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Experimental Neurology 1

Effects of the perivascular space on convection-enhanced delivery of

liposomes in primate putamen

Michal T. Krauzea, Ryuta Saitoa, Charles Nobleb, John Bringasa, John Forsayetha,

Tracy R. Mcknightc, John Parkb, Krystof S. Bankiewicza,*

aDepartment of Neurological Surgery, Laboratory of Molecular Therapeutics, University of California, 1855 Folsom Street,

Room 226, San Francisco, CA 94103, USAbDivision of Hematology-Oncology, University of California, San Francisco, CA 94103, USA

cDepartment of Radiology, University of California, San Francisco, CA 94103, USA

Received 3 May 2005; revised 11 July 2005; accepted 14 July 2005

Available online 16 August 2005

Abstract

Convection-enhanced delivery has recently entered the clinic and represents a promising new therapeutic option in the field of

neurodegenerative diseases and treatment of brain tumors. Understanding of the principles governing delivery and flow of macromolecules

within the CNS is still poorly understood and requires more investigation of the microanatomy and fluid dynamics of the brain. Our

previously established, reflux-free convection-enhanced delivery (CED) technique and real-time imaging MR method for monitoring CED

delivery of liposomes in primate CNS allowed us to closely monitor infusions of putamen. Our findings indicate that CED in putamen is

associated with perivascular transport of liposomes, throughout CNS arteries. The results may explain side effects seen in current clinical

trials using CED. In addition, they clearly show the necessity for a monitoring technique for future direct delivery of therapeutic agents to the

human central nervous system. Based on these findings, we believe that the physiological concept that the perivascular space serves as a

conduit for distribution of endogenous molecules within the CNS also applies to interstitially infused agents.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Convection-enhanced delivery; MRI; Primate; Liposomes; Perivascular space; Putamen

Introduction

Direct delivery of therapeutic agents to the human central

nervous system (CNS) is one of the most challenging new

approaches for gene and molecular therapy for neurological

diseases. Mechanisms and pathways by which drugs and viral

vectors are distributed in the CNS are still under investigation

and remain an unpredictable factor for clinical studies. A

clearer understanding of how the microanatomy and fluid

dynamics of the brain govern delivery and flow of macro-

molecules within the CNS still requires more investigation.

In the present study, we have investigated the distribution

of liposomes delivered by CED in non-human primate

putamen. We have previously established a reflux-free CED

0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2005.07.009

* Corresponding author. Fax: +1 415 514 2177.

E-mail address: kbank@itsa.ucsf.edu (K.S. Bankiewicz).

technique and real-time imaging MR method for monitoring

CED delivery of liposomes in primate CNS (Saito et al.,

submitted for publication). This allowed us to monitor

liposomal distribution in real time in the putamen nucleus.

In addition to observing the robust distribution of liposomes

in the putamen, the most important finding was liposomal

‘‘leakage’’ along primate cerebral arteries, a phenomenon

independent of the cannula placement. This finding suggested

the existence of a transport mechanism further to the pressure

gradient provided by CED.

The concept that the perivascular space serves as a conduit

for the distribution of molecules within the CNS has been

explored for the last 30 years or so (Cserr and Ostrach, 1974;

Rennels et al., 1985, 1990). Perivascular spaces are extensions

of the subarachnoid spaces that accompany vessels that

penetrate the brain down to the level of capillaries. Physio-

logical and anatomical studies in a variety of mammalian

96 (2005) 104 – 111

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111 105

species have emphasized the immunological role of perivas-

cular spaces in the brain similar to lymphatic drainage

pathways (Kida et al., 1995; Knopf et al., 1995; Weller et

al., 1992, 1996). Tracers injected into rat gray matter drain

along perivascular spaces which are lined by immunocompe-

tent perivascular cells (Cserr et al., 1992; Kida et al., 1993).

This lymphatic drainage pathway plays a key role in the

immunology of the central nervous system, perhaps explaining

why removal of cervical lymph nodes significantly reduces

immune reactions in the brain mediated by both B and T

lymphocytes (Cserr et al., 1992; Harling-Berg et al., 1989;

Knopf et al., 1995; Phillips et al., 1995, 1997; Weller et al.,

1996). Previous studies of peri-arterial spaces in the human

cerebral cortex have shown that arteries are surrounded by a

layer of leptomeninges that is subtended from the pia mater

(Zhang et al., 1990) and, by this anatomical arrangement, the

perivascular spaces of the intra-cortical arteries are contiguous

with the peri-arterial domains in the sub-arachnoid space. The

lack of similar coating of leptomeningeal cells around veins in

the cerebral cortex suggests that peri-venous spaces are

contiguous with the sub-pial space (Weller, 1995; Zhang et

al., 1990) and probably do not function as lymphatic drainage

pathways in the same way as peri-arterial spaces (Pollock et

al., 1997). Further studies revealed that arteries in the basal

ganglia are surrounded by 2 distinct layers of leptomeninges

separated by a perivascular space contiguous with the peri-

arterial, sub-arachnoid space (Pollock et al., 1997). The

anatomy of the peri-arterial spaces in the basal ganglia differs

significantly from those in the cerebral cortex where there is

only a single peri-arterial layer of leptomeninges. Pollock and

colleagues have suggested that differences seen in the structure

of peri-arterial spaces in different sites in the brain may be

reflected in differing relative efficiencies in the drainage of

interstitial fluid (Pollock et al., 1997). Drainage in perivascular

spaces has been examined with various markers and is clearly

affected by cardiac pulsation of cerebral arteries (Rennels et

al., 1985).

In this study, we tested the hypothesis that perivascular

spaces of lateral striate arteries in the putamen, and in large

sub-arachnoid arteries, are capable of transporting therapeu-

tic agents throughout the CNS. This hypothesis is based

upon previously described physiological and anatomical

properties of perivascular spaces and observations of MR-

monitored liposomal infusions as well as histological

analysis of infused animals. Transport of liposomes in the

peri-arterial space could have implications for gene and

molecular therapy for neurological diseases and might

explain some side effects seen in clinical trials.

Methods

Liposome preparation

Separate liposomes were prepared for detection by MRI

and by histological examination. Liposomes that contained

the MRI contrast agent were composed of 1,2-dioleoyl-sn-

glycero-3-phosphocholine (DOPC)/cholesterol/1,2-dis-

tearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(po-

lyethylene glycol)-2000] (PEG-DSPE) with a molar ratio of

3:2:0.3. DOPC and PEG-DSPE were purchased from Avanti

Polar Lipids (Alabaster, AL) and cholesterol was purchased

from Calbiochem (San Diego, CA). The lipids were

dissolved in chloroform/methanol (90:10, vol/vol), and then

the solvent was removed by rotary evaporation, resulting in

a dried lipid foam. The lipid foam was hydrated with a

commercial United States Pharmacopeia solution of 0.5 M

gadoteridol (a gadolinium complex of 10-(2-hydroxy-

propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,

Prohance; Bracco Diagnostics, Princeton, NJ) by 6 succes-

sive cycles for rapid freezing and thawing. Residual

chloroform was removed from the hydrated lipid by a brief

treatment with rotary evaporation. Unilamellar liposomes

were formed by extrusion (Lipex; Northern Lipids, Vancou-

ver, Canada) with 15 passes through double-stacked

polycarbonate membranes (Whatman Nucleopore, Clifton,

NJ) with a pore size of 100 nm, resulting in a liposome

diameter of 85.1 T 37.5 nm as determined by light scattering

(N4Plus particle size analyzer, Beckman Coulter, Fullerton,

LA). Unencapsulated gadoteridol was removed with a

Sephadex G-75 (Sigma, St. Louis, MO) size-exclusion

column eluted with HEPES-buffered saline (5 mM HEPES,

135 mM NaCl, pH 6.5, adjusted with NaOH).

Liposomes, loaded with sulforhodamine B for histolo-

gical studies, were formulated with the same lipid compo-

sition and preparation method as the gadoteridol-containing

liposomes except that the lipids were hydrated directly with

20 mM sulforhodamine B (Sigma) in HEPES-buffered

saline (pH 6.5) by 6 successive cycles of rapid freezing and

thawing. The sulforhodamine B liposomes had a diameter of

79.0 T 34.3 nm (used for co-infusion with the gadoteridol-

containing liposomes). Both of the final liposomal con-

structs consist of an ionic, water soluble, small molecule

indicator that is contained within the aqueous liposome

interior by the organized hydrophobic membrane bilayer.

Each liposome exterior surface has a slight negative net

charge from the presence of the PEG-DSPE at 10% (mol/

mol) of the phospholipid (6.0% of the total lipid).

Quantification of liposome-entrapped gadoteriodol by

magnetic resonance imaging

The concentration of gadoteriodol entrapped in the lip-

osomes was determined from nuclear MR relaxivity measure-

ments. The relationship between the change in the intrinsic

relaxation rate imposed by a paramagnetic agent (DR), also

known as ‘‘T1 shortening,’’ and the concentration of the agent

is defined by the equation: DR = r1[agent], in which r1 =

relaxivity of the paramagnetic agent and DR = (1/T1observed1/T1intrinsic). As gadoteriodol was encapsulated within the

liposome, we corrected for the change in the observed T1

imposed by the lipid by measurement of the T1 of solubilized

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111106

liposomes with and without gadoteriodol by means of an

iterative inversion-recovery MRI sequence on a 2-T Brucker

Omega scanner (Brucker Medical, Karlsruhe, Germany). The

relaxivity of gadoteriodol had been empirically derived

previously on the same system and had a value of 4.07

mM�1 s�1. The concentration of the encapsulated gadoter-

iodol was then calculated with the following equation:

[gadoteriodol] = [(1/T1wGado) � (1/T1w/oGado)]/4.07.

Experimental subjects

The protocol was reviewed and approved by the Institu-

tional Animal Care and Use Committees at the University of

California San Francisco (San Francisco, CA). Adult male

Cynomolgus monkeys (Macaca fasicularis, n = 3, 3–5 kg)

were individually housed in stainless steel cages. Each animal-

room was maintained on a 12-h light/dark cycle and room

temperature ranged between 64-F and 84-F. Purina Primate

Diet was provided daily in amounts appropriate for the size

and age of the animals. This diet was supplemented with fruit

or vegetables daily. Also, small bits of fruit, cereal or other

treats were provided as part of the environmental enrichment

program. Tap water was freely available to each animal

through an automatic watering device or an attached water

bottle. Prior to assignment to the study, all imported animals

underwent at least a 31-day quarantine period as mandated by

the Centers for Disease Control and Prevention (Atlanta, GA).

Liposome infusion procedure

Infusion of gadoteriodol/rhodamine-loaded liposomes

during real-time magnetic resonance imaging monitoring

Primates (n = 3, 3–5 kg) received a baseline MRI scan and

underwent neurosurgical procedures to position MRI-compat-

ible guide-cannulas in the left putamen. Each customized

guide cannula was cut to a specified length and stereotacti-

cally guided to its target through a burr-hole created in the

skull. The guide-cannula was secured to the skull with dental

acrylic, and the tops of the guide-cannula assemblies were

capped with stylet screws for simple access during the

infusion procedure. Animals recovered for at least 2 weeks

before starting liposome infusions. Under isoflurane anes-

thesia, the animal’s head was placed in an MRI-compatible

stereotactic frame and a baseline MRI scan was performed.

Vital signs, such as heart rate and PO2, were monitored

throughout the procedure. Infusions were performed accord-

ing to previously established CED techniques for non-human

primates (Bankiewicz et al., 2000). Briefly, the infusion

system consisted of a fused-silica needle cannula that was

connected to a loading line (containing liposomes) and an oil-

infusion line. A 1-ml syringe (filled with oil), mounted onto a

micro-infusion pump (BeeHive; Bioanalytical Systems, West

Lafayette, IN), regulated the flow of fluid through the system.

Based on MRI coordinates, cannula was mounted onto a

stereotactic holder, and manually guided to the targeted region

of the brain through a guide-cannula previously secured. The

length of each infusion cannula was measured to ensure that

the distal tip extended approximately 3 to 4 mm beyond the

length of the respective guide. This created a stepped design at

the tip of the cannula to maximize fluid distribution during

CED procedures. Following secure placement of the needle

cannula, the animal’s head was repositioned in the MRI gantry

and CED procedures were initiated while MRI data were

continuously acquired. Putamen infusion volume was 99 Al in2 primates and 300 Al for the third primate. An initial infusion

rate of 0.1 Al/min was applied and increased at 10-min

intervals to 0.2, 0.5, 0.8, 1.0 Al/min and 1.5 Al/min in the 99-

Al primates and up to 3 Al/min in the 300-Al primate. Each

animal received a mixture of liposomal Gd and rhodamine

liposomes; the approximate concentration of liposomes

injected corresponded to a formulated concentration of 10

mM phospholipids and 5 mM gadoteriodol. Approximately

15 min after infusion, the cannula was withdrawn from the

brain. Each animal was infused up to 3 times with at least 4-

week intervals between each infusion procedure. Immediately

after the last intracranial CED procedure, the animal was

euthanized with an overdose of pentobarbital. The brain was

then harvested and coronally sectioned into 3 to 6 mm blocks.

Each brain slice was immediately frozen in cooled isopentane/

dry-ice, and processed for histological analysis.

MRI and MRA acquisition

T1-weighted images of primate brains were acquired on a

1.5-T Signa LX scanner (GE Medical Systems, Waukesha,

WI) with a 5-in. surface coil. Prior to insertion of infusion

cannulas, baseline spoiled gradient echo (SPGR) images

were taken: repetition time (TR)/echo time (TE)/flip angle =

28 ms/8 ms/40-, number of excitations (NEX) = 4, matrix =

256 � 192, field of view (FOV) = 16 cm � 12 cm, slice

thickness = 1 mm. These parameters resulted in a 0.391 mm3

voxel volume. Once the cannulas were inserted and infusion

commenced, SPGR scans were taken consecutively

throughout the infusion. The scan-time was dependent on

the number of slices needed to cover the extent of infusion

and ranged from 9.73 min to 11.88 min.

MRA was performed using UCSF Radiology standard

imaging protocol for 2D MR Angiography of CNS.

Signal quantification and three-dimensional reconstruction

from MR images

The Signal of gadoteriodol liposome distribution for each

infused primate putamen was quantified with BrainLabRsoftware (Heimstetten, Germany). MR Images acquired

during the infusion procedure were correlated with volume

of infusion at each series started during an infusion

procedure. BrainLab software reads all data specifications

from MR images. After the pixel threshold value for

liposomal signal is defined, the software calculates the signal

above a defined threshold value, and establishes the volume

of distribution from primate brain. This allows volume of

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111 107

distribution to be determined at any given time-point and can

be reconstructed in a three-dimensional image.

Results

MRI monitored leakage out of non-human primate striatum

after liposomal infusion

We established a method to monitor in real time the

infusion of liposomes loaded with a surrogate marker. We then

used this system to infuse various anatomical structures in

non-human primate brain including putamen. CED of up to

300 Al of liposomes was performed in non-human primate

putamen, and subsequent distribution was monitored (Figs. 1

and 2). Placement of cannula in primate putamen was verified

for each animal by MRI prior infusion of liposomes. MRI was

used to monitor CED of liposomes throughout the infusion

procedure and reflux-free delivery was established to ensure

optimal convection parameters. After starting the primate

putamen infusion procedure, signal enhancement was detected

in the perivascular space of the medial cerebral artery (MCA)

(Figs. 1a, c, d, e, f). At the lateral putamen border, lateral

striate arteries (LSA) also showed signal enhancement (Fig.

1b). Volume infused into each animal at which MCA signal

enhancement was first seen on MRI was as follows: A—50 Al(Figs. 1a, b), B—20 Al (Figs. 1c, d) and C—15 Al (Figs. 1e, f).Signal enhancement continued to spread in the perivascular

space along branches of MCA (Figs. 1a, c, f). Increasing

signal enhancement in the Sylvian fissure and insular region

was also visible, while infusion of liposomes into putamen

Fig. 1. Coronal images showing robust distribution of CED delivered liposomes

MCA to finally accumulate in the sylvian fissure and insula cortex area (a– f). Sa

Axial view showing accumulation of liposomes in sylvian fissure after putamen

continued with perivascular MCA signal present (Figs. 1a, e).

No signal in the external capsula bordering on insular cortex

was seen throughout the infusions (Figs. 1a, b, e).

MRA (Magnetic Resonance Angiography) of non-human

primate cerebral vessels

The signal seen in primate cerebral arteries (Figs. 2d–f)

after performing MRA shows the luminal MCA signal in

coronal, axial and sagittal views. This signal location

exactly matched liposomal MRI signal seen after putamen

infusions in same anatomical views (Figs. 2a–c). Results of

this study confirmed the (perivascular) arterial origin and

perivascular transport of the liposomal signal seen during

intra-putaminal infusions. Post-mortem examination con-

firmed localization of LSA with respect to perivascular

transport of liposomes seen during MRI (see Figs. 4 and 5).

Reconstruction in three dimensions of putamen infusion and

leakage pathway

To understand special relationship between localization of

the vessels and the pattern of perivascular transport of

liposomes 3-dimensional (3-D) reconstruction was performed.

3D reconstruction is seen from an anterior superior view, after

delineating the liposomal signal seen after putamen infusion

(Figs. 3a, b). Although MCA leakage was seen first after 50 Alinfusion (Animal A), data for delineation was taken at the

150-Al infusion volume in order to visualize the complete

leakage pathway. This reconstruction enabled us to demon-

strate clearly distribution in the putamen, and leakage of

in primate putamen (a, b). Liposomal signal is visible to drain along LSA,

gittal view of putamen infusion including perivascular signal in MCA (d).

infusion (e).

Fig. 2. Coronal section with liposomal leakage shown on top with the corresponding MRA below (a–f). MRA identifies LSAvessels in the putamen and MCA

in horizontal part (d). Axial view of primate liposomal perivascular MCA enhancement on top (b) compared to axial MRA of MCA vessel below (e). Sagittal

view of liposomal enhanced MCA (c) and the corresponding MCA signal in MRA.

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111108

liposomes, in relation to MRI data (Fig. 3a). Digital

subtraction of MR image allowed further detailed analysis

of the leakage pathway in relation to primate brain anatomy

(Fig. 3b). The following landmarks are shown: liposomal

putamen distribution, anterior lateral leakage following

perivascular space of LSA connecting to perivascular space

of medial cerebral artery and termination of signal in the

Sylvian fissure and insular cortex (Fig. 3b). Insula and sylvian

Fig. 3. 3-dimensional re-construction of putamen distribution and leakage. Liposo

distribution components and perivascular transport in relation to anatomical landm

fissure again display accumulation of liposomal signal seen in

MR imaging during the infusion procedure.

Analysis of anatomical structures and fluorescence along

the leakage pathway

In order to correlate data obtained in vivo with post-

mortem examination, MRI data were compared with data

mal distribution overlapped with MR imaging (a). Detailed analysis of all

arks (b).

Fig. 4. Primate putamen hematoxylin and eosin histology section showing

branches of lateral striate arteries along lateral putamen border penetrating

into putamen nucleus.

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111 109

from histological analysis of non-human primate brains.

Vessels of the lateral striate arteries with perforating

branches, that are the arterial supply for the putamen, are

seen at the lateral putamen border in a more ventral section

seen from infusion site (Fig. 4).

Co-infusion of gadoteridol- and sulforhodamine B-

loaded liposomes allowed us to perform primate histology

for fluorescent marker (Fig. 5), thus making comparison

between MRI and histological demonstration of perivascular

transport possible. Our previous experience with these

Fig. 5. Fresh frozen coronal primate histology section with putamen, LSA and MC

seen after infusion of 300 Al liposomes in basal areas of primate brain (b, 5� ma

formulations of liposome-loaded surrogate markers has

demonstrated the surrogate to be retained within, and

accurately represent the location of the liposome carrier.

Minimal leakage followed by subsequent dilution and rapid

clearance of the hydrophilic markers diminishes the signal

contribution of extraliposomal gadoteridol and sulforho-

damine B. Structures that were enhanced on MRI were

histologically analyzed for fluorescence to confirm the

perivascular origin of transport. A histological section that

contained putamen, LSA vessels and MCA vessel was

analyzed to demonstrate structures involved in leakage

pathway (Fig. 5a). Clear fluorescent signal can be seen at

the basal areas of brain surrounding the perivascular space

of a MCA vessel (Fig. 5b).

Discussion

In the light of current clinical trials and efforts to design

safe clinical trials in the future, controlled distribution of

therapeutic agents within the CNS is essential for any

approach to gene or molecular therapy. The concept of a

perivascular pump as an integral part of physiological

drainage mechanisms in the CNS has been known for years,

but is still controversial. What role it plays in the movement

of interstitially infused therapeutics is still unclear. Although

the distribution of different molecules depends on their

specific physicochemical properties, such as size, charge

and affinity for cellular receptors, our results indicate that a

distinct perivascular space pathway in the putamen might

apply to every interstitially applied agent. Anatomical

differences in perivascular ultrastructures in putamen and

A (a, 1.5� magnification). Intense MCA and LSA vessel wall fluorescence

gnification).

M.T. Krauze et al. / Experimental Neurology 196 (2005) 104–111110

sub-arachnoid arteries might explain the rather unusual

transport of liposomes out of putamen infusion site. The 2

distinct layers of leptomeninges in the putamen very likely

allow perivascular liposomal transport against the blood

flow in the LSAwhereas further perivascular transport in the

single leptomeningeal-coated MCA clearly shows transport

in direction of blood-flow. Postmortem histological analysis

shows the high vessel density in the ventral putamen and the

strong fluorescent vessel signal seen from MCA to sylvian

fissure. Diffusion of liposomal dye into cerebral cortex of

the insula region also suggests leakage of liposomes into

CSF during perivascular transport.

Liposomes are a good marker to visualize transport

throughout the CNS. However, it is currently not possible to

predict, based on the putamen cannula placement, the

amount of infusate needed to achieve leakage. A large part

of the putamen relies on blood flow that is mainly supplied

by the LSA vessels that arise from the MCA. This anatomy,

together with our histological findings, explains why no

liposomal signal is seen in other sub-arachnoid arteries. As

the diameter of LSA vessels is rather small compared to the

MCA, perivascular liposomes might give less signal on

MRI in LSA vessels, since the MCA signal seems to appear

earlier in the infusion. These MRI findings were not

confirmed by histology as fluorescent liposomal signal

was seen along the entire leakage pathway.

Our liposomal findings suggest that every therapeutic

given into putamen should be closely monitored for

distribution since the possibility of side effects increases

greatly with leakage out of infusion site. It is unclear

whether placement of the cannula in different parts of the

putamen would allow a greater volume of infusion

without corresponding perivascular transport along ves-

sels. Adding our findings to the body of knowledge

regarding the immunological role of perivascular spaces

might explain the antibodies reported in some clinical trial

subjects after GDNF infusion into putamen nucleus (Hood

and Sherer, 2004). The fast transport through the

perivascular space after infusion of up to just 300 Al ofliposomes shown here strongly suggests that infusion of 8

ml of GDNF in PD patients (Gill et al., 2003; Slevin et

al., 2005) most likely followed the same route and ended

up in the regions that are remote form the infusion site,

including CSF. Clearance pathways of liposomes in the

CNS strongly suggest perivascular transport occurs over

the entire CNS if very large volumes are infused. This

also raises the possibility of therapeutics being transported

away from their intended site of action, with concomitant

side effects.

In summary, our study indicates the potential hazards and

challenges for future human gene and molecular therapy.

Furthermore, it advances the importance of monitoring

strategies during delivery of therapeutics to CNS. The

importance of perivascular transport after intracranial

delivery warrants further investigation, as our understanding

of the latter is still poor.

Acknowledgments

Special thanks to BrainLAB for support and help with

iPlanR software.

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