Download Delivery of Neurotherapeutics Across the Blood Brain Barrier in Stroke

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Current Pharmaceutical Design, 2012, 18, 3704-3720
Delivery of Neurotherapeutics Across the Blood Brain Barrier in Stroke
Xiaoming Hua,b, Meijuan Zhangb, Rehana K Leakc, Yu Gana,b, Peiying Lia,b, Yanqin Gaoa,* and Jun Chena,b,d,*
a
State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University, Shanghai, China; bDepartment of
Neurology and Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
c
Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, Pittsburgh, PA, 15282; dGeriatric Research,
Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA 15261
Abstract: Stroke is a devastating disease with few therapeutic options. Despite our growing understanding of the critical mechanistic
events in post-stroke brain injury, the clinical translation of these findings has been less effective. A monumental hurdle to the field has
been the inability of many systemically applied therapies to efficiently cross the blood brain barrier (BBB) and enter brain cells. Over the
last two decades, however, significant technological achievements have overcome this obstacle to facilitate central nervous system (CNS)
drug delivery. Noninvasive drug carriers, especially cell penetrating peptide (CPP) show great potential to deliver neurotherapeutics
across the BBB for the treatment of ischemic brain injury. This review begins with a brief introduction to the BBB in relation to drug delivery and then provides an overview of the development of drug carriers for neurotherapeutics, with a focus on CPP-mediated transduction. We discuss recent advances and limitations in this field, as well as mechanisms underlying CPP-mediated brain targeting. We also
summarize the application of CPPs in stroke research. Continuing modifications and improvements of CPPs are expected to enhance both
their feasibility in clinical stroke management and their specificity towards particular cell types.
Keywords: Cell penetrating peptide, TAT protein transduction domains, blood brain barrier, stroke, neuroprotection.
INTRODUCTION
Stroke is one of the most common causes of death and the leading cause of serious long-term adult disability in the United States.
Currently approved therapies for ischemic stroke are limited by a
short temporal window of opportunity and low success rate. The
development of new therapeutic strategies for stroke is imperative.
In recent years, an increasing number of molecules have been identified as potential therapeutic agents for central nervous system
(CNS) diseases such as stroke. However, the blood brain barrier
(BBB) prevents the free passage of many of these substances into
the CNS and has stymied drug development in this area. For example, nearly all peptide- or protein-based drugs fail to penetrate into
brain tissue to an appreciable extent and have led to very few clinical applications. Thus, the development of drug delivery and targeting strategies to overcome the obstacle of the BBB is more urgent
now than ever before. Here, we discuss recent developments in
drug carriers for neurotherapeutics, with particular focus on cell
penetrating peptide (CPP)-mediated transduction. We review the
mechanisms, recent achievements, and limitations of CPP-mediated
delivery and the application of CPP to stroke research.
BLOOD BRAIN BARRIER: AN OBSTACLE TO EFFECTIVE CNS DRUG DELIVERY
The presence of a barrier between the blood and the brain was
first observed by Paul Ehrlich and Edwin Goldman a century ago
with intravenous versus intracranial infusions of dyes such as Trypan Blue (for reviews see [1, 2]). However, it is Lewandowski that
is often credited by researchers for coining the term “Bluthirnshranke” (BBB in German) and Lina Stern for coining the term
“hematoencephalic barrier” [1, 2]. Although there is also a bloodcerebrospinal fluid barrier, an arachnoid barrier, and a blood-retinal
barrier, the study of the BBB for the past century has overshadowed
that of all the others. As a whole, these studies confirm that the
*Address correspondence to these authors at the Department of Neurology,
University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA;
Tel: 412-648-1263; Fax: 412-383-9985; E-mail: [email protected] OR
State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China; E-mail: [email protected]
1381-6128/12 $58.00+.00
BBB is a highly effective physical and metabolic barrier segregating CNS parenchyma from the blood circulation. It prevents the
passage of non-CNS cells, various foreign substances and microbes
from the bloodstream to the CNS. As a result of its efficacy in protecting vulnerable post-mitotic neurons and resident glia from potentially harmful peripheral substances, it also acts as a natural obstacle for the systemic delivery of neurotherapeutics. The BBB is
composed principally of specialized capillary endothelial cells
sealed by highly restrictive tight junctions not found elsewhere in
the circulation. These endothelial cells are, in turn, surrounded by
pericytes that cover 32% of the brain capillaries [3]. Pericytes synthesize most elements of the basement membrane and share this
membrane with the endothelial cells. Astrocytes embrace the outer
surface of the endothelium and respective basement membrane with
their end-feet, also known as the glia limitans perivascularis. Altogether, these densely packed cells and structure as whole maintains
and stabilizes the BBB Fig. (1). In general, a CNS-penetrating drug
has to be hydrophobic or small enough to cross this impressive
fortification effectively. However, most newly-discovered neuroprotective substances are hydrophilic peptides or proteins that cannot cross the BBB on their own.
In addition to its restrictive physical properties, the BBB has an
active enzymatic system to curtail the effective half-life of perceived toxins and to remove infiltrating foreign substances Fig. (1).
For example, the enkephalin metabolizing enzymes, aminopeptidase M, angiotensin converting enzymes and neutral endopeptidase
are all active enzymatic barriers to prevent the free passage of many
peptides through the BBB [4].
Finally, the BBB is also reinforced by a group of active efflux
transport proteins Fig. (1), including P-glycoprotein (P-gp),
multidrug resistance protein (MRP), and breast cancer resistance
protein (BCRP), all of which belong to the ATP-binding cassette
protein family [5]. A high concentration of P-gp is expressed in the
luminal membranes of the cerebral capillary endothelium. It is the
most important efflux system and actively removes many compounds from the endothelial cell cytoplasm before they penetrate
the brain parenchyma. BCRP and several members in the MRP
family (MRP-1, MRP3 and MRP5) are also expressed in the BBB
where they help to maintain brain homeostasis and remove toxic
compounds.
© 2012 Bentham Science Publishers
Delivery of Neurotherapeutics
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
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Fig. (1). Blood brain barrier as an obstacle for CNS drug delivery. The BBB is a physical and metabolic barrier segregating the CNS parenchyma from
blood circulation. BBB is composed principally of specialized capillary endothelial cells joined by highly restrictive tight junctions. The endothelial cells are
surrounded by pericytes, which synthesize most elements of the basement membrane and share it with the endothelial cells. Astrocytes embrace the outer
surface of the endothelium and respective basement membrane by their end-feet. All these densely packed cells and structure form a membranous barrier to
maintain and stabilize the BBB. In addition to its restrictive physical property, the BBB is reinforced by an active enzymatic system and a group of active
efflux transport proteins.
DRUG CARRIERS TO ENABLE BBB PENETRATION
To promote the passage of therapeutic agents into the CNS, a
variety of approaches have been attempted, including disruption of
the BBB [6], bypassing the BBB [7], blocking efflux transporters
[8], and using drug carriers such as cell-penetrating peptides (CPPs)
[9], antibodies or ligands based on receptors on the BBB [10],
liposomes [11], and nanoparticles (NPs) [12]. The current review
will focus on non-invasive drug carriers, with a special emphasis on
the CPPs.
CELL-PENETRATING PEPTIDES DERIVED FROM PROTEIN TRANSDUCTION DOMAINS FOR CNS DRUG DELIVERY
Analysis of naturally occurring proteins that are able to circumvent membranous barriers has led to the discovery of several protein sequences known as protein transduction domains (PTD). CPPs
are small peptides derived from PTD that retain the cell-membranepermeable properties of the native proteins. When attached to the
molecule of interest via either chemical or genetic means, CPPs
confer their cargo with the ability to cross cellular membranes and
gain access to CNS targets.
CPPs are derived from various sources (Table 1). Naturally
derived CPPs, including the TAT peptide from Human Immunodeficiency Virus (HIV) transcription-activating factor [13] and penetratin peptide from the Drosophila antennapedia protein [14] are
prototypical CPPs and have been used extensively. Synthetic CPPs,
such as homoarginine peptides and transportan, are designed with
predetermined structures and are easily modified with different
chemical entities, which give rise to novel and more efficient thera-
peutic agents [15, 16]. Chimeric CPPs, such as the sequence signalbased peptide, combine sequences with different functions to
achieve synergistic translocation efficiency [17].
Although heterogeneous in size and sequence, CPPs share a few
common properties. (1) All CPPs are small peptides between 6 to
30 amino acids long. (2) Nearly all CPPs are positively net charged
at physiological pH due to the high content of basic amino acids
(eg. arginine and lysine) in their protein sequences. This positive
charge seems to be critical for the cellular uptake of CPPs. The
substitution of basic residues in CPPs with neutral alanines reduces
cell penetration, while substitution of neutral residues with arginines actually enhances internalization [18, 19]. The cationic nature
itself, however, is not sufficient for optimal translocation of CPPs.
Other features, such as the number of arginine residues are also
important for their translocation activity [20]. (3) A vast majority of
CPPs are characterized by an amphipathic structure while showing
overall hydrophobicity. Of course, a certain degree of hydrophobicity can help the cationic peptide sequences enter target cells, but a
highly hydrophobic peptide would be trapped in the membrane
rather than being internalized [21]. In short, an amphipathic structure is critical for the internalization of CPPs.
In addition to its BBB permeability, several other advantages of
the CPP delivery system have made it one of the most widely exploited drug carriers in CNS studies. First, CPP-mediated delivery
is surprisingly independent of cargo size. Molecules range in size
from small peptides, proteins, oligonucleotides, imaging agents to
massive structures such as nanoparticles and liposomes have all
been successfully delivered into the brain using CPPs. Second, the
3706 Current Pharmaceutical Design, 2012, Vol. 18, No. 25
Table 1.
Hu et al.
Selected Cell Penetrating Peptides
CPP
Sequence
Source
Refs
Naturally-derived
TAT
GRKKRRQRRRPPQ
HIV transcription-activating factor
[22]
Penetratin
RQIKIWFQNRRMKWKK
Drosophila homeoprotein antennapedia
[23]
VP22
DAATATRGRSAASRPTERPRAPARSASRPRRPVD
HSV VP22 protein
[24]
SynB1
RGGRLSYSRRRFSTSTGR
Protegrin
[25]
pVEC
LLIILRRRIRKQAHAHSK
Murine vascular endothelial cadherin
[26]
SAP
(VRLPPP) 3
Modified maize -zein-related sequence
[27]
hCT
LGTYTQDFNKFHTFPQTAIGVGAP
Human calcitonion
[28]
BagP
MKKKTRRRST
Human protein bag-1
[29]
HP4
RRRRPRRRTTRRRRR
Herring portamine
[19]
Lactoferrin (hLF)
KCFQWQRNMRKVRGPPVSCIKR
Human milk protein lactoferrin
[30]
Nucleolar targeting peptides
YKQCHKKGGKKGSG
Crotamine, a toxin in venom of the South American rattlesnake
[31]
Cytosol Localizing Peptide-1 (CyLoP-1)
CRWRWKCCKK
Crotamine, a toxin in venom of the South American rattlesnake
[32]
gH625
HGLASTLTRWAHYNALIRAFGGG
glycoprotein gH of herpes simplex type I virus
[33]
Model amphipathic peptide (MAP),
KLALKLALKALKAALKLA
Synthetic
[34]
Homoarginine peptides
RRRRRRR(RR)
Synthetic
[20]
C105Y
CSIPPEVKFNKPFVYLI
Synthesized based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin
[35]
M918
MVTVLFRRLRIRRACGPPRVRV
Synthetic
[36]
S41
CVQWSLLRGYQPC
Synthetic
[37]
CADY
GLWRALWRLLRSLWRLLWRA
Synthetic
[38]
MPG
GALFLGFLGAAGSTMGAWSQPKSKRKV
A hydrophobic domain from the fusion sequence of HIV gp41 and a
hydrophilic domain from the NLS of SV40 T-antigen
[39]
Signal sequence- based
peptide
MGLGLHLLVLAAALQGAWSQPKKKRKV
A hydrophobic domain from Caiman crocodylus Ig(v) light chain and
a hydrophilic domain from the NLS of SV40 large T antigen
[17]
Transportan 10
GWTLNSAGYLLGKINLKALAALAKKIL
12 amino acids from the N-terminal of neuropeptide galanin and 14
amino acid long wasp venom peptide, mastoparan, connected via a
lysine residue
[40]
PEP-1
KETWWETWWTEWSQPKKKRKV
a hydrophobic tryptophan-rich motif and a hydrophilic lysine-rich
domain derived from the nuclear localization sequence (NLS) of simian virus 40 (SV-40) large T antigen, separated by a spacer domain
(SQP)
[41]
S4(13)-PV
ALWKTLLKKVLKAPKKKRKV
a 13 amino acid cell-penetrating sequence derived from the dermaseptin S4 peptide and the NLS of SV40 large T antigen
[42]
Synthesized
Chimeric
Delivery of Neurotherapeutics
in vivo systemic delivery of CPPs lead to rapid expression of cargo
molecules in the CNS cells. For example, certain proteins constructed using TAT-PTD technology can cross the BBB and transduce into CNS cells in a matter of mere hours after systemic administration [9, 43]. Finally, peptide-mediated delivery of macromolecules is superior to other commonly used delivery systems in
many aspects, such as relatively low toxicity, high delivery efficiency, and the possibility for peptide backbone modifications.
Hence, the use of CPPs opened a new gateway for CNS drug delivery and is a promising approach for treating neurological diseases.
In this section, select CPPs used frequently for BBB penetration
and their application in CNS diseases will be discussed.
HIV TRANSCRIPTION-ACTIVATING FACTOR TAT PEPTIDE
The transactivator of transcription protein of the HIV is an 86
amino acid protein that plays an essential role in virus replication.
In 1988, two research groups independently discovered that the
HIV TAT protein can penetrate cells efficiently when added exogenously in tissue culture [13, 44]. It was also found that the transduction property could be attributed to the amino acid counterpart of
the gene sequence position 37-72 [44]. A subsequent study [22]
more precisely located the 11-amino acid sequence (YGRKKRR
QRRR at amino acids 47–57), now known as the TAT peptide as
the essential CPP for TAT protein.
It was then demonstrated in 1994 that several large proteins,
when conjugated to TAT transduction peptides at amino acids 1-72
or 37-72, could enter various types of cells in culture [45]. For example, TAT-conjugated -galactosidase was shown to distribute to
cells of multiple organs, but not the brain, after systemic delivery
[46]. Later on, by constructing fusion proteins with the essential
TAT-PTD at their amino- or carboxy-terminus, an improved TAT
delivery system was shown to mediate an efficient and rapid expression of fusion protein in various tissues after intraperitoneal
injection [9]. Spectacularly, the cargo protein activity was detected
in brain neurons without impairment of the BBB, as manifested by
the lack of Evans blue dye brain infiltration [9]. This attraction of
TAT-PTD for the brain and non-dividing neurons has particularly
important implications in neuroscience research and in CNS drug
design. In recent years, different modifications of TAT-CPP have
successfully enhanced its efficient transduction into cultured cells
and increase its bioavailability and activity in vivo [47-49]. The
application of the TAT-based delivery system has extended from
peptides/proteins to a wide variety of cargoes (Table 2). In the later
section, we will discuss in detail the application of TAT in stroke.
PEP-1
Pep-1 is another CPP frequently used in CNS studies. It is a
chimeric CPP with an amphipathic sequence. It contains a hydrophobic tryptophan-rich motif (KETWWETWWTEW), which is
responsible for the interaction with proteins and cell membranes, a
hydrophilic lysine-rich domain derived from the nuclear localization sequence (NLS) of simian virus 40 (SV-40) large T antigen
(KKKRKV), which improves solubility, and a spacer domain in
between (SQP), which improves the flexibility and the integrity of
the other two domains [41]. When mixed with target proteins (GFP,
-gal), Pep-1 peptide led the nondenatured target protein into cultured cells.
A number of studies have demonstrated that Pep-1 is able to
deliver a variety of proteins and peptides into the brain. A fusion
protein of antioxidant enzyme Cu,Zn-superoxide dismutase (SOD)
and Pep-1 can cross the BBB after intraperitoneal injection and
transduce into neurons [50]. A series of studies reported that this
Pep-1-SOD fusion protein protected against transient forebrain
ischemia [50-52] and spinal cord injury [53, 54]. Pep-1 also successfully delivered other neuroprotective proteins into the brain,
including copper chaperone for SOD (CCS) [55], ribosomal protein
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
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S3 (rPS3) [51], HSP27 [56], catalase [57] and Frataxin [58] to reduce post-ischemic injury.
PENETRATIN
Penetratin is derived from Drosophila homeoprotein Antennapedia (Antp) and belongs to a class of trans-activating factors that
are critical in several morphological processes including neuronal
morphogenesis [14, 59]. These homeoproteins bind DNA through a
specific sequence of 60 amino acids called the homeodomain. It
was found that the homeodomain of Antp has the capacity to translocate across the neuronal membrane [14]. Structurally, the homeodomain consists of three -helices with one turn between
helix 2 and 3 [60]. Subsequent studies identified a 16-amino-acid
sequence (KETWWETWWTEWSQPKKKRKV), so-called penetratin, in the third helix of the Antp homeodomain that is involved
in the translocation process and is the minimal CPP of Antp [23].
The ability of penetratin to deliver cargoes across the BBB was
first shown by Rousselle and colleagues. In their study, penetratin
CPP enabled a covalently coupled chemotherapeutic agent to cross
the cellular membranes of the BBB with high efficiency without
compromising BBB integrity [25]. It has also been shown that
penetratin can help small proteins, such as C-terminal structure
BIR3-RING of X-linked inhibitor of apoptosis protein (XIAP) [61]
and calbindin D [62], enter rat brains after systemic application and
thereby decrease transient middle cerebral artery occlusion
(MCAO) induced cell apoptosis. Penetratin was also conjugated
with polyethylenimine (PEI) and polyamidoamine (PAMAM) to
prepare Antp-modified DNA-loaded nanoparticles, which enhances
the transfection and expression efficiency of DNA/PAMAM
nanoparticle in brain capillary endothelial cells [63].
SYNB VECTORS
The SynB peptide family is derived from a natural antimicrobial peptide called protegrin 1 (PG1) [64]. PG1 is 18 amino acids
long (RGGRLCYCRRRFCVCVGR), forming an antiparallel sheet structure constrained by two disulfide bridges [65]. It interacts
with and disrupts the lipid matrix of bacterial membranes [66].
SynB1 is a linear analogue of PG1 with the four cysteine residues
replaced by serines and two valines replaced by threonines [25].
SynB1 is able to cross cell membranes efficiently and is devoid of
any cytolytic effect of PG-1. Further optimizations have led to the
development of shorter SynB peptides with improved translocation
properties [67].
Several drugs conjugated to SynB vectors have demonstrated
increased brain uptake and in vivo activities [25, 68, 69]. For example, the BBB non-permeable doxorubicin can gain access to the
brain after systemic application when it is covalently linked to
SynB1 peptide [25]. Interestingly, this delivery strategy may reduce
the cardiotoxicity of doxorubicin, as the heart had significantly
lower SynB1-doxorubicin levels. The SynB vectors have also been
used to deliver peptide molecules such as enkephalin and streptavidin to the brain [70]. A recent study designed a novel delivery
system that contains SynB1 and a thermally responsive elastin-like
polypeptide (ELP). It was shown that this SynB-ELP polypeptide
could enhance thermal-based therapeutics targeting to various regions of the brain, particularly the cerebellum [71].
OTHER CPPs FOR CNS DELIVERY
Several other CPPs can penetrate the BBB after systemic application. For example, a synthetic arginine-rich CPP ((RXRRBR)XB)
facilitated the brain uptake of antisense morpholino oligonucleotides [72], which can induce alternative pre-mRNA splicing and
thereby offer a potential therapeutic tool for neurogenetic disorders
such as ataxia-telangiectasia [73]. In another study, a basic amino
acid-rich nucleolar localization signal in LIM kinase 2 (LIMK2
NoLS) showed potential to transport peptides and proteins across
3708 Current Pharmaceutical Design, 2012, Vol. 18, No. 25
Table 2.
Hu et al.
Cargoes Delivered Across Blood Brain Barrier by TAT Cell Penetrating Peptides Delivery System in Stroke and other
CNS Disease Models
Cargo
Formula
Route
Animal Model
Biological Effect
Refs
TAT-Bcl-x(L)
fusion protein
iv or ip
Mouse, Ischemia
reperfusion (I/R)
Anti-apoptotic; attenuates ischemia-induced brain injury;
increases survival of neural precursor cells
[43, 97,
102, 103]
ip
Rat, neonatal HI
Anti-apoptotic; decreases cerebral tissue loss
[126]
ip
Gerbils, transient
forebrain ischemia
Prevents delayed neuronal death in the hippocampus
[102]
Peptide and Protein
Bcl-x(L)
X chromosome-linked
inhibitor of apoptosis
(XIAP)
TAT-XIAP
fusion protein
iv
Mouse, permanent
ischemia
Anti-apoptotic; efficiently targets the lesion, reduces infarct
volumes, reverses long-term impairments
[100]
Glial-cell-line-derived
neurotrophic factor
(GDNF)
TAT-GDNF
fusion protein
iv
Mouse, I/R
Neurotrophic factor; prevents neuronal death and reduces
infarct volume
[99]
ip
Mouse, MPTP model
of Parkinson's disease
Neurotrophic factor; reaches dopaminergic neurons but
elicits no protection
[127]
Erythropoietin (EPO)
TAT-EPO
fusion protein
ip or iv
Mouse,
TAT enhances the capacity of EPO to cross the BBB at least
twofold when given IP and up to five-fold when given IV,
TAT-EPO protects against ischemic brain injury.
[108]
Inhibitor of PKC
(V1-1)
V1-1-TAT
fusion protein
ip
Rat, I/R
Improves microvascular pathology, increases cerebral blood
flow, reduces infarct size
[101]
-Receptors for
Activated C Kinase
(RACK)
TAT-RACK fusion
protein
iv
Rat, Global cerebral
ischemia
Activation of PKC by TAT-- RACK protects the brain
against ischemic damage by regulating cerebral blood flow
[114]
c-Jun N-terminal
kinase-inhibitor (DJNKI1)
TAT-DJNKI1
fusion protein
ip
Rat/mouse, permanent ischemia
JNK inhibitor; reduces lesion volume and improves neurological function
[115-117]
JNK-inhibitor JBD
TAT-JBD
fusion protein
ip
Rat, neonatal HI
Reduces neonatal HI brain damage with long-lasting anatomical and behavioral improvements
[128]
Postsynaptic density95 (PSD95) inhibitor
Tat-NR2B9c
fusion protein
iv
Rat, I/R and permanent ischemia
Blocks NMDAR-PSD95 signaling, reduces infarct volumes
and improves long-term neurobehavioral functions
[119, 120]
vein.
infusion
Rat, status epilepticus
Reduces cell loss in hippocampus
[129]
I/R
Neuroglobin
TATNeuroglobin
fusion protein
iv
Mouse, I/R
Efficiently transduces into neurons and protects the brain
from ischemia
[97]
Nogo-A extracellular
peptide residues 1-40
(NEP1-40)
TAT-NEP140 fusion
protein
ip
Mouse/rat, I/R
Inhibits neuronal death, promotes axonal regrowth and
functional recovery
[122, 123]
Hsp70
TAT-Hsp70
fusion protein
iv
Mouse, I/R
Chaperone; reduces infarct volume and increases survival of
neural precursor cells
[124]
ip
Mouse, MPTP model
of Parkinson's disease
Chaperone; prevents neuronal cell death
[130]
ip
Rat, I/R
Increases SOD activity in the brain and protects the brain
from I/R injury
[125]
Superoxide dismutase
TAT-SOD
fusion protein
Delivery of Neurotherapeutics
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
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(Table 2) Contd.....
Cargo
Formula
Route
Animal Model
ip
Gerbils, transient
forebrain ischemia
Biological Effect
Refs
Prevents neuronal death in hippocampus
[131]
Apaf-1-inhibitory
protein (AIP)
TAT-AIP
fusion protein
ip
Rat, neonatal hypoxia-ischemia (HI)
Anti-apoptotic; decreases brain damage and improves neurological functions
[132]
NF-B essential
modulator (NEMO)
binding domain
(NBD)
TAT-NBD
fusion protein
ip
Rat, neonatal HI
NF-B inhibitor; causes reduction in brain tissue loss and
improvement in sensorimotor and cognitive functions
[133]
Sensitive to apoptosis
gene (SAG)
TAT-SAG
fusion protein
ip
Gerbils, transient
forebrain ischemia
Prevents neuronal death in hippocampus, decreases lipid
peroxidation in the brain
[135]
Leptin
TAT-leptin
ip
Mouse, fed with
high-fat diet
Increases leptin in hippocampus, reduces body weight
[136]
Lipoamide dehydrogenase (LAD, E3)
TAT-LAD
fusion protein
iv
E3-deficient mice
Causes significant increase in the enzymatic activity of the
mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and brain
[137]
Ubiquitin c-terminal
hydrolase L1 (UchL1)
TAT-HAUch-L1 fusion protein
s.c
Mouse, APP/PS1
Alzheimer model
Restores spine density
[138]
Ciliary neurotrophic
factor (CNTF)
TAT-CNTF
Ip; per
rectum
Mouse, A-induced
dementia
Neurotrophic factor, reduces? learning and memory impairments
[139]
Choline acetyltransferase
TAT-choline
acetyltransferase fusion
protein
iv
Mouse, A-induced
dementia
Neurotransmitter, improves memory and cognitive dysfunction
[140]
Cy5.5
Cy5.5-TAT
iv
Rat, experimental
autoimmune encephalomyelitis
Labels and tracks T lymphocyte
[141]
CdS ratio Mn/ZnS
quantum dots (Qdots),
TAT-Qdots
Intraartery
Rat
Reaches the brain parenchyma for imaging
[142]
FITC doped silica
nanoparticles
TAT-FSNPs
Intraartery
rat
Used for brain imaging
[143]
TAT conjugated with
cholesterol
for doxorubicin- loaded
liposome
iv
Rat, Brain glioma
Increases suvival time of glioma-bearing rats; distribution of
doxorubicin increases in the brain and decreases in heart
[144]
Coumarinloaded
MPEG-PCL TAT
Intranasal
Rat
Nano-sized micelles modified with Tat facilitate direct
intranasal brain delivery
[145]
Imaging agents
Liposomes
Doxorubicin-loaded
liposome
Nanoparticles
Coumarin-loaded
methoxy
poly(ethylene glycol)
(MPEG)/poly(caprolactone) (PCL)
3710 Current Pharmaceutical Design, 2012, Vol. 18, No. 25
Hu et al.
(Table 2) Contd.....
Cargo
Formula
Route
Animal Model
Biological Effect
Refs
Cholesterolconjugated antimicrobial peptides G(3)R(6)
nanoparticle
Cholesterolconjugated
G(3)R(6)TAT
iv
Rabbit.
C.neoformans meningitis model
Antimicrobial; Crosses the BBB and suppresses yeast
growth in brain tissues
[146]
Ciprofloxacin-loaded
cholesterol-conjugated
poly(ethylene glycol)
(PEG)
TAT-PEG-bColCiprofloxacin
iv
Rat
Ritonavir-loaded Poly
(L-lactide) (PLA)
nanoparticle,
RitonavirPLA-TAT
iv
Mouse
the BBB [74]. With rapid advancements in technology, an increasing number of CPPs will be discovered, designed, or modified to
enable brain delivery of therapeutic biomolecules.
RECEPTOR-MEDIATED SPECIFIC TRANSCYTOSIS
Receptor-mediated specific transcytosis is a delivery method
based on the physiological receptors expressed on endothelial cells
forming the BBB. The molecule of interest is conjugated with the
monoclonal antibodies or ligands to these physiological receptors,
which lead to the translocation of fusion products through the BBB.
So far, several receptors, including the insulin receptor and transferrin receptor, have been exploited for delivering compounds across
the BBB in experimental stroke studies.
TRANSFERRIN RECEPTOR
Serum transferrin functions as a transporter carrying iron from
the site of intake to various tissues and cells. Transferrin draws
wide attention in the drug delivery area due to its nonimmunogenicity, low toxicity, as well as biodegradability [75]. In addition, the
high expression levels of transferrin receptors (TfR) on CNS endothelial cells relative to other organs makes this vector an ideal candidate for CNS-targeted delivery [75]. One disadvantage, however,
is that the transferrin vector applied in vivo has to compete with
large amounts of endogenous transferrin for BBB TfR binding sites,
which significantly diminishes its delivery efficiency [76]. To overcome this problem, a monoclonal antibody against the TfR named
OX26 was generated. This antibody binds to a distinct site on TfR
from the binding site for endogenous transferrin and thus cannot be
outcompeted. Using this approach, nerve growth factor (NGF) was
found to penetrate the BBB in a dose-dependent fashion following
intravenous injection and prevented the degeneration of cholinergic
striatal neurons in a rat model of Huntington’s disease [10]. In another study, a single intravenous injection of BDNF-OX26 protected neurons in rats subjected to 60-min MCAO [77]. In order to
further strengthen the binding between the drug and OX26 as well
as to reduce the drug modification, the high affinity of streptavidin
for the biotin molecule was exploited in this system. Briefly, a conjugate of OX26 and streptavidin was produced in parallel with the
monobiotinylated drug [78]. The subsequent conjugate of biotinylated FGF and OX26-streptavidin was shown to be effective in
reducing brain infarct volume and in improving neurological deficits in rats subjected to permanent MCAO [79].
INSULIN RECEPTOR
Small amounts of circulating insulin can cross the BBB into the
CNS through insulin receptors on endothelial cells. The monoclonal
antibody to human insulin receptor (HIRMAb) thus acts as a Trojan
horse to ferry a molecule of interest across the BBB when this
Antimicrobial; crosses the BBB and enters the brain
Enhances the CNS bioavailability of the encapsulated PI
and maintains therapeutic drug levels in the brain for a
sustained period
[147, 148]
[149]
molecule is fused to the carboxyl terminus of the heavy chain of
HIRMAb. Exemplary HIRMAb fusion proteins used for the uptake
of various drugs into the brain are listed in (Table 3). Among these
fusion proteins, HIRMAb-EPO and HIRMAb-GDNF have been
shown to cross the BBB after intravenous injection in rhesus monkeys [80] and both protect against cerebral ischemia following intracerebral administration in rat model of stroke [81, 82].
Although many studies observed an increase in brain uptake of
the receptor ligand/antibody-conjugated proteins from the periphery
compared to the corresponding unmodified proteins and reported
the effectiveness of these conjugated proteins in models of neurological diseases after systemic application, several issues remain
poorly addressed. For instance, it is unclear if and how fusion with
antibodies or ligands alters the activity of the modified protein. In
addition, given that all these targeted BBB receptors are also expressed on peripheral organs and play important physiological
roles, this raises the concern that the effective dosage to enter brain
parenchyma may lead to unbearable peripheral side effects. Furthermore there is also the possibility that chronic repeated injection
of the fusion protein in vivo may cause the down-regulation of targeted receptors during the therapeutic period.
NANOPARTICLE-MEDIATED DELIVERY
Recently, various types of nanoparticles (NPs) infused with
therapeutic agents have been developed as efficient, site-specific
and control-released modes of drug delivery. NPs used for drug
delivery consist of different materials such as polymers, lipids or
metals. In principle, the biodegradable and stable polymers such as
poly (alkyl cyanoacrylate) (PBCA), poly (lactide-co-glycolide)
(PLGA), human serum albumin [83] are excellent candidates.
Drugs can either be integrated into the matrix of the particle or
attached to the particle surface. These NPs allow peptide cargo to
gain access to the surface of the endothelial cells forming the BBB
and to penetrate cell membranes. Coating the NPs with certain surfactants such as polysorbate 80 (PS 80) [84] or attaching the NPs
with certain ligands such as transferrin [85] or lipoprotein [86]
could redistribute the NPs and enhance their accumulation in the
cerebral parenchyma. These pre-coating modified NPs also reduce
kidney and liver toxicity because of their targeted distribution [87].
This system has already been effective against in vivo models of
ischemic stroke [88], brain tumors [87, 89], Alzheimer’s disease
[90], and Parkinson’s disease [91].
The internalization of NPs depends on the cell type, the size of
the particles, the surface charge, and the hydrophobicity of the particles [92]. For instance, small particles below 200 nm are probably
internalized through clathrin-mediated endocytosis, while the uptake of larger particles up to 500 nm may be caveolae-mediated. It
Delivery of Neurotherapeutics
Table 3.
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
3711
Insulin Receptor-Mediated CNS Delivery
Fusion protein
Route
Animal Model
Functions
Refs
HIRMAb-EPO fusion protein (Erythropoietin
conjugated to human insulin receptor monoclonal
antibody )
iv
rhesus monkeys
HIRMAb-EPO fusion protein is rapidly transported
into brain
[80]
HIRMAb-EPO fusion protein
icv
rat permanent
MCAO
Reduces infarct volume and reduces neurological
deficits
[81]
HIRMAb-GDNF (Glial-derived
factor bind to HIRMAb)
neurotrophic
icv
rat permanent
MCAO
The fusion protein is active in vivo and reduces infarct volume
[82]
HIRMAb-IDS fusion protein
sulfatase conjugated to HIRMAb)
(iduronate-2-
iv
Mucopolysaccharidosis
[150] Type II
Produces therapeutic concentrations of IDS in the
brain
[151]
HIRMAb-TNFR fusion protein (type II TNF
receptor conjugated to HIRMAb )
iv
rhesus monkeys
Fusion protein selectively targets brain more than
peripheral organs; 3% of the injected dose is taken up
by the primate brain
[80]
HIRMAb-ScFv (a single chain Fv antibody
against the Abeta peptide conjugated to HIRMAb)
iv
rhesus monkeys
Can be transported across the primate BBB in vivo
[152]
has also been reported that PS 80 coated PBCA NPs have the ability to adsorb apolipoprotein E and B in the plasma after intravenous
injection and therefore cross the BBB through low-density lipoprotein receptor-related protein (LRP) [93].
IMMUNE CELL-BASED DELIVERY
Resent research suggests that immune cells might also be feasible drug delivery vectors [94, 95]. This approach exploits the natural ability of peripheral immune cells such as macrophages to pass
the BBB and home to sites of neuronal injury or degeneration to
endocytose cellular debris. For example, transplantation of genetically modified macrophages overexpressing GDNF protects against
MPTP-induced dopaminergic neurodegeneration [94]. Another
study demonstrated that macrophages could deliver the vectors of
NPs into gliomas for photothermal ablation [95]. Finally, it is well
known that the infiltration of peripheral immune cells into the
ischemic core and penumbra occurs immediately after stroke and
remains prominent for several days [96]. In this regard, immune
cell-based delivery is a highly promising approach to rapidly deliver neurotherapeutics to the ischemic brain for an early rescue of
injured neurons. A distinct advantage of this approach is its cellular
specificity, as the immune cells are not likely to target uninjured
sites, either in the brain or the periphery.
TAT-MEDIATED CNS DELIVERY OF THERAPEUTIC
PEPTIDES AND PROTEINS IN PRE-CLINICAL STROKE
RESEARCH
Despite our growing understanding of the critical mechanistic
events in post-stroke brain injury, there has been little clinical translation of these findings. One important limiting factor is the inability of systemically applied strategies to efficiently cross the BBB
and enter brain cells. Several viral vectors, especially the adenoassociated virus (AAV), have shown great promise to achieve robust transgene expression in the brain. However, given the relatively acute nature of cell death after stroke and the small time window of opportunity for stroke treatment, gene delivery, which necessitates a relatively long time for protein expression, is not a feasible option. Additionally, the expression of viral vector-delivered
genes relies on host machinery for protein synthesis, which is often
severely compromised during ischemic injury. Thus far, the application of viral vectors is therefore mostly in the research arena.
An alternative approach for CNS delivery of neurotherapeutics
after stroke is to directly send the gene product itself into the brain.
CPP is a commonly used strategy under this category. As a nonviral
system, the in vivo delivery of CPP does not require a prolonged
incubation period to achieve gene expression. This advantage allows for its therapeutic application in acute CNS diseases such as
stroke. According to most studies, TAT coupled proteins are detected in the brain as early as 4 hours after systemic injection [43,
97-99], a timeframe that is within the therapeutic window for
stroke. More intriguingly, some of these proteins have proven protective even when applied after ischemic onset [43, 98-101]. All of
these advantages encourage the translation of these treatments into
clinical use. Quite a few peptides and proteins have been successfully delivered into the brain by CPP for the treatment of stroke and
other neurological disorders (Table 2). In this section, we will use
TAT as a prototypical CPP and review several specific TAT fusion
proteins that cross the BBB and exert neuroprotection against cerebral ischemia.
ANTI-APOPTOTIC MOLECULES
In 2002, three independent research groups [43, 83, 98] reported that systemically administered TAT-Bcl-xL penetrated the
brain and protected neurons against focal ischemia/reperfusion
injury. Our study [43] showed that intraperitoneally applied TATHA-Bcl-xL could be detected in brain tissue by Western blot at 4
hours after injection. Dose-dependent increases in the amount of
TAT-HA-Bcl-xL were detected by either anti-HA or anti-Bcl-xL
antibody in various brain regions including the cortex, caudoputamen, hippocampus, cerebellum and spinal cord. In contrast, the
HA-Bcl-xL without TAT could not achieve detectable protein
transduction. Quantitative ELISA confirmed that transduced Bcl-xL
reached concentrations of 4 ng/mg in cortex tissue. Importantly, this
TAT-HA-Bcl-xL fusion protein was protective against 90-min transient focal ischemia even when applied 45 min after ischemia onset.
Similarly, Kilic and colleagues showed that intravenously injected
TAT-Bcl-xL entered the brain within 4 hours. This TAT-Bcl-xL
greatly reduced infarct size and improved neurological performance
after 90-min of MCAO [98]. TAT-Bcl-xL also exerted neuroprotec-
3712 Current Pharmaceutical Design, 2012, Vol. 18, No. 25
tion against a 30-min or a 2-hour focal ischemia. In another study, a
derivative of Bcl-xL with a more flexible and mobile pore-forming
domain was generated and designated as FNK [83]. FNK has a
higher protective activity than wild-type Bcl-xL. The TAT-mycFNK fusion protein accessed brain neurons after intraperitoneal
injection and prevented delayed neuronal death in the hippocampus
caused by 5-min global ischemia. Subsequent studies using TATBcl-xL fusion protein demonstrated that this protein not only inhibits ischemia-induced cell death, but also enhances the survival of
neural precursor cells and improves hippocampal neurogenesis after
transient ischemia [102, 103].
X chromosome-linked inhibitor of apoptosis (XIAP), a potent
inhibitor of both caspase-3 and apoptosis, is a 62-kDa protein that
does not cross BBB by itself. TAT fused XIAP, however, could
transduce cortical cells even when applied topically. Shorter constructs could successfully target brain lesions after intravenous
delivery and significantly reduced infarct volumes induced by distal
occlusion of the MCA [100].
TROPHIC FACTORS
Many trophic factors have shown high potential for neuroprotective applications. However, they usually do not readily cross the
BBB. For example, glial cell line-derived neurotrophic factor
(GDNF) is a potent neurotrophic factor that promotes neuronal
survival [104]. Kilic and colleagues showed robust GDNF transduction in the brain of TAT-GDNF-treated animals 4 hours after intravenous application [99]. TAT-GDNF administered either before or
30 minutes after onset of ischemia was neuroprotective in a MCAO
model of ischemic stroke, suggesting that this protein may be a
powerful tool for the immediate treatment of stroke.
It is now widely known that erythropoietin [41] is not only a
haematopoietic factor, but also a multifunctional trophic factor with
neuroprotective effects in animal models of ischemia [105] and in
stroke patients [106]. However, the BBB permeability of EPO is
very low as only 0.5-1% of systemically administered EPO crosses
the BBB [107]. To reach therapeutic concentrations in the brain,
either large amounts or multiple doses of EPO have to be applied,
which may induce undesired side effects such as polycythemia and
secondary stroke [105, 106]. Research from our group reported that
a TAT–derived peptide enhanced the capacity of EPO to cross the
BBB in animals to at least two fold higher levels than unmodified
EPO when intraperitoneally administered and up to five fold higher
levels when intravenously administered. Not surprisingly, the
therapeutic dose of the TAT fused EPO was about ten fold lower
than regular EPO to achieve equivalent neuroprotection in terms of
reducing infarct volume induced by MCAO in mice [108].
PROTEIN KINASES
PKC is known to be activated by ischemia/reperfusion stress
in multiple cell types of the brain [109] and is involved in cellular
responses such as cell apoptosis, mitochondrial dysfunction, and
oxidative stress [110, 111]. TAT-mediated delivery of V1-1, a
peptide inhibitor selective for PKC, was shown to be protective
even when given intraperitoneally 6 hours after onset of reperfusion
following 2-hour MCAO [101]. Furthermore, TAT-V1-1 improved
microvascular pathology, characterized as an increased number of
patent microvessels and increased cerebral blood flow following
acute focal ischemia [101]. Indeed, TAT-V1-1 fusion protein (also
named KAI-9803) has already shown promise in a clinical trial of
myocardial ischemia [112]. Clinical trials are now underway to test
the effect of TAT-V1-1 fusion protein on ischemic stroke.
In contrast to PKC, the activation of PKC, a different member of the PKC family, was documented to be neuroprotective
against cerebral ischemia [113, 114]. Intravenous pretreatment with
a fusion protein of TAT--Receptors for Activated C Kinase
(RACK), a PKC-selective peptide activator, 30 min prior to global
Hu et al.
cerebral ischemia conferred protection to the CA1 region of the rat
hippocampus by regulating cerebral blood flow [114].
Protection against permanent or transient cerebral ischemia was
also achieved when D-JNKI1 (also named XG102), a fusion protein
containing a TAT sequence and a 20-amino acid sequence of the cJun N-terminal kinase (JNK) binding motif of Islet-Brain-1/JNKinteracting protein-1 (JIP-1), was applied intraventricularly or systemically with a 6-hour therapeutic window [115]. D-JNKI1afforded neuroprotection is associated with caspase-3 activation and
potent inhibition of phospho-c-Jun [116]. A subsequent study further showed that D-JNKI1 could also protect against ischemic damage in the presence of tPA [117].
POSTSYNAPTIC DENSITY-95 (PSD95) INHIBITOR
It has been known for a long time that activation of the NMDA
receptor by pathologically high levels of excitatory neurotransmitter
glutamate causes excitotoxicity and plays a key role in ischemic
brain injury. However, direct targeting of the NMDA receptor is not
practical for stroke treatment because blocking NMDA receptors
causes substantial side effects in humans. Recent studies revealed
that the postsynaptic density protein PSD95 is a prominent organizing protein coupling the NMDA receptor with other intracellular
proteins and signaling enzymes including nNOS [118, 119]. A TAT
coupled peptide (TAT-NR2B9c) targeting the NMDA receptor
binding site on PSD95 has been shown to perturb the PSD95NMDA receptor interaction. Intravenously or intraperitoneally injected TAT-NR2B9c crossed the BBB in both rats and mice [119]
and potently alleviated ischemic brain damage in rats even when
applied 3 hours after onset of permanent or transient MCAO. Behavioral studies showed that the reduction in infarct size was accompanied by improved long term neurological functions [120].
NEUROGLOBIN
Neuroglobin is an oxygen-binding protein found in cerebral
neurons. Neuronal expression of neuroglobin enhances neuronal
survival upon hypoxic challenge [121]. However, like most other
protein neurotherapeutics, neuroglobin is too big to cross the BBB.
Although intracerebral administration of a neuroglobin-expressing
AAV vector has been shown to reduce infarct volume and improve
functional outcome after focal ischemia, its invasive application and
delayed expression after transduction render it clinically unfeasible.
Recently, a TAT-fused neuroglobin has been developed and tested
in the MCAO model of stroke [97]. TAT-neuroglobin fusion protein was delivered efficiently (at least 10-fold higher levels than
endogenous neuroglobin) into mouse brain 4 hours after intravenous injection. Pretreatment with TAT-neuroglobin led to prominently reduced post-ischemic brain damage and improved neurological functions. Post-treatment with TAT-neuroglobin immediately after reperfusion, however, did not show significant protection
against 2-hour ischemia.
OTHERS
Several other proteins such as Nogo-A extracellular peptide
residues 1-40 (NEP1-40) [122, 123], heat shock protein 70 (HSP70)
[124], and superoxide dismutase (SOD) [125] were show to penetrate the brain when fused with TAT and exert neuroprotective
effects in models of cerebral ischemia. With ever-growing knowledge on the mechanism of neuronal damage in ischemic stroke and
further improvements in the TAT transduction technique, greater
numbers of therapeutic proteins or peptides will likely be tested
against stroke treatment and eventually brought into the clinic for
use in humans.
MECHANISMS OF CPP INTERNALIZATION
The mechanisms by which CPPs cross cell membranes are not
completely understood. So far, three main entry mechanisms have
been proposed: (1) direct penetration across the membrane; (2)
Delivery of Neurotherapeutics
endocytosis-mediated entry; and (3) translocation through inverted
micelle formation.
DIRECT PENETRATION
Early studies on the mechanism of CPP internalization suggested an energy-independent mechanism involving the direct
translocation of CPPs across the cell membrane. Lowering the incubation temperature to 4°C or depletion of cellular ATP did not
inhibit the rapid transduction of TAT into cells, suggesting that
TAT-mediated protein transduction is endocytosis independent [22,
153, 154]. Other arguments in favor of an energy-independent
mechanism include the absence of effect of metabolic or endocytosis inhibitors on CPPs internalization and the failure of radiolabeled
CPPs to label endocytotic vesicles [155]. Moreover, structureactivity studies indicate that the internalization was equally effective whether the CPPs were in inverse or retro forms, which implies
receptor-independent recognition [156]. A direct penetration
mechanism was thus proposed which emphasizes the positive
charges on most CPPs including TAT and Antp. It was suggested
that the unfolded fusion protein interacts with the membrane
through electrostatic interactions between the basic amino acids on
CPPs and the negatively charged cell membrane polysaccharides or
phospholipids, and then nucleates a pore, which enables protein
translocation across the membrane [156-159]. According to this
theory, once inside the cell, the fusion protein is refolded into its
active form with the help of the endogenous chaperone system.
However, several studies have challenged the energy- and receptor-independent CPP translocation theory. It has been argued
that artifactual uptake of CPPs arose following fixation procedures
used for fluorescence microscopy. In addition, CPPs such as TAT
or penetratin strongly bind to the plasma membrane and cannot be
removed by the standard washing protocol used in FACS analysis.
These membrane-bound peptides might be mistaken by FACS as
internalized peptide, leading to questionable conclusions with respect to direct CPPs penetration [72]. The application of procedures
avoiding these experimental artifacts yielded an alternative inter-
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
3713
nalization mechanism favoring endocytosis-mediated entry (discussed below). However, there are still experiments supporting the
direct penetration of TAT peptide across the membrane even in
conditions devoid of the artifacts described above. For example,
Ter-Avetisyan and colleagues demonstrated that a low temperature
did not abrogate TAT uptake or change its intracellular distribution
in live unfixed cells. They also showed that genetically engineered
cells incapable of performing caveolin or clathrin-mediated
endocytosis showed an equal capacity to take up TAT as control
cells [160].
ENDOCYTOSIS-MEDIATED ENTRY
Endocytosis is a process of cellular ingestion by which patches
of plasma membrane invaginate to engulf substances into the cell.
Richard and colleagues demonstrated in their fluorescence microscopy study that live unfixed cells exhibit characteristic endosomal
distribution of TAT or (Arg)9. Flow cytometry analysis further
indicated that the kinetics of TAT uptake were similar to the kinetics of endocytosis. These data are all consistent with the involvement of endocytosis in the cellular internalization of CPPs [72].
Endocytosis is now believed to be the major route for CPP internalization. There are three types of endocytosis: clathrinmediated, caveolae-mediated and macropinocytosis Fig. (2). Studies on TAT internalization with chemical inhibition of specific endocytotic pathways point to the involvement of multiple mechanisms. In support of lipid raft-mediated endocytosis involving
caveolae, Fittipaldi and colleagues showed that the internalization
of TAT-EGFP endosomes was resistant to nonionic detergents and
colocalized with caveolae markers. Moreover, both the disruption
of lipid rafts and the disturbance of caveolar trafficking impaired
the internalization of TAT-GFP [161]. They also showed that conjugated TAT was not internalized in clathrin-coated endosomes. In
contrast, using unconjugated TAT, Richard and colleagues showed
that specific inhibitors of clathrin-dependent endocytosis partially
inhibit TAT peptide uptake, while inhibitors of raft/caveolindependent pathway or genetic deficient of caveolin-1 expression
Fig. (2). Endocytosis-mediated CPP internalization. Three types of endocytosis: clathrin-mediated, caveolae-mediated and macropinocytosis, could mediate
the internalization of CPPs.
3714 Current Pharmaceutical Design, 2012, Vol. 18, No. 25
could not inhibit the internalization of TAT [162]. Taken together,
these results suggest that unconjugated TAT peptide might follow a
clathrin-mediated endocytic pathway, whereas conjugated TAT
follows a caveolae-mediated endocytic pathway. Furthermore, lipid
raft-dependent macropinocytosis, yet another form of endocytosis
that is independent of either caveloe or clathrin, was also implicated
in the endocytosis of large TAT-fusion proteins in excess of 30kD
[163] as well as unconjugated TAT [164]. Dose-dependent inhibition of TAT uptake was observed when cells were pretreated with
amiloride, an inhibitor of the Na+/K+ exchanger required for
macropinocytosis and when cholesterol was removed from the cell
membrane [163, 164]. Indeed, the co-existence of different routes
of endocytosis is not unique for TAT but true for many other CPPs
such as homoarginine peptide [150, 165] and penetratin [134, 166].
Although endocytosis is an important mechanism for CPPmediated cargo internalization, it is not surprising that this process
of heterophagic internalization usually ends up with lysosomal
delivery and significant degradation of CPPs and their cargoes. As a
result, only a small fraction of the cargo reaches the cytoplasm in
active form. In order to avoid such endosomal trapping, invasive
approaches such as electroporation and microinjection were reported decades ago [167, 168]. Noninvasive methods were developed later to achieve efficient endosomal escape without physical
damage to the targeted cells. One of these methods is to add peptides that can be activated in the acidic environment in the endosome and lyse the endosomal membrane to release entrapped
CPP-cargoes. For example, HA2-TAT chimeric peptide, which
consists of TAT and the pH-sensitive HA2 fusion peptide from
influenza virus, has recently been engineered to enhance both
macropinocytosis and endosomal release/delivery of TAT fused
proteins and other macromolecules [163, 169].
INVERTED MICELLE FORMATION
An inverted micelle refers to an aggregate of colloidal surfactants where the hydrophilic groups are concentrated inside and the
hydrophobic groups extend outward. The inverted micelle model
was first proposed for the internalization of penetratin [156]. The
model proposes that the binding of CPPs to negatively charged
lipids in the cell membrane leads to the disruption of the lipid bilayer, resulting in the formation of inverted micelles that entrap the
peptide in a hydrophilic environment. Further interaction of the
peptides with membrane components leads to the inverse process,
resulting in the release of peptides into the cell [156, 170]. Subsequent studies showed that inverted micelles are also involved in the
internalization of arginine-rich CPPs [171].
Although endocytosis is now recognized as the predominant
route of CPPs internalization, mounting evidence suggested that
several different mechanisms may work in concert. It is possible
that different CPPs utilize different internalization mechanisms
depending on their specific biophysical properties, the nature of
their cargo and the experimental conditions. Furthermore, one type
of CPP may use several different pathways simultaneously for efficient internalization.
ADVANCES IN CPPS’ DELIVERY SYSTEM
Organelle-targeted Delivery
Organelle-specific CPPs are under rapid development to provide a means to target therapeutic agents not only to the cell, but
also to a specific subcellular compartment to achieve optimal activity and reduce non-specific effects. So far, the nucleus and the mitochondria have been successfully targeted with CPPs for gene
therapies and anti-cancer chemotherapies.
Nucleus-targeted delivery is important for gene therapies. A
common strategy to improve nuclear delivery of CPP-fused cargoes
is to add a nuclear localization signal (NLS) to the conjugates.
NLS-conjugates are highly cationic short peptides. The sequence
PKKKRKV derived from SV40 large T antigens is the most often
Hu et al.
used NLS. For example, this NLS sequence is a component of MPG
chimeric CPP and has been shown to improve the nuclear uptake of
plasmid DNA and oligonucleotides without necessitating nuclear
membrane breakdown [172]. The NLS-mediated nuclear delivery
system has been mainly tested in tumor cell lines or in vivo tumors
because a large number of anticancer drugs are DNA toxins that
must bind nuclear DNA or its associated enzymes for cytotoxic
effects. Recently, a nucleolin binding peptide (NBP) was reported
to deliver macromolecules including fluorophores, recombinant
protein and DNA to the nuclei of ocular tissues in vivo. Pegylated
NBP nanoparticles could further improve nuclear delivery and the
expression of transgenes. This NBP might be a useful tool for
therapeutic delivery to the eyes [173]. Future studies are warranted
to evaluate the potential of nuclear-targeted CPPs in gene therapies
against brain diseases, such as gene therapy for enzyme/neurotransmitter replacement in neurodegenerative diseases, antisense gene
therapy for brain cancers, and anti-apoptotic/anti-oxidative gene
therapy for other brain injuries such as stroke.
Mitochondria are another subcellular compartment specifically
targeted by modified CPPs. Mitochondria are a primary source of
reactive oxygen species (ROS) in the cell, and release critical apoptotic signaling molecules when damaged. Mitochondria-specific
CPPs may thus provide a platform for anti-apoptotic or antioxidant
therapies for many common diseases. One strategy to target mitochondria is to incorporate a mitochondrial signal sequence, such as
mitochondrial malate dehydrogenase (mMDH) signal sequence into
a CPP fusion protein [174]. For instance, it has been shown that
TAT-mMDH-GFP was enriched in mitochondria in vitro and detectable throughout fetal and neonatal pups when injected into
pregnant mice [174]. Recently, mitochondria penetrating peptides
(MPP) were designed by Horton and colleagues. They examined a
panel of mitochondrial transporters-synthetic cell-permeable peptides and observed efficient mitochondrial penetration in a variety
of cell types [175]. According to their studies, MPP efficiency can
be optimized by altering lipophilicity and charge. The application
of mitochondria-specific delivery to neuronal cells was reported by
Zhao and colleagues, who described that a novel mitochondriatargeted CPP antioxidant SS-31 (D-Arg-Dmt-Lys-Phe-NH2; Dmt =
2',6'-dimethyltyrosine) protected against oxidant-induced mitochondrial dysfunction and apoptosis in two neuronal cell lines
[176].
The Combined Use of Peptides and other Transportation
Mechanisms
Many new formulations of CPPs combined with other transportation mechanisms have increased CNS delivery capacity. For example, liposomes formulated with TAT-conjugated cholesterol
were recently shown to exhibit high delivery efficiency across the
BBB [144]. In addition, NPs conjugated with low-molecular-weight
protamine (LMWP), a CPP with high cell-translocation potency,
exhibited enhanced cellular accumulation compared to unmodified
NPs and increased the efficiency of brain delivery of coumarin after
intranasal administration [177]. In another study, TAT was used to
facilitate transport of fluorescently labeled silica NPs into the brain
[143]. These studies suggested that CPP-conjugated NPs are effective carriers for brain drug delivery. With the increased interest in
peptide-based drugs for the treatment and diagnosis of CNS diseases, CPP-conjugated peptidic NPs may become increasingly utilized as nanoplatforms to improve transportation across the BBB.
LIMITATIONS
Lack of Tissue and Cell Selectivity
The lack of both tissue and cellular phenotype specificity for
cargo delivery represent important caveats for using CPPs technology in the nervous system, where inappropriate targeting could lead
to dire consequences for neurological function and animal behavior.
Although there is a tendency for TAT-fusion proteins to favorably
Delivery of Neurotherapeutics
transduce neurons over glial cells [43], this delivery system is still
unable to convey proteins specifically to a targeted cell type within
the CNS or even to the CNS as a whole. In this regard, a doubletargeted delivery system simultaneously capable of extracellular
accumulation at the appropriate cellular location through specific
receptors or enzymes, and intracellular penetration through CPPs
may overcome this shortcoming and render CPPs capable of distinguishing between non-target and target cells. For example, lipoplexes modified with monoclonal anti-myosin monoclonal antibody
specific toward cardiac myosin and with TAT for cell penetrating
can achieve targeted gene delivery to ischemic myocardium [178].
Targeted CPP delivery system to CNS cells, however, has not yet
been developed, but will be essential before clinical translation is
feasible.
Uncertainty in Fusion Protein Activity
Another caveat of using CPP as the delivery system is that there
is no guarantee that the transduced fusion protein will gain its natural biological activity in target cells. Once inside the cells, it has
been proposed that the CPP-fusion protein is properly refolded by
chaperone proteins such as HSP 90 [179]. However, we found that
the TAT system may not deliver all classes of proteins with equal
success. For example, proteins that are only biologically active in
their multimeric state may not be good candidates for this approach.
Even the delivery of native monomeric subunits cannot ensure the
proper assembly of the subunits into a functional enzyme. In this
regard, the CPPs delivery system has not yet rendered the viral
vectors for delivery of gene products obsolete. This is because, in
many instances, the viral system utilizes host machinery for endogenous folding, enzyme assembly or post-translational modifications in a more efficient and appropriate manner.
Stability
For successful in vivo delivery, the CPPs must also not be metabolically degraded until they send their cargo to the appropriate
destination. However, concerns about the stability of these peptide
vectors still plague the field. For instance, the free TAT CPP has six
potential trypsin cleavage sites [180] and thus has a short half-life
of just a few minutes in the presence of trypsin [181]. Several structure-activity relationship features, including amino acid sequences,
amino acid three-dimensional configurations, incorporation of non-amino acids, and type of linkage between CPPs and cargoes, have
been shown to affect the stability of CPP conjugates in human serum and within cells. Generally, the D-configuration of the peptide
is less sensitive to degradation by proteases and remains intact in
serum and cells for a much longer time than the naturally occurring
L-amino acids [182]. The replacement of protease-sensitive amino
acids with their stable synthetic mimics [183, 184] or the insertion
of -alanines [182] also increase CPP stability. A recent study
showed that the shielding of TAT through TAT conjugated
poly(ethylene glycol)-phosphatidyl ethanolamine block copolymer
(TAT-PEG-PE) incorporation into PEG-PE micelles could protect
TAT against trypsinolysis with an over 100-fold increase in halflife compared to free TAT [181]. With all our efforts to stabilize
CPPs, one must also keep in mind that the metabolic cleavage of
the CPPs after internalization is a prerequisite for the release of
their cargo. Another tradeoff of gaining enhanced metabolic stability is an impairment in the clearance of CPPs and a subsequent
increase in their toxicity. Therefore, a thorough evaluation of each
modified CPP, with an emphasis on safety profiles, as discussed
below, is essential for the eventual translation of these CPPs into
the clinic.
Toxicity and Immunogenicity
Various CPPs have toxic effects on cultured cells. A comprehensive characterization of the metabolic effects of CPPs was done
by Kilk et al [185]. Their study suggested that transportan treatment
changed the GSH redox ratio and significantly decreased cellular
Current Pharmaceutical Design, 2012, Vol. 18, No. 25
3715
redox potential, while other four tested peptides (TAT, Antp, Arginine9 and MAP) showed minimal effect on metabolic profiles.
Cardozo and colleagues showed that although up to 100 μM TAT
(48–57) alone was essentially harmless, TAT-peptide conjugates
triggered significant cytotoxicity when used at concentrations
higher than 10 μM [186]. In comparison, free penetratin exhibits
intrinsic toxicity and penetratin conjugates showed more cellular
toxicity than TAT conjugates. Although all the factors that determine the cytotoxicity of CPPs are still not completely clear, peptide
length, peptide concentration, cargo molecule and coupling strategy
have all been suggested as contributing factors [187].
The immunogenicity of CPPs is not frequently reported. However, since most CPPs are derived from non-human proteins, they
have the potential to induce immune responses when used in a
clinical scenario [188]. Therefore, further examinations of immune
responses to CPPs are highly warranted.
CONCLUSIONS
On the one hand, the BBB is essential to maintain the integrity
of the brain and the spinal cord, and yet it also presents a vexing
barrier to getting drugs to their neural destinations in human CNS
diseases. What was first observed by Paul Ehrlich as an inability of
vital dyes to cross into the brain has therefore motivated many researchers in the past century to overcome this formidable obstacle
to CNS drug delivery. Relative to previous years, the past decade
witnessed significant achievements in successfully penetrating the
BBB and facilitating targeted CNS drug delivery to injured neurons, to tumors, and even to specific subcellular organelles. This is
in large part due to the noninvasive drug carriers, especially CPPs,
which show great potential to deliver various neurotherapeutics
across the BBB for the treatment of ischemic brain injury, among
other conditions. However, it behooves us to retain a balanced view
of these new techniques because certain weaknesses, in particular
the lack of tissue and cell type specificity, still restrict their clinical
application. Future modifications of CPP sequences or the combinations of CPPs with other drug carriers are expected to improve the
specificity of CPPs as well as reduce the side effects associated
with the delivery of cargoes to unintended organs. With such modifications in hand, CPP research may lead the way to novel avenues
of non-invasive stroke management in humans.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts
of interest.
ACKNOWLEDGMENTS
XIAOMING HU is supported by a grant (10POST4150028)
from the American Heart Association. JUN CHEN is supported by
National Institutes of Health/NINDS Grants NS36736, NS43802,
NS56118, and NS45048. YANQIN GAO is supported by Chinese
Natural Science Foundation grants 30670642, 30870794, and
81020108021. We thank Pat Strickler for excellent secretarial support.
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Received: January 11, 2012
Accepted: January 24, 2012
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