# Targeting ischemic penumbra Part II: selective drug delivery using liposome technologies

Shimin Liu, H Richard Winn, Steven R Levine

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Shimin Liu, H Richard Winn, Steven R Levine. Targeting ischemic penumbra Part II: selective drug delivery using liposome technologies. Journal of Experimental Stroke & Translational Medicine, 2010. [hal-04363816](https://hal.science/hal-04363816v1)

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# Targeting ischemic penumbra
## Part II: selective drug delivery using liposome technologies

Shimin Liu, M.D., PhD¹*; Steven R. Levine, M.D²; H. Richard Winn, M.D³*

1 Department of Neurology, Boston University School of Medicine, Boston, USA

2 Department of Neurology, Health Science Center, SUNY Brooklyn, New York, USA

3 Department of Neurosurgery, Hofstra North Shore-LIJ School of Medicine, New York, USA

## Abstract

In the present review (part II), we discuss the challenges and promises of selective drug delivery to ischemic brain tissue by liposome technologies. In part I of this serial review, we proposed "selective drug delivery to ischemic brain tissue" as a technique for neuroprotective treatment of acute ischemic stroke. To be effective, drugs must pass a series of barriers to arrive at ischemic brain. Brain ischemia results in metabolic and structural changes in the ischemic region, which cause additional obstacles for drug delivery. Liposome drug delivery system can pass these barriers and selectively target ischemic tissue by utilizing ischemia-induced changes in metabolism and molecular structure.

**Keywords:** Penumbra; drug delivery; liposome; pH-sensitive; blood-brain barrier.

Acute ischemic stroke causes irreversible injury in the ischemic core, but reversible injury in the peri-focal zone, the penumbra. (Astrup et al. 1981; Ebinger et al. 2009; Memezawa et al. 1992) Conventional drug delivery methods cause unwanted drug exposure to other tissue or brain regions. Because of limited blood supply to ischemic penumbra region, systemic high dose is often required for a drug to reach therapeutic levels in ischemic tissue, which may cause severe side effects and toxicity. Advanced drug delivery system using liposome techniques may achieve effective regional drug level without increasing systemic dose. Liposomal nanocarriers facilitate penetration of biological membrane, protect drugs from enzymatic and chemical degradation and albumin binding, and minimize drug exposures to non-target tissue. (Jain 2007; Tiwari and Amiji 2006) Following paragraphs discuss the challenges of selective drug delivery to ischemic brain tissue and the promises of liposome technologies in stroke therapy.

## 1. The blood brain-barrier

The blood brain barrier (BBB) consists of an endothelial enclosure and periendothelial accessories, which include pericytes, astrocytes, and a basal membrane. BBB strictly limits exchanges of sub-

stance between blood and the brain. The endothelial cells of the BBB lack fenestrations and have both tight junctions and adherens junctions between the cells (Bazzoni and Dejana 2004; Kniesel and Wolburg 2000; Schulze and Firth 1993). BBB prevents passive diffusion of hydrophilic molecules and allows diffusion of uncharged small molecules (Grieb et al. 1985; Levin 1980). A thin basement membrane (i.e. basal lamina) surrounds the endothelial cells and pericytes supporting the abluminal surface of the endothelium. Astrocytes further encase this structure with endfeet interspersed with pericytes. (Hellstrom et al. 2001; Kacem et al. 1998). The BBB protects brain from foreign substance, but it also strictly limits therapeutic agents entering the brain.

Because of the lipidic nature of all biological membranes, including brain endothelial cell membrane, lipophilic molecules can diffuse across BBB whilst hydrophilic molecules utilize transporter-facilitated systems. (Pardridge 1999) Active transport of some endogenous or exogenous hydrophilic molecules crossing BBB can be in either influx or efflux directions. (Ohtsuki and Terasaki 2007) Some of these transporters, such as for amino acids, monocarboxylic acids, organic cations, hexoses, nucleosides, peptides, have been well studied and molecularly identified.

<footnote>* Correspondence should be sent to: (Size 10 arial font, bold, italic)</footnote>

<footnote>Shimin Liu, M.D., Ph.D., Department of Neurology, Boston University School of Medicine, 715 Albany street, C329, Boston, MA02118; Tel: 617-638-7776; Fax: 617-638-5354; Email: lius@bu.edu.</footnote>

<footnote>H. Richard Winn, M.D., Director of Neurosurgery, 130 East 77th Street, Lenox Hill Hospital, New York, New York 10075; Tel: 212-434 3673; Fax: 212-434 2787; Email: hrwinn@Lenoxhill.net.</footnote>

<footnote>Copyright © 2011 SFES 1939-067X/11</footnote>
fied. We refer the reader to the excellent review articles (Tamai and Tsuji 2000; Tsuji 2005) for more information of transport- assisted passage of BBB.

### BBB during cerebral ischemia

Cerebral ischemia causes BBB damage and temporary BBB disruption. However, this temporary BBB opening causes edema, hemorrhagic transformation, and additional brain injury, therefore, it cannot be used for neuroprotective purpose. The area of the BBB disruption depends on injury intensity, injury type, and presence or absence of reperfusion. The BBB opening during ischemia in the absence of reperfusion occurs when the ischemic damage is already irreversible. For example, gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) extravasations was found grossly visible between 4.5 and 6 h after permanent MCAO (Kastrup et al. 1999). At this time, ischemic brain injury has already happened and is mostly irreversible. Reperfusion induced BBB disruption occurs after 3 and 48 hours of 2-h MCAO with reperfusion, with maximal opening at 48 hours and returns to normal by 14 days (Rosenberg et al. 1998). Gelatinase A (MMP-2) and TIMP-2 are associated with reperfusion induced BBB disruption (Rosenberg et al. 1998). After transient 20-min MCAO BBB disruption peaks at day 7 and resolves at day 14 after ischemia (Abulrob et al. 2008). BBB disruption during reperfusion stage is believed to be a precursor to hemorrhagic transformation (HT) and poor outcome (Latour et al. 2004), which should be treated with adjunctive therapies.

### Methods for transducing the blood-brain barrier

Traditional methods for opening BBB are invasive, inefficient, and non-selective. Transient osmotic opening of the BBB by arabinose or mannitol (Shinohara et al. 1979; Siegal et al. 2000), shunts (Zlotnik et al. 2008), or implanting microspheres (Beduneau et al. 2007; Emerich et al. 1999) to deliver therapeutic agents are invasive methods and have potential side-effects. Osmotic agents can cause accumulation of mannitol in the cerebral tissue and increase edema, whilst implantations require surgery (Kaufmann and Cardoso 1992; Maioriello et al. 2002). These methods do not selectively open BBB for drug delivery or selectively target tissue.

More advanced methods for CNS drug delivery include altering the molecular structure of drugs to increase their membrane permeability, fusing the drug molecule with cell penetrating peptides, and encapsulating drugs into liposomal nanocarriers. Drugs can also compete to use a carrier system if their structure mimics that of an endogenous molecule. Drugs that cannot use an endogenous transport system may be fused to a molecule, such as transferrin or glucose (Rajkumar et al. 1995), which can be effectively

transported across the BBB. In addition, monoclonal antibodies can be utilized to transport some drugs across the BBB via a receptor-mediated transport system (Lee et al. 2000; Pardridge 2005). In a similar manner, many cell penetrating peptides (CPPs) have been used for CNS drug delivery by conjugating to functioning therapeutic agents for cerebral ischemia (Esneault et al. 2008; Eum et al. 2004; Fan et al. 2006). Nanoliposomes and other nanoparticles can be utilized to deliver genes, proteins/peptides, and many other agents across the BBB. Nanoliposomes are engineered to enclose hydrophilic or ambiphilic agents within one or two lipid bilayers, similar to biological membranes.

The nanoliposome crosses the BBB by one of three methods: passive diffusion, endocytosis, or assisted passage with brain-targeting ligands (Beduneau et al. 2007; Chen et al. 2010; Markoutsa et al. 2011; Tamaru et al. 2010; Wong et al. 2010). These advanced methods for CNS drug delivery transduce BBB for a particular therapy, but they are not readily tissue-selective or cell type-selective. It is possible to achieve significant improvements on the delivery specificity and efficiency by extensive modification and optimization of these methods. Selective BBB transduction for drug delivery that targets the diseased tissue will improve delivery efficiency and reduce adverse effect, and potentially can be used in ischemic stroke and other CNS disorders.

## 2. Compromised blood supply and shrinkage of extracellular space

In addition to the challenge of BBB, ischemic brain tissue also has compromised blood flow that can further limit efficient drug delivery through blood supply. For example, the regional cerebral blood flow (rCBF) in the inner penumbra is only about 15 ml/100g/min (Murphy et al. 2006; Ohashi et al. 2005). Moreover, the extracellular space (ECS) is also decreased during ischemia (Thorne and Nicholson 2006; Zoremba et al. 2008). Ischemia causes cytotoxic edema that increases cell volume by 12% and reduces ECS by 50%. (Homola et al. 2006) Theoretically, large dose will be needed for reaching an effective regional drug concentration; this increased dose may lead to severe drug toxicity. Many neuroprotectants have been tested effective in animal studies, but they cannot be used in stroke patients at effective dose because of drug-related severe toxicity.

## 3. The advantages of liposomal drug delivery system

Advanced technologies, such as pH-sensitive nanoliposomes with enhanced BBB penetration and tissue selectivity, hold promises for ischemic stroke therapies. The lipid layer affords nanoliposomes BBB permeability for their encapsulated contents. Moreo-
ver, these liposomal nanocarriers can acquire addi-
tional features through further modification, such as
long circulating time, enhanced BBB penetration and
ischemic tissue selectivity. The advantages of using
nanoliposomes also include: 1). maintaining drugs in
active state when being encapsulated inside the na-
noliposome; 2). minimizing exposure to non-target
tissue when tissue-selective liposomes being used;
3). providing a protective shell for the encapsulated
contents, which prevents drugs from non-specific
binding and enzymatic digestion; 4). being able to
afford a high intraliposomal drug concentration.
Therefore, nanoliposomes could be used as a basic
means for drug delivery to ischemic stroke penum-
bra.(Jain 2007; Tiwari and Amiji 2006)

4. Possibility of selective drug delivery to pe-
numbral tissue

Brain ischemia causes a metabolic shift towards anaerobic glycolysis, resulting in a lower intracellular pH in the ischemic brain tissue. Targeting at this property of ischemic brain tissue, liposomal nanocarriers may be optimized to selectively release their contents under acidic condition (Collins et al. 1989) similar to the microenvironment of ischemic brain tissue (pH<6.75) (Anderson et al. 1999). The fusogenic property of dioleoylphosphatidylethanolamine and transactivator of transcription (TAT) peptide will facilitate liposomes escaping from endosomes/lysosomes and normal cells (Boomer et al. 2009; Hatakeyama et al. 2009). These liposomes will release their cargos when they reach cytosols of ischemic cells that have a pH of around 6.75. The pH difference between extracellular and intracellular space in ischemic tissue is usually less than 0.5 unit in normoglycemic rats (Nedergaard et al. 1991), therefore, a portion of pH-sensitive liposomes will deliver their cargos in the interstitial space.

These liposomal nanocarriers can gain additional
features by incorporating functional molecules onto
their membrane surfaces. Stroke-homing peptide
(Hong et al. 2008) may be useful for selective drug
delivery to ischemic brain tissue.

5. The strategies for liposomal drug delivery to ischemic penumbra

Currently there are no specially tailored nanolipo-
somes available for targeting ischemic stroke pe-
numbra. A nanosized pH-sensitive liposome com-
monly has 3-5 structural components, which include
a basic lipid, a balancer, a stabilizer, optional function
peptides, and an optional tracer. The basic lipid pro-
vides the sensitivity to acidic environment. The ba-
lancer is used to titrate pH-sensitivity to the desired
level. The stabilizer protects the nanoliposome from
unwanted leakage and fusion. Optional function pep-
tides may provide the nanoliposome additional abili-

ties, such as enhanced BBB penetration and specific
tissue targeting. Optional tracer can be a fluorescent
lipid or fluorescent peptide.

The basic lipid DOPE

There are two basic lipids that provide nanolipo-
somes a sensitivity to acidic environment, the phos-
phatidylethanolamines (PE), and the dioleoylphos-
phatidylethanolamine (DOPE) (Drummond et al.
2000). PE based nanoliposomes tend to release their
contents in a more acidic environment that may mim-
ic ischemic core. DOPE based nanoliposomes can
be optimized to release contents at mild acidic envi-
ronment that may mimic ischemic penumbra. DOPE
also has fusogenic property that facilitates liposomes
escaping from endosomes and lysosomes (Boomer
et al. 2009; Hatakeyama et al. 2009). The mild acidic
activation and fusogenic property of DOPE plus resi-
dual blood flow in penumbral region give DOPE-
based liposomes a higher chance of drug delivery to
ischemic penumbra.

Balancers and stabilizers:

N-succinyldioleoylphosphatidylethanolamine (suc-
DOPE), oleic acid (OA), palmitoylhomocysteine
(PHC), cholesteryl hemisuccinate (CHEMS), and 1,2-
dioleoyl- or 1,2-dipalmitoyl-sn-3-succinylglycerol
(DOSG or DPSG) are a few of the stabilizers that
have been used to prepare pH-sensitive liposome
formulations. CHEMS-balanced pH-sensitive nanoli-
posomes have been demonstrated for efficient intra-
cellular delivery. (Costin et al. 2002) Poly (ethylene-
glycol) (PEG) with different molecular weights is the
most commonly used stabilizer and compatible with
pH-sensitive nanoliposomes.

PEG coated pH-sensitive nanoliposomes for long
circulating time.

Conventional or plain nanoliposomes are rapidly re-
moved from circulation by the reticuloendothelial sys-
tem (RES) and accumulate in the liver and spleen
(Allen 1994; Cowens et al. 1993; Rahman et al.
1990), which limits their clinical applications. Recent
progress in microencapsulation showed that introduc-
ing poly(ethylene-glycol) (PEG) onto nanoliposome
surface can prevent nanoliposomes from opsoniza-
tion (Allen et al. 1991; Blume and Cevc 1990; Blume
and Cevc 1993; Torchilin et al. 1994), resulting in a
long circulating time (Klibanov et al. 1990). PEG
coated pH-sensitive nanoliposomes can be con-
structed using low pH-cleavable PEG or DSPE-PEG
of a proper molar ratio. The working mechanisms of
PEGylated pH-sensitive nanoliposomes have been
well illustrated. (Romberg et al. 2008; Sawant et al.
2006) The low pH-cleavable PEG is linked to its anc-
hor by some special linkers, which include Dior-
thoester, Orthoester, Vinylether, Phosphoramidate,
Hydrazone, b-thiopropionate. (Romberg et al. 2008)
The DSPE-PEG coating is commonly used for con-
structing pH-sensitive liposomes. (Hong et al. 2002;
Simoes et al. 2001; Slepushkin et al. 1997) The fea-
ture of pH-dependent release is determined by
DOPE/CHEMS ratio and PEG percentage. When
DSPE-PEG is anchored on to pH-sensitive liposome
with a proper molar ratio, it may be randomly ex-
tracted and shed from liposome surface allowing up-
take by cells when its anchor, the base lipid, be-
comes unstable in low pH environment. The fusogen-
ic property of DOPE lipid (Boomer et al. 2009; Hata-
keyama et al. 2009) may work synergically with TAT
peptide to initiate internalization and intracellular deli-
very. The methodologies for construction and purifi-
cation of DSPE-PEG-coated nanoliposomes have
been well established. (Liang et al. 2004).

Functional peptides

Functional peptides for nanoliposomes can be classi-
fied into three categories: 1). cell-penetrating pep-
tides (CPPs); 2). pH-dependent fusion peptides; and
3). special function peptides. Here we just list the
names of these peptides. More detailed and con-
densed information is available in related review pa-
pers(Deshayes et al. 2005; Drummond et al. 2000).
CPPs include TAT, octarganine/R8, penetratin/
AntpHD, FGF/MTS, VP22, PEP-1, DynA, transportan,
MAP, pegelin/SynB, pVEC, KALA, ppTG20, P1, MPG,
trimer, and PrP. pH-dependent peptides and toxins
include HA2, E1, G1/G2, G1/G2, G, E, gp36,
TH8/TH9, GALA, SFP, Poly(Glu-Aiba-Leu-Aib,
EGLA-I, EGLA-II, JTS1, VP-1, INF3, INF5, INF7,
INF8, INF9, INF10, HA peptide, D4, E5, E5L, E5NN,
E5CC, E5P, E5CN, AcE4K, poly(I-lysine), poly(I-
histidine), poly(acrylic acid) derivatives, succinylated
poly(glycidol)s, and copolymers of N-
isopropylacrylamide (NIPAM). It may be practical to
use CHEMS for titrating nanoliposomes to a desired
pH-sensitivity and leave these pH-sensitive peptides
as alternative approaches. Lysosomes-disrupting
peptides have been reported to enhance intracellular
delivery efficiency (Baru et al. 1998). Stroke-homing
peptide is a special peptide that has been reported
recently being able to targeting ischemic brain tissue.
This peptide has the sequence "CLEVSRKNC"

TAT peptide targeted pH-sensitive nanoliposomes for
BBB transduction. The human immunodeficiency vi-
rus-type 1 trans-activating transcriptional activator
(HIV-1 TAT protein, an 86-mer polypeptide) (Frankel
and Pabo 1988; Green and Loewenstein 1988) has
been shown the ability for inducing cell transduction.
Certain small regions of such proteins (10–16-mers)
called protein transduction domains (PTDs) are re-
sponsible for TAT protein induced cell transduction.
The minimal PTD of the TAT protein comprises 11
amino acids (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-

Arg-Arg) functioning as a cell penetrating peptides
(CPP). Fluorescently labeled TAT was visible
throughout the brain 20 minutes after intraperitoneal
(IP) administration in mice (Schwarze et al. 1999).
Many other CPPs have also been discovered and
synthesized (Deshayes et al. 2005). TAT peptide is
the most widely used CPP among other CPPs for
enhancing BBB penetration and for intracellular drug
delivery (Herve et al. 2008). Moreover, relatively
large 200-nm plain or polyethylene glycol-coated
(PEGylated) nanoliposomes can be delivered into
various cells by multiple TAT peptide molecules at-
tached to the nanoliposome surface. (Hyndman et al.
2004; Levchenko et al. 2003; Torchilin 2002; Torchilin
et al. 2001)

Stroke-homing peptide. Recently, the CLEVSRKNC (single letter sequence for Cys-Leu-Glu-Val-Ser-Arg-Lys-Asn-Cys) peptide has been demonstrated to be selective for ischemic brain tissue. (Hong et al. 2008)This peptide was screened from a phage peptide library based on a T7 415-1b phage vector displaying CX7C (C, cysteine; X, random peptides)(Pasqualini and Ruoslahti 1996). The CLEVSRKNC-phage preferentially targeted ischemic stroke tissue after intravenous administration in a rat MCA occlusion model. Because the CLEVSRKNC peptide is displayed on the phage membrane, theoretically it should be compatible with nanoliposome membrane layers. This means that CLEVSRKNC peptide may be incorporated onto nanoliposomes layers in ways similar to TAT peptide (Hyndman et al. 2004; Levchenko et al. 2003; Torchilin 2002; Torchilin et al. 2001) for localized drug delivery to ischemic brain tissue.

Lipid and peptide tracers

Lipids and peptides can be conjugated with some
fluorescent dye to trace the distribution of nanolipo-
somes in cells or tissue. Rhodamine and Tetramethyl
Rhodamine (TRITC) are commonly used for this pur-
pose, and the synthesis and conjugating process are
commercially available.

Liposome size for penumbra drug delivery

Liposome size is one of the factors that influence *in vivo* liposome distribution and drug delivery. The *in vivo* extracellular space width in rat brain is estimated 38-64 nm (Thorne and Nicholson 2006), at least 2-fold greater than estimates from fixed tissue. Liposomes for brain drug delivery have been used in sizes of 50-150 nm (Chapat et al. 1991; Madhankumar et al. 2009), 64-73 nm (Xie et al. 2005), 120-147 nm (Puisieux et al. 1994), and 134-143 nm (Ko et al. 2009). Some of these liposomes sizes were bigger than extracellular space width. In such case, liposomes may mainly depend on endocytosis and transtellular route for drug delivery, similar to the me-
chanism that allows their passage through the BBB (Patel et al. 2009). Because of the shrinkage of extracellular space (Thorne and Nicholson 2006; Zoremba et al. 2008), relatively small-sized liposomes may facilitate interstitial diffusion in ischemic tissue; but a smaller size also increases the risk of potential pulmonary toxicity (Nel et al. 2006).

**6. Concluding remarks**

The inability to selectively deliver S therapeutic agents to brain tissue, especially during ischemic, has been a limitation in the treatment of stroke. However, advanced drug delivery tools provide a me-

chanism to overcome these difficulties in stroke therapy. Because ischemia causes an acidic environment and residual blood flow exists in ischemic penumbral region, liposomal delivery system with a low pH-dependent release feature can be used for targeting ischemic penumbra. The DOPE/CHEMS/DSPE-PEG system holds promises as a basic formula of pH-dependent liposomes for stroke treatment. Other peptides, such as TATp and stroke homing peptide (STROKEp) may provide added features for such system. The proposed working mechanism is depicted in Fig. 1.

<figure><img src="image_2.png" /><figcaption>**Fig.1 Diagram of selective drug delivery to ischemic penumbra by pH-sensitive liposomes.** TAT pep-tide (TATp) and stroke-homing peptide (STROKEp) are conjugated with PEG-PE on the surface of liposomes (TATp-PEG-PE and STROKEp-PEG-PE, respectively). TATp and the lipid layer facilitate drug-loaded liposome passage of the BBB and arrival at interstitial space. Liposomes remain stable in neutral pH environment, but they become destabilized in ischemic region due to low pH. STROKEp helps liposomes targeting ischemic cells. TATp and lipid layer help liposomes uptaken by ischemic cells and escaping from endosomes. Destabilized pH-sensitive liposomes release drugs in the cytosol of ischemic cells. A portion of liposomes may release drugs in extracellular space.</figcaption></figure>

**Acknowledgement**

This work was supported by NIH grant 7R21NS065912-02, 5T32NS051147-02 and NS 21076-24.

**Conflict of interest**

None

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