of obstacles have to be overcome to achieve this goal. As
Comments
Tenofovir is a NtRTI. Effective and well-tolerated; new standard of NRTI/NtRTI components in HAART
Comparable efficacy to tenofovir + emtricitabine in patients with low to moderate viral load
Previous standards of NRTI components
Standard of care; available as a once-daily fixed dose with tenofovir + emtricitabine
Lopinavir new standard of care. Atazanavir and fosamprenavir have comparable efficacy
Superior to lopinavir in patients with viral load ≥ 100,000 HIV RNA copies/mL
Comparable efficacy, but more frequent dosing required
506 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517

reflected by the pharmacokinetic parameters (shown in Table 2), the CNS. For examples, even though PIs typically exhibit a high degree of
ARVs often exhibit high non-specific binding, and do not reside long in lipophilicity (log10P = 2.9–5.2) [55], their CSF levels are extremely low
the blood plasma [44–47]. The share of the administered dose of the (Table 2). While the large (typically 600–750 Da) molecular weight of
drug that can reach the brain is consequently quite limited. The CNS these PIs likely contributes to their low CNS bioavailability, it must be
penetration by ARVs is further compromised by the presence of the pointed out that smaller, neutral PIs such as amprenavir (505.6 Da) still
BBB and BCSFB [48]. This is reflected by the low values of CSF-plasma exhibit a poor CSF-plasma ratio. It is obvious that in addition to the
ratio observed in most ARVs, especially the PIs (Table 2). anatomical features, the BBB and BCSFB have other mechanisms to
Both BBB and BCSFB are equipped with specialized anatomical control passage of drugs into the CNS. Indeed, there are several
structures which dramatically prevent access of several exogenous membrane transporters located at these two barriers which mediate
compounds to the CNS compartment [49,50]. The BBB is formed mainly their efflux from the CNS compartment back to the blood. Most of these
by the brain capillary endothelium. It serves as the primary interface transporters belong to the superfamily of ATP-binding Cassette (ABC)
between the CNS and the peripheral circulation, separating the brain membrane transporters [52]. Furthermore, cerebral blood flow and
parenchyma from the bloodstream [51,52]. There are a number of degree of local inflammation can also affect drug CNS permeability.
structural features unique to brain capillaries when compared to other
blood capillaries such as a lack of fenestration and minor pinocytosis. 2.2. ABC transporters at the BBB
The tight junctions between these cells are extensive, continuous and
provide a very high electrical resistance (over 1500 Ω cm−2) across the 2.2.1. ABC transporters
brain capillary endothelium. Together, these properties significantly The ABC family is among the most ubiquitously expressed and
limit the paracellular transport of hydrophilic molecules [53]. To allow largest membrane-associated protein superfamily known to date. ABC
access of the brain by hydrophilic compounds such as glucose, amino members are involved in the translocation of both endogenous and
acids and proteins that are essential for brain functioning, specific cell exogenous substrates and metabolites against their concentration
surface processes such as solute transporters and receptor-mediated gradient [56]. The energy to transport substrates is provided by
endocytosis are present at this barrier [50,51]. hydrolysis of ATP at the nucleotide binding domains. In humans, 50
The BCSFB serves as the secondary barrier against drug penetra- ABC genes have been identified and are classified according to seven
tion into the CNS. It is formed by the choroid plexus epithelial cells subfamilies based on the organization and sequence of their ATP-
which like the brain microvessel endothelial cells present tight binding domain(s) [57]. ABC-transporters can also be classified into
junctions [49,53]. These epithelial cells have well-developed apical full or half transporters. A full ABC-transporter consists of two
brush border and basolateral interdigitations, as well as numerous transmembrane domains and two ATP-binding domains, whereas a
mitochondria, all of which may be important in fluid and solute half transporter consists of only one of each [58].
transport [50]. The choroid plexus consists of a single layer of cuboidal Several ABC transporters, specifically P-glycoprotein (P-gp), multi-
epithelial cells surrounding a rich vascular network from which the drug resistance-associated proteins (MRP) isoforms, and ABCG2 are
epithelial cells are separated by a loose stroma. CSF secretion is about known to be involved in the cellular extrusion of a broad range of drug
350 μl/min in an adult male which represents a turnover rate of about molecules. P-gp, a full transporter also known as MDR1 protein, ABCB1
0.4%/min [54]. The production of CSF serves to keep the concentration or CD243, is probably the most studied and characterized ABC member.
of compounds that passively diffuse into the brain lower than that It was first found as a 170-kDa ATP dependent membrane glycoprotein
found in plasma, a phenomenon known as the CSF sink effect [54]. that acts as a drug efflux pump [59]. Up to date, a diversity of structurally
The penetration of a drug molecule across these two barriers and functionally distinct biomolecules and chemical compounds have
depends on a number of its physicochemical properties including been identified as the substrates of P-gp [60]. These substrates are
lipophilicity, size, and degree of ionization, Although in principle, typically hydrophobic, amphipathic compounds, with many of them
uncharged lipophilic compounds smaller than 400–600 Da should possessing weakly basic or cationic groups. MRPs belong to the ABCC
easily cross these barriers by passive diffusion across the cell subfamily [60,61]. Multiple isoforms of MRP (MRP-1 to MRP-8) have
membranes, many of these compounds are restricted from access into been discovered [62]. They are full transporters normally expressed in
the canalicular part of the hepatocyte where they play a crucial role in
Table 2 biliary transport [63]. Many MRP substrates are also lipophilic, basic or
Clinical pharmacokinetic properties of commonly prescribed ARVs. cationic compounds, but unlike P-gp, MRP substrates also include
Antiretroviral Plasma protein Elimination CSF-plasma ratio
neutral or mildly anionic molecules [63,64] . Many of these substrates
drugs binding (%) half-life (h) are drug conjugates of lipophilic anions (e.g. glucuronate or glutathione
Abacavir 50 1.54 0.18-0.33
conjugates). ABCG2, also known as breast cancer resistance protein, is a
Didanosine b5 1.5 0.21 half ABC-transporter first identified in breast cancer [65]. It was later
Lamivudine b36 1.42 0.06–0.31 found in other normal tissues, including placenta, liver canaliculi, small
Emtricitabine b4 8–10 Unknown intestine, colon, the bronchial epithelial layer in the lung, and brain
Stavudine Negligible 1.2–1.4 0.2
capillary endothelial cells [66]. Many P-gp substrates are also substrates
Zalcitabline⁎ Negligible 1.2–2 0.1–0.37
Zidovudine b38 1.1 0.17–0.60 of ABCG2.
Delavirdine 98 5.8 0.004
Efavirenz 96-99 52-76 0.02-0.1 2.2.2. Role of ABC-transporters in ARVs delivery to the brain
Nevirapine 60 45 0.45 Many ARVs are large, lipophilic compounds with fairly high
Amprenavir⁎⁎ 90 7.1–10.6 b 0.01
molecular weights. They are therefore likely candidates to be ABC
Atazanavir 86 5.28 0.0021–0.0226
Indinavir 60 1.8 0.15 transporter substrates. Several studies in polarized and P-gp over-
Lopinavir 98-99 5.6 Negligible expressing cell lines have indeed shown that various PIs (e.g.
Nelfinavir N98 3.5-5 Negligible amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir) are
Ritonavir 98-99 3-5 0.01–0.05
substrates of P-gp [67–71]. As shown by a number of in vitro studies,
Saquinavir 97 2.5 0.01–0.02
many PIs are also substrates of MRPs, particularly MRP1 and MRP2
⁎Zalcitabine was discontinued in 2006. [67,68,72,73] . Furthermore, PIs have been shown to be competitive
⁎⁎ Amprenavir was discontinued in 2004; a prodrug version (fosamprenavir) is
currently available.
blockers of P-gp and MRPs, as shown by their inhibitory activities
Data obtained from: Oldfield and Plosker, 2006; Perry et al., 2005; Swainston and Scott, against the transport of established P-gp and MRP substrates [74–79].
2005; Wynn et al., 2002. [44–47]. Studies in human embryonic kidney cells and in MDCKII cells stably
 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
transfected with ABCG2 have shown that while PIs are probably not
ABCG2 substrates, they are good inhibitors of ABCG2-mediated
mitoxantrone or pheophorbide A transport [80,81]. A few recent
studies have further demonstrated that NRTIs and NNRTIs are also
capable of interacting with ABC membrane transporters. For
instances, delavirdine is a P-gp blocker, whereas abacavir and
stavudine are substrates of MRP4 and MRP5, respectively [82–84].
There are other lines of evidence supporting the frequent interactions
of ABC transporters and ARV compounds. For further detail, please
refer to the reviews by Dallas et al and Ronaldson et al [62,85].
P-gp, MRP isoforms and ABCG2 have been identified at the BBB and
BCFSB and found to play a significant role in regulating the levels of
ARVs, most notably PIs, in the brain compartment [85]. Fig. 1 presents
the proposed distribution of these transporters at the BBB. Studies in
isolated animal brain microvessels have shown the potent inhibition
of P-gp and MRP-mediated transport by saquinavir and ritonavir [86].
Our group has also shown that indinavir, saquinavir and ritonavir can
inhibit the accumulation of digoxin, a P-gp substrate, in an
immortalized rat astrocyte cell line system (CTX TNA2), a rat brain
microvessel endothelial cell line (RBE4) and primary cultures of rat
astrocytes [75,87]. Strong interactions between PIs and P-gp and
MRPs were observed. Further evidence is provided from in vivo
studies using mdr1a (−/−) knockout mice. Significant enhancement
(4–36 fold) in brain accumulation of indinavir, nelfinavir, ritonavir,
and saquinavir has been shown in the knockout mice compared to the
mdr1a (+/+) wild-type controls [71]. In comparison, the role of
ABCG2 in BBB transport of ARVs is more controversial. Studies using
ABCG2-deficient mice indicate that ABCG2 does not play a significant
role in limiting the CNS distribution of zidovudine and abacavir [88].
Further work is required to clarify the role of this transporter in vivo.
2.3. Strategies to improve ARVs penetration across the BBB and BCFSB
Knowing the anatomical and molecular characteristics of the BBB
and BCFSB, a number of strategies have been proposed to improve the
permeability of ARVs across these barriers. Each of these strategies
presents strengths and limitations.
2.3.1. Inhibition of ABC transporters
In the last decade, a number of chemical entities capable of
blocking specific ABC-transporters have been developed. Some
success in using these blockers to improve ARVs availability to the
CNS has been shown in various animal studies using different types of
blockers. For example, the CNS levels of several PIs (i.e., amprenavir,
indinavir, nelfinavir, saquinavir) in mice were significantly enhanced
Fig. 1. Proposed localization of major ATP-binding cassette (ABC) membrane transporters at the blood-brain barrier (BBB). AP: apical side; BL: basolateral side. (Adapted from
Ronaldson et al., 2008).
507
after treatment of mice with a P-gp inhibitor LY-335979 [89]. Multiple-
fold increases in the brain accumulation of saquinavir were similarly
observed in animals treated with GF120918 and MK571, a P-gp/ABCG2
blocker and MRP inhibitor, respectively [90]. In another study, a 9-fold
increase in nelfinavir concentration in the brain of mdr1a/1b (+/+)
wild-type mice versus P-gp knockout mdr1a/b (−/−) mice was
obtained when GF120918 was co-administered [91]. However, it should
be noted that the nelfinavir levels in other vital organs were also
increased by multiple-fold. Considering the ubiquitous presence of ABC
transporters, these blockers are unlikely to be CNS-specific. The
resulting overhaul of the global pharmacokinetics is generally undesir-
able as it can easily lead to elevated risks of drug toxicity and
unpredictable drug-drug interactions.
2.3.2. Hyper-osmotic opening of the BBB
It is known that an hypertonic solution of mannitol or urea can
shrink the capillary endothelial cells by inducing water efflux and
subsequently opening the tight junction network momentarily [92,93].
As a result, the paracellular flow can be considerably increased allowing
more efficient BBB passage by drug compounds. This strategy has been
applied in animals with some success for increasing the BBB
permeability [92]. Unfortunately, it is a risky procedure. Seizures were
observed in some subjects and unpredictable long-term neurological
complications can occur. This strategy is therefore reserved as the last
resort [94].
2.3.3. Pharmacological disruption of BBB
Cytotoxic agents, especially alkylating agents such as etoposide
and cisplatin, may disrupt tight junctions and create openings
between the endothelial cells [95]. Similarly, vasoactive agents such
as bradykinin, peptidase inhibitors and angiotensin II may also render
the BBB permeable temporarily [96–98]. Improved brain accumula-
tion of drug molecules and even drug carriers has been observed after
administration of these agents. However, many of these compounds
are very toxic [99–101]. Long-term use of this strategy for brain
delivery of ARVs is likely not appropriate.
2.3.4. Drug modification approach
Molecules with good lipophilicity can cross the cell membrane of
the endothelial cells by passive diffusion. Improved BBB passage can
thus be achieved by conjugating ARV molecules with suitable side
branch or functional group(s) to form “prodrugs” with more favorable
lipophilicity. These prodrugs, after gaining access to the endothelial
cells, can be hydrolyzed and release the parent ARVs [102]. Since a
prodrug is often officially recognized as a distinct chemical entity,
508 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
substantially more drug purification steps, screening tests and clinical
studies are expected, which is usually not cost-effective.
2.3.5. Focused ultrasound and microbubble approach
Microbubbles are fine gas bubbles of less than 50 μm in diameter.
When exposed to ultrasound, these microbubbles serve as the cavitation
nuclei to focus and transduce the acoustic energy into mechanical
power [103–105]. Studies have shown that this combinational approach
was able to induce transient disruption of the BBB [103,105] This may
lead to enhanced delivery of therapeutic compounds into the brain
compartment. However, there are several concerns regarding the safety
of this strategy [105]. More work is required to establish the optimal
conditions for extensive use of this method.
2.3.6. Nanotechnology for ARVs delivery to the brain
ARVs can be effectively delivered to the brain using drug carriers of
nanometer or submicron scale [106–109]. Depending on the type of
nanocarriers, chemical modifications of ARVs are often not required for
efficient loading and delivery. There is a broad range of nanocarriers
available, including liposomes, polymeric systems, nanoparticles, and
micelles, many of them clinically used before. Their versatility allows
them to carry diverse ARVs. The following sections will discuss the use of
nanocarriers for ARVs delivery to the brain in details.
3. Nanotechnology to improve ARVs delivery to the brain
3.1. General principles of brain delivery using nanocarriers
3.1.1. Rationale
The use of nanocarriers can improve brain delivery of ARVs in
several ways. The availability of ARVs to the CNS compartment can be
improved. Numerous studies using distinctively different nanocar-
riers for delivering a broad range of therapeutic or diagnostic agents
have generally reported enhanced in vitro and in vivo BBB perme-
ability and drug accumulation in the brain [106–109]. Table 3 provides
some examples of brain delivery of various therapeutic compounds
using diverse types of nanocarriers. Many of the agents delivered are
well-established substrates of ABC transporters. These include P-gp
substrates, e.g. doxorubicin, digoxin, rhodamine, vinblastine, and MRP
substrates, e.g. methotrexate, fluorescein [60,62]. Although they
usually present poor BBB permeability, with the use of nanocarriers
these compounds were able to achieve the desired therapeutic levels
in the CNS [see details and references in Table 3].
It is believed that with significantly higher levels of ARVs reaching
the CNS, it is possible to reduce their doses and shorten the length of
therapy. This may translate into reduced risks of peripheral adverse
drug effects. In addition, nanocarrier systems are known for their
flexibility and versatility. They can be made of different biocompatible
materials, and most carriers can be engineered to obtain more
desirable pharmacokinetic and biodistribution profiles for optimal
treatment of the CNS [106]. For example, the dosing frequency can be
reduced by using a carrier that releases the ARV in a sustained
manner. The circulation time can be prolonged and non-specific tissue
binding reduced by coating a nanocarrier with polyethylene glycol
(PEG) [110]. Because only the carrier itself is engineered without the
need to modify the drug molecules, dramatic alterations of the drug
pharmacology can often be avoided.
3.1.2. Overview of nanocarrier-mediated drug delivery across the BBB
Fig. 2 presents a proposed scheme depicting how nanocarriers can
be used to improve drug transport across the BBB. Overall, nanocarriers
can enhance brain delivery by three major pathways, which include:
i) increasing the local drug gradient at the BBB by passive targeting,
ii) allowing drug-trafficking by non-specific or receptor-mediated
endocytosis and iii) blocking drug efflux transporters at the BBB.
In brief, by carefully choosing the biomaterials and adjusting the
formulation parameters of the nanocarriers, these can be prepared with
physicochemical properties desirable for interaction with the barrier
structures of the CNS (further details in Section 4.1). This is sometimes
known as passive targeting. As a result, a high local level of drug-loaded
nanocarriers can accumulate at the brain capillary endothelium, pro-
ducing a high local concentration gradient to drive the drug penetration
rate by passive diffusion.
In addition to simply staying on the endothelium surface to release
the loaded drug, some nanocarriers can enter cells by endocytosis, a
pathway that allows “drug-trafficking” [111]. This can occur via non-
receptor mediated or receptor-mediated mechanisms. One example of
the non-receptor mediated endocytosis is macropinocytosis. Macropi-
nocytosis is a relatively non-specific process which allows cellular uptake
of large particles up to the micron size range [112]. This is likely a useful
pathway for nanocarriers such as solid lipid nanoparticles, which are
frequently 200 to 300 nm in diameter. Macropinocytosis is also related to
the uptake of Tat-peptides [113]. Receptor-mediated endocytosis, on the
other hand, is triggered by receptor–ligand interaction. By choosing a
receptor that is strongly and specifically expressed on the surface of the
cells to be targeted, and tagging the nanocarrier surface with the ligand
molecules that match the receptor type, the delivery process can be made
more selective and efficient [111]. Many receptor-mediated endocytotic
pathways involve the formation of clathrin-coated pits, which envelop
the nanocarriers to be transported and eventually form vesicles, detach
from the cell surface and carry the nanocarriers into the cytosolic
compartment [114]. This is a highly regulated and energy-dependent
process, but may allow the whole nanocarrier and the loaded drug to go
through the BBB, even bypassing the drug efflux transporters.
Finally, some efflux transporters expressed at the barrier structures
can be inhibited by the nanocarrier itself or the inhibitor blocking agent
loaded into the nanocarrier (see Sections 3.2.3 and 4.4.3). This results in
local inhibition of the drug efflux and opens up a window where ARV
molecules can permeate.
3.2. Current use of nanocarriers for brain delivery of ARVs
So far, the research on nanocarrier-based delivery of ARVs to the CNS
largely remains at the experimental or pre-clinical stage. A number of
nanocarrier systems have been studied in in vitro or animal models
[106–109,115,116]. These nanocarriers generally fall into a few broad
categories: polymer/dendrimer-based, lipid-based or micelle-based.
Despite the vast number of biomaterials eligible for nanocarrier
preparation, only a few of them are suitable for brain delivery. The
complexity of the CNS calls for conservative choices of biomaterials with
solid track records of safety, particularly considering the duration of ARV
therapy, which usually takes years. Nanocarriers must be non-toxic and
fully biodegradable, producing well-characterized and harmless degra-
dation products only. Many lipids exist physiologically and are relatively
non-toxic. Polymers that fit these criteria include the acrylic polymers
and polyesters that contain lactide units. Pluronic block co-polymers/
surfactants have also been widely used for preparing brain-targeting
micellar systems [108].
3.2.1. Polymer or dendrimer-based nanocarriers
Poly(butyl cyanoacryalate) (PBCA), an acrylic polymer, has so far
been the most studied polymer for brain delivery. PBCA nanoparticles
have demonstrated good accumulation in both brain tissues and
cerebrospinal fluid without physical disruption of the BBB integrity
[117]. PBCA is biodegradable and its lipophilicity facilitates efficient
encapsulation of diverse types of neutral and weak base compounds
such as dalargin, loperamide, amitriptyline, methotrexate and doxoru-
bicin [117–119]. However, their in vivo biodegradation is generally too
fast and potentially harmful formaldehyde by-products can be formed
during the degradation [120]. Their loading capacity is also relatively
low, particularly for polar or ionic compounds. Charged acrylic co-
 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517 509

Table 3
Examples of nanocarrier brain delivery systems for therapeutic compounds.

Type Materials Therapeutic agents Surface coating Comments

Polymeric PBCA Methotrexate [118] Polysorbate 80 Significant increase in methotrexate levels in brain and cerebrospinal
nanoparticles fluid. Size b 100 nm penetrated BBB better.
MMA-SPM; PCBA Zidovudine (AZT)
Lamivudine (3TC) [121] PBCA np increase BBB permeability of AZT & 3TC 8–20 and 10–18 folds,
respectively; MMA-SPM np increase BBB permeability of both drugs
by 100%; MMA-SPM loads AZT better
PBCA Rhodamine [117] 20-fold increase in uptake by brain endothelial cells after Tween-80
coating
PBCA Dalargin [119] 3-fold increase in dalargin BBB penetration; dalargin has to be pre-
adsorbed on to PBCA np for enhanced BBB penetration
PLA Vasoactive intestinal peptide [125] PEG, agglutinin Nasal administration of np led to 5.6–7.7-fold increase in the brain
accumulation with agglutinin coating
PLGA Dexamethasone [126] Embedded in alginate PLGA np embedded in alginate matrices were administered from neural
electrode; dexamethasone released slowly in 2 weeks to reduce
inflammation of surrounding glial cells
albumin Loperamide [157] Apo-lipoprotein E Loperamide-loaded np induced antinociceptive effects after iv injection;
interaction with lipoprotein receptors required
Chitosan 99 m-Technetium [193] Polysorbate 80 5-fold increase in brain concentration in mice
Liposomes Phospholipids Phenytoin [136] N/A Improved local action against epilepsy
GABA [136] Decreased penicillin induced epileptic activity
Horseradish peroxidase [194] Transferrin Increased in vitro passage across BCEC culture
Amphotericin [140] PEG-RMP-7 Multiple fold increase in brain uptake
Micelle Pluronic P85 DPDPE, biphalin, morphine [146,148] N/A P85 enhanced the analgesic profile of biphalin, DPDPE, and morphine,
both above and below the critical micelle concentration.
Doxorubicin, digoxin, ritonavir, Drug permeability in monolayered BBB model by P-gp substrates
taxol, vinblastine, increased from 1.6 to 19.0 fold; no statistical difference in digoxin
rhodamine 123 [147,149] concentration in the brain of mdr1 knockout mice with the
addition of Pluronic P85

BBB: blood-brain barrier; BCEC: brain capillary endothelial cells; DPDPE: [D-Pen2,D-Pen5]-enkephalin; PBCA: Poly(butyl cyanoacryalate), MMA-SPM methylmethacrylate-
sulfopropylmethacrylate; np: nanoparticles; PLA: polylactide; PLGA: poly(D,L-lactide-co-glycolide).

polymers such as methylmethacrylate-sulfopropylmethacrylate (MMA-
SPM) were therefore studied as a substitute. Their negative charges
grant them a higher loading capacity for polar compounds including
zidovudine when compared to PBCA [121]. This MMA-SPM nanocarrier
system was able to increase the permeability of zidovudine and
lamivudine across an in vitro BBB model of bovine brain-microvascular
endothelial cells by 8–20 and 10–18 folds, respectively [121]. Further in
vivo studies will help to establish whether this increase in brain
permeability will improve delivery to infected brain cells such as brain
mononuclear macrophages.
Polyesters such as poly(D,L-lactide-co-glycolide) (PLGA) and poly-
lactide (PLA) have several qualities that make them appealing for brain
delivery. Their degradation products (e.g. water and carbon dioxide) are
metabolic by-products and relatively non-toxic [122]. Because of their
safety profiles, PLGA and PLA are two of the few polymers officially
approved for clinical use. In addition, these polyesters are known for

Fig. 2. Major pathways used by nanocarrier systems to improve antiretroviral penetration across the blood-brain barrier. (1) increasing the local drug gradient at the BBB by passive
targeting, (2a) allowing drug-trafficking by endocytosis (non-specific or receptor-mediated), (2b) blocking drug efflux transporters. (–): inhibitory effect.
510 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
their versatility. Their molecular weights, hydrophilicity, degradation
rate and hence the release kinetics can be conveniently tailored by
adjusting the composition [123]. They also easily form hydrolysable
bonds with diverse therapeutic molecules [122–124] and targeting
moieties such as lectin [124]. Drug loading into PLGA/PLA nanocarriers
and modification of these systems for brain targeting are therefore quite
convenient. Multiple-fold increases in brain drug concentration were
indeed observed in PLGA/PLA systems (e.g. vasoactive intestinal peptide
on PLA, dexamethasone on PLGA) [125,126], both administered via
intranasal route. Even large molecules such as peptides have been
shown capable of crossing the BBB in animal models [125]. However, up
to date the use of PLGA/PLA based nanocarrier specifically for ARVs
delivery to brain has not been reported. This is clearly an area for further
study.
Like regular polymers, dendrimers also consist of repeating
monomer units, but dendrimers are characterized by their repeatedly
branched molecular structures. These highly branched molecules, when
precisely engineered, can form monodispersed globular or spheroidal
nanostructures of 1 to over 10 nm in diameter [127]. Some of these
nanostructures may contain internal void spaces or surface functional
groups for encapsulation or conjugation of drug molecules, and can be
used as nanocarriers for drug delivery. They have been shown to
increase the BBB permeability of therapeutic agents such as DNA and
methotrexate [128,129]. Recently dendrimers have been evaluated for
CNS delivery of ARVs. Polyamidoamine dendrimers loaded with
lamivudine, a NRTI commonly used in HIV treatment, were evaluated
for their in vitro antiviral activity in MT2 cells infected with HIV-1. When
loaded on dendrimeric nanocarriers, a 21-fold increase in cellular
lamivudine uptake and 2.6-fold reduction in the viral p24 levels were
observed when compared to the group treated with free drug solution
[130]. Despite these promising results, it should be noted that the drug
release kinetics of dendrimers are sometimes inconsistent, and their
long-term safety profiles are relatively less established than polymers
like PLGA. More in vivo data are needed to further validate the use of
dendrimeric nanosystems for ARVs delivery to the CNS.
3.2.2. Lipid-based nanocarriers
Lipid-based nanocarriers hold strong promise for delivery of ARVs to
the CNS. There are a wide range of physiological lipids and phospho-
lipids available for lipid nanocarriers [please see reviews [131–133].
These materials are by nature biocompatible and biodegradable. A
number of lipid-based formulations (e.g. liposome, lipoplex) are already
commercially available and all of them have solid track record of clinical
safety. The technology of their production on industrial scale has also
been well-established. Because lipophilic materials have the natural
tendency to target the BBB, it is expected that lipid-based nanocarriers
will be useful for CNS delivery of ARVs. There are several classes of lipid-
based nanocarriers available, including liposomes, micro- or nanoemul-
sion and solid lipid nanoparticles (SLN).
Liposomes are vesicles made of one or more phospholipids bilayers.
They are probably the most studied and used lipid-based nanocarriers
[132]. In fact, a number of liposomal systems have been developed and
evaluated for the treatment of various brain illnesses, such as cerebral
ischemia by citicholine [134], brain tumors by cisplatin [135], and
epilepsy by phenytoin [136]. Overall, significant improvement in brain
drug levels were observed in these studies. Although there are a few
liposomal formulations for delivery of ARVs, e.g. stavudine and
zidovudine [137,138], relatively few of them are designed for HIV-
associated CNS illnesses. Foscarnet is an antiviral used as a salvage
therapy for late-stage HIV patients with multidrug resistance. Liposomal
foscarnet was able to increase the drug level in rat brains by 13-fold
when compared to the free foscarnet solution [139]. Another drug,
amphotericin B, is commonly used to treat the opportunistic fungal
infections in HIV patients. However, amphotericin B does not cross the
BBB. The use of liposomes coupled with brain targeting peptides for
amphotericin B delivery significantly increased the drug transport
across the simulated BBB model formed by rat brain endothelial cells
[140]. These studies further support the use of liposomes for more
effective treatment of HIV-associated CNS complications.
Nanoemulsions and microemulsions are usually oil-in-water
formulations in which the oil phase is highly dispersed to droplets
of submicron size and stabilized by surfactants and co-surfactants.
This type of formulations is especially suitable for highly lipophilic
HIV drugs such as PIs. Recently, saquinavir, the first PI marketed for
HIV treatment, was evaluated for brain delivery in an oral formulation
of flaxseed oil-based nanoemulsion [141]. The average oil droplet size
was around 100 to 200 nm in diameter. Use of saquinavir nanoemul-
sion instead of its free drug solution resulted in a three-fold increase
in the saquinavir concentration in the systemic circulation and three-
and five-fold increase in the area-under-the curve (AUC) values and
maximum saquinavir concentration in the brain, respectively, of male
balb/c mice. This study shows that in addition to enhancement of BBB
permeability, the small size of the nanoemulsion may also help bypass
other barriers such as the gastrointestinal tract when used as oral
formulations. There is clearly untapped potential in this nanocarrier
class.
SLN are a relatively new class of lipid-based nanocarriers [142]. They
are made of one or more lipids with melting points higher than body
temperature, so the carriers remain in solid state after administration.
The low solubility of nanocarrier biomaterials probably contributes to
the high tolerability of this formulation. A study showed that SLN in fact
caused less non-specific cell toxicity even compared to nanoparticles
made of PLGA [143], which has long been the standard for biocompat-
ible materials. In addition, by getting immobilized within a lipophilic
environment, the loaded drugs can be more adequately protected from
degradation. Our group has investigated the use of SLN for atazanavir
delivery [144]. Using a human brain microvessel endothelial cell line
(hCMEC/D3) representative of the BBB, a significantly improved
accumulation of [3H]-atazanavir was obtained when the drug was
delivered by SLN. Cytotoxicity experiments indicate that SLN exhibit no
toxicity in hCMEC/D3 cells up to a concentration corresponding to
200 nM of atazanavir. It was also noted that rhodamine-123, a well-
established P-gp substrate, delivered by the same system also resulted
in higher cellular accumulation, demonstrating that the P-gp efflux
activity at brain endothelial cells can be bypassed using SLN formula-
tions. SLN evidently hold strong promise for brain delivery of ARVs,
especially PIs which are mostly lipid soluble.
3.2.3. Micelle-based nanocarriers
A micelle is an aggregate formed by typically 50–100 amphiphilic
molecules (e.g. surfactants, block-copolymers) when dispersed in a
liquid phase [145]. In aqueous solution the amphiphilic molecules
aggregate and expose their hydrophilic heads outside and hide their
hydrophobic segments in the inner core region. This structure facilitates
solubilization of hydrophobic drug compounds within the micelle core.
The size of a micelle usually falls in the range of 5 to 20 nm in diameter.
The small size and good drug solubilization properties make micelles
potentially valuable nanocarriers.
Pluronic micelles were shown highly effective for BBB drug transport
enhancement in vitro and in vivo [145–147]. Using bovine brain
microvessel endothelial cell monolayer model, the effect of Pluronic
P85 on the permeability of a broad range of structurally unrelated
compounds was examined by Kabanov's group [147,148]. Increases in
the drug permeability up to 19-fold were detected. This permeability
enhancement was particularly strong with P-gp substrates such as
paclitaxel, vinblastine as well as ritonavir [148], a PI frequently used in
HAART regimens as a booster. In animal models, Pluronics increased the
drug delivery to the brain of wild-type mice, but the same benefit was
not observed in mdr1a/b knockout mice, indicating that the drug
permeability enhancing effect by Pluronics is mediated at least in part by
P-gp inhibition at the BBB [145]. It was suggested that this P-gp
suppressive effect could be mediated by ATP depletion, membrane
 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
fluidization by the co-polymer, or a combination of both mechanisms
[147–149].
4. Recent trends to optimize nanotechnology use for ARV
brain delivery
Despite promising data, the use of nanocarriers for ARVs delivery
to the brain remains at the experimental stage. Different strategies
have been proposed to further improve the various aspects of this
novel therapeutic approach, from efficiency, safety and specificity.
4.1. Optimization of nanocarrier properties to improve passive brain
targeting
The physicochemical parameters of nanocarriers such as their size,
surface charge and hydrophilicity can be optimized to favor the non-
specific, passive form of brain targeting. Using methotrexate-loaded
PBCA nanoparticles with sizes of 70, 170, 220, 345 nm, respectively, it
was shown that the 70 nm nanoparticles achieve significantly higher
peak drug levels in the cerebrum, cerebellum and cerebrospinal fluid
than the particles of larger sizes [129]. The authors suggested that
particles smaller than 100 nm could better mimic the membrane
receptors (e.g. low density lipoprotein receptors) and be more
efficiently transcytosed via receptor-mediated pathways. In another
study, using liposomes administered by the convection enhanced
delivery technique, not only the smaller liposomes (40 nm and
80 nm) crossed the BBB more easily than the larger ones (200 nm) to
achieve higher overall brain levels but they also penetrated deeper in
the brain tissues (0.79 mm average radius of penetration for the 80 nm
liposomes vs. 0.64 mm for the 200 nm liposomes) [150]. Overall,
carriers of size smaller than 100 nm are likely suitable for BBB passage.
The effect of carrier surface charge on brain delivery is less
conclusive. BBB is inherently negative in charge, so in theory cationic
carriers should lead to largest extent of brain delivery. However, a
study by Lockman et al., performed in rodents found that cationic SLN
administered by in situ brain perfusion did not effectively permeate
the BBB [151]. It is possible that the strong cell binding may prevent
the carriers from penetrating. In the same study it was noticed that
anionic SLN were able to permeate the BBB about 1 to 2-fold better
than the neutral or cationic formulations even at low SLN concentra-
tion range, and there was no sign of damage to the endothelial tight
junctions [151]. This increase in the BBB permeability was therefore
more likely a result of improved cell internalization rather than
leakage via the paracellular pathway. It was suggested that this
unexpected behaviour could be derived from the strong binding of the
anionic SLN to the low-density lipoprotein (LDL) receptors at the BBB,
which induced receptor-mediated endocytosis in both in vitro and in
vivo models [151,152]. However, it should be noted that neutral
carriers were found to exhibit the strongest in vivo stability. Overall,
carriers with neutral or anionic charge will probably result in optimal
in vivo brain targeting.
A drug can passively diffuse through the BBB in a more efficient
manner after it is converted into a more lipophilic prodrug. The same
principle can be applied to brain targeting by delivering drugs on
nanocarriers with enhanced lipophilicity. Fenart et al showed that
when polysaccharide nanoparticles were coated with a lipid bilayer, a
3 to 4-fold improvement in brain uptake without disruption of the
BBB integrity was observed [152]. The study also demonstrated a 27-
fold increase in the uptake of albumin when it was coated with the
same lipid bilayer. However, nanocarriers with high surface lipophi-
licity may favor non-specific tissue binding, and are also likely to
become a target of the reticuloendothelial system (RES) so they will
be eliminated from the circulation before it reaches the brain [153]. To
reduce the interactions with the phagocytic cells in the RES located
mainly in the spleen, liver and lymph nodes, many researchers coat
their colloidal carriers with PEG. It is well reported that PEG coating on
511
nanocarriers may minimize their removal by the phagocytic cells,
thereby prolonging their circulation time to typically 8 to 10 h [153].
This will create a larger time window for the drugs to reach the CNS.
4.2. Development of specific brain targeting strategies
To further improve the efficiency and specificity for brain delivery,
various biomolecules expressed at the BBB can be targeted. Overall,
these measures typically fall into two categories — indirect targeting
and direct receptor targeting. With indirect targeting, nanocarriers are
made of materials that bind to specific molecules in human body,
which have high affinity with the receptors at the BBB. With direct
targeting, nanocarriers are surface-grafted with ligand molecules that
specifically target those receptors at the BBB.
Polysorbates (also known as Tweens) are a commonly used class of
non-ionic surfactants. They have very low toxicity and are officially
approved for intravenous use. Polysorbates may serve as micellar
nanocarriers when used alone, and can also form the surface coating of
other nanocarrier systems to confer these systems the BBB permeabiliz-
ing properties [154]. It was found that the brain targeting properties of
the aforementioned PBCA nanoparticles were mostly derived from their
polysorbate coating [155]. Studies revealed that polysorbate, particu-
larly polysorbate-80, can increase the concentration of apolipoprotein E
(apoE) adsorbed on the nanoparticle surface, and these apoE-enriched
nanoparticles probably exploit the LDL receptor-mediated endocytotic
pathway at the brain endothelial cells [155]. More evidence supporting
this hypothesis was later provided by Kreuter et al, which showed that
the drug has to be loaded in the nanoparticles to gain passage across the
BBB, and therefore a nanoparticle-mediated drug transport process
must be involved [156].
LDL-receptor targeting can also be achieved by the direct
approach. Instead of using polysorbate as the linker moiety to adsorb
apoE, a recent study directly conjugated the apoE molecules to their
albumin-based nanoparticles by covalent linkages [156]. Significantly
improved delivery to the mouse brains was achieved. The same study
compared this direct approach (covalently linked apoE) to the
indirect approach (apoE adsorbed by polysorbate-80), and showed
moderately longer therapeutic effect in the former group (157).
Other receptors have been studied for brain targeting by immuno-
liposomes. Huwlyer et al. [158] have developed immunoliposomes for
delivery of the antineoplastic agent daunomycin to the rat brain. The
monoclonal antibody (mAb) used in these studies is the OX26 mAb to
the rat transferrin receptor [159], which in vivo is selectively enriched in
the brain microvascular endothelium [160]. Significant improvement in
brain uptake of [3H]daunomycin was achieved when the drug was
delivered using OX26 immunoliposomes versus the standard liposomes
without OX26 mAb. Brain targeting of immunoliposomes was not
observed when immunoliposomes were conjugated with a mouse IgG
(2α) isotype control. This technology has been extensively studied
[161–164] with development of a monoclonal antibody to the human
insulin receptor [165] and could be extended to immunoliposomes
loaded with ARVs for targeted delivery to the brain.
The specific receptor targeting strategy is particularly needed for
PEG-coated nanocarriers. The hydrophilicity of PEG molecules can
reduce the cell-carrier interaction and may be counterproductive to
brain targeting. Researchers developed PEGylated immunoliposomes
(PIL) to solve this issue [166,167]. In PIL, approximately 1 to 2% of PEG
molecules are conjugated to targeting peptidomimetic monoclonal
antibodies. This helps trigger receptor-mediated transcytosis of the
PIL across the BBB. Using PIL, the therapeutic agents that normally
cannot enter the brain, such as antisense oligomers and peptide drugs,
can be made available to the brain [166,167].
It was found that single domain antibodies alone, without attaching
to a nanoparticle or liposome, can also serve as a vector to deliver
therapeutic peptides across the BBB. For instance, novel single domain
antibodies such as FC5 were shown able to transmigrate across human
512 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
cerebral microvessel endothelial cells in vitro and the BBB in vivo [168].
By attaching horseradish peroxidase-tagged IgG to FC5 their transmi-
gration across cerebral endothelial cells was significantly enhanced
[168]. Pretreatment of human brain endothelial cells with wheat germ
agglutinin, sialic acid, alpha (2,3)-neuraminidase or Maackia amurensis
agglutinin which recognize alpha (2,3)-sialoglycoprotein receptor
significantly reduced FC5 transcytosis. The data suggest that FC5 binds
luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-
mediated endocytosis. This antibody-based vector may provide a new
brain-targeting drug delivery platform for HIV treatment [169].
Overall, the potential drawback of this class of specific receptor-
targeting platforms lies in their cost. To obtain large quantity of
monoclonal antibodies of clinically useful grade at sufficiently low cost
poses a major obstacle.
4.3. Cell penetrating peptides
Trans-activator of transcription (Tat) is a peptide derived from HIV-
1. It is able to substantially promote the level of transcription of the HIV
DNA [15,170]. Infected cells can produce Tat to activate the uninfected
cells to initiate the HIV gene production. Tat contains a basic region
consisting of six arginine and two lysine residues [171]. Their strong
cationic charges facilitate interaction with the normally negatively
charged cell surface, trigger permeabilization of the cell membrane via a
receptor/transporter independent pathway which results in endocyto-
sis of the sequence [172]. It was found that by tagging particulate
subjects with Tat, these particles can gain entrance into cells using the
same uptake mechanism. For instance, Tat-conjugated fused proteins
were able to efficiently bypass the BBB [173]. These findings led to a
number of studies on Tat-based brain targeting systems for treatment of
HIV-associated CNS complications [174–176]. Nanoparticles tagged
with intact or scrambled Tat sequences can interact better with cell
membrane of vascular endothelial cells, and significantly increase the
bioavailability of ritonavir to the brain when compared to the
nanoparticles without Tat. The potency of Tat is quite high. Use of
nanograms of Tat was sufficient to achieve a therapeutic level of
ritonavir in the CSF.
Other cell penetrating peptides such as antennapedia and penetratin
have also been studied [177,178]. It must be noted the long-term risks of
toxicity or immune responses associated with these products have not
been fully established. Their target specificity is also inconclusive [179].
Regardless, considering the potential return this novel strategy may lead
to, it is still an exciting area to further explore.
4.4. Other nanotechnology-based strategies to improve ARVs brain delivery
There are experimental strategies based on nanotechnology that
have not been extensively tested for ARV delivery to brain, but are
highly novel and show enormous potential.
4.4.1. Use of macrophages for BBB passage
A novel therapeutic strategy was developed based on the finding
that the BBB of HIV-infected patients can be easily penetrated by
macrophages by the aforementioned Trojan horse effect. Indinavir was
homogenized into very fine nanocrystals and coated with phospholipids
[180]. These nanocrystals were allowed to be internalized into bone-
marrow-derived macrophages and the indinavir-loaded macrophages
were injected into a mouse model of HIV-infection. It was found that the
macrophages migrated into the brain and delivered therapeutic dose of
indinavir. The antiretroviral effects, as determined by the decrease in
HIV-1 p24 viral levels, were sustained for weeks after a single injection
of the formulation [180].
4.4.2. Alternative route for nanocarriers administration
Recently, the use of intranasal administration for brain targeting has
raised significant interest. Extensive studies have been conducted on the
transport of large molecules from the nasal cavity to the brain. For
details please refer to the reviews by Illum L [181]. This approach was in
fact inspired by the brain entrance mechanisms of a number of viruses,
which enter the olfactory lobe of the brain and the cerebrospinal fluid
via the nasal passages. This implies that particulate matters from
proteins to viruses should be able to use this shortcut route to reach the
brain. The major barrier is in the passage of the carriers through the
nasal mucosal membrane. Because this membrane has plenty of lectin
receptors, PLA-PEG nanoparticles targeting lectin receptors were
recently studied [182], and 2-fold increase in brain uptake was observed
when compared with the nanoparticles without the lectin coating. Use
of this route to deliver anti-HIV peptides was also tested [183],
improvement of cognitive functions in tested subjects was observed.
Convection enhanced delivery is a novel delivery technique to
bypass the BBB and administer therapeutic agents directly into targeted
brain parenchyma or tissue. This technique involves one or more
catheters to be stereotactically placed through cranial burr holes into the
brain. Therapeutic agents such as liposomes are subsequently admin-
istered by microinfusion pump [150]. Although this method is too
drastic to be used in most HIV-infected patients, it may be reserved for
late-stage patients when the worsening of mental conditions is out of
control.
4.4.3. Advanced delivery of ABC-transporter blockers
The use of ABC-transporter blockers to enhance CNS delivery of ARVs
is limited by their risks of side effects and drug interactions. These
limitations can possibly be overcome by encapsulating and delivering
these agents with drug carriers. By choosing nanocarriers with high
brain affinity, strong localized blocking effect on the ABC-transporters
may be obtained. This approach has been first adopted for the treatment
of cancer expressing P-gp [184,185]. Polymer-lipid hybrid nanoparticles
co-loaded with doxorubicin (cytotoxic compound) and verapamil or
GF120918 (P-gp blockers) were prepared. It was found that this co-
loaded system resulted in significant accumulation of doxorubicin in the
P-gp overexpressing MDA435/MDR1 breast cancer cell line. The
doxorubicin-mediated cell kill was improved by nearly a log10 order.
This same strategy for BBB targeting has been evaluated in a few studies.
For example, PSC833, a non-immunosuppresise, cyclosporine-A analog,
P-gp blocker, was encapsulated in liposomes made of intralipids for in
vivo inhibition of P-gp at the BBB in primates. The AUCbrain/AUCblood
ratio of 11C-verapamil (a P-gp substrate) radioactivity was increased
2.3-fold with the addition of PSC833-liposomes [186]. As discussed in
the previous sections, Pluronics and polysorbates do not just serve as
micelles to carry drugs, these amphiphilic molecules also possess
intrinsic ABC-transporter blocking activities. In the future, it is expected
that more nanocarrier systems will incorporate biomaterials or a
secondary drug with ABC-transporter blocking properties to improve
the delivery of ARVs to the CNS.
5. Future perspectives and conclusion
Since the advent of HAART, it is no longer unrealistic to achieve
reasonable control of the HIV viral load in the periphery. However,
without effective measures to deliver the ARVs to the brain, the viral
load that persists at this site can lead to various debilitating neurological
complications and pose a strong threat to drug resistance and latent
reinfection. Current use of HAART regimens to clear the CNS viral load is
not only ineffective and risky, but also too costly for widespread use in
the developing nations. Studies have demonstrated that nanocarriers
can significantly increase the CNS penetration of several ARVs. Although
most studies remain at the experimental stage, they have already
established the feasibility of this approach. The next step will be to
optimize this general strategy, in terms of efficiency, safety and
specificity. To achieve this goal, future research in this field should
focus on the following issues.
 H.L. Wong et al. / Advanced Drug Delivery Reviews 62 (2010) 503–517
5.1. Improve understanding of the barrier structures
The knowledge of the various biomolecular events that occur at
the BBB allows us to develop the more active and specific forms of
brain targeting strategy. It is expected that more and more receptors
will be identified for specific brain targeting. In fact, receptors such as
transferrin receptor and insulin receptor have been targeted and
improved BBB passages were demonstrated [187,188]. The discovery
of cell penetrating peptides such as Tat also opens up exciting
opportunities, although issues like specificity and safety need to be
addressed in a more conclusive manner. Development of nanocarriers
for the delivery of ABC-transporter blockers is another valuable
option. The main goal should be to minimize excessive interference of
the ABC-transporters in other organs and tissues.
5.2. Tailor-made nano-formulations for brain delivery
With a better understanding of the architectures of the BBB and
BCFSB, nanocarriers should be tailor-made with the suitable physico-
chemical properties that will allow at least adequate passive brain
targeting. This means careful choice of the biomaterials and
formulation parameters. Lipophilic nanocarriers below or near
100 nm in diameter will probably be most useful. In addition, the
biomaterials selected need to possess very low toxicity and are fully
biodegradable to avoid damages to the CNS. Overall, the more
established materials such as lipids, phospholipids, PLGA and a few
selected non-ionic surfactants are likely good candidates to build
these nanocarrier platforms.
5.3. Use of better experimental models
The lack of appropriate in vivo models to simulate the changes
in BBB integrity in HIV infection is a major obstacle to further
development of brain delivery strategies. There has been increasing
evidence that indicate structural and functional alterations of the
BBB during HIV infection [189,190]. In particular, key membrane
proteins (e.g. occludin and zona occludens-1) forming the tight
junctions of brain capillary endothelium are significantly down-
regulated in HIVE. For detailed mechanisms of the BBB disruption
in HIV-infection, please see the review by Toborek et al., [191]. It
has been well documented that in cancer nanotechnology, increased
tumor penetration of nanoparticles can be caused by the leaky
vasculature and poor lymphatic drainage in the solid tumors [192].
Similarly it could be hypothesized that alterations in the BBB
integrity could allow easier passage of nanocarriers across the BBB
and thereby permitting enhanced delivery of ARVs. With more
realistic in vivo models, novel strategies based on therapeutically or
pathophysiologically induced changes in the drug permeability of
the BBB can be more thoroughly studied to maximize delivery of
ARVs using nanocarriers.
When in vitro models are used, their limitations should be
considered in order to avoid unsubstantiated conclusions. For
example, while a nanocarrier system can be efficiently taken up by
brain microvessel endothelial cells, this does not necessarily translate
into high BBB permeability. Trapping of nanocarriers in endosomes/
lysosomes can frequently occur following endocytotic processes (e.g.
clathrin-mediated endocytosis) [50]. Unless the loaded ARVs can be
released or the nanocarrier can trancytosed, the drugs will likely
accumulate in the brain capillary endothelium. Furthermore, success-
ful passage of ARVs across a simulated BBB model is not fully
predictive of its therapeutic effect. The drugs still need to access
the HIV-infected cells in the brain and remain biologically active.
Caution should be exercised in using the data generated from these
models.
513
5.4. Refine targets and endpoints of ARV delivery
It must be noted that in many of the previously discussed in vivo
animal studies, the whole brain was often considered as a single,
homogeneous target. Drug levels in different brain cell types were
seldom differentiated. As a result, uninfected cell types such as the
brain capillary endothelial cells may accumulate high ARV levels,
whereas the cells that are actually HIV-infected (e.g. microglia and
brain macrophages) may not receive adequate ARV treatment even
though, overall, the brain drug levels are high. In the future, studies
should involve differentiation of the brain cell types. Use of
technology such as flow cytometry to sort out the CD4+ cell
subpopulations may be an example of a method which could be
applied in animal models.
In addition, most of the current studies in this field focus on
measurement of distribution and accumulation of ARVs in the brain.
Only very few studies evaluate pharmacodynamic endpoints that are
more directly linked to the anti-viral effects (e.g. p24 level [130]). Future
studies should cover both: the pharmacokinetic and pharmacodynamic
endpoints to provide more solid evidence of the therapeutic effects of
novel therapies.
Ultimately, it is critical to ensure that the nanocarrier systems
developed for clinical use can be manufactured on an industrial scale at
reasonable cost. Nanocarrier types such as SLN and liposomes may fit
these criteria. Further studies will help explore their full potential. Given
the immense versatility and flexibility of many nanocarrier systems, it is
not unrealistic to foresee the clinical application of nanotechnology in
the future to treat HIV infection of the brain in an efficient and yet cost-
effective manner.
Acknowledgements
This work is supported by grants from the Canadian Institutes of
Health Research and the Ontario HIV Treatment Network (awarded
to RB). NC is supported by a U.S Army DOD BCRP predoctoral fellow-
ship (W81XWH-08-1-0519) and a CIHR Vanier Canada Graduate
Scholarship.
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