PEG Minocycline-Liposomes Ameliorate CNS

Autoimmune Disease
Wei Hu1., Josbert Metselaar2., Li-Hong Ben1, Petra D. Cravens1, Mahendra P. Singh1, Elliot M.
Frohman1,3, Todd N. Eagar1,4, Michael K. Racke5, Bernd C. Kieseier6*, Olaf Stüve1,4,6,7*
1 Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 2 Department of Pharmaceutics, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, 3 Department of Ophthalmology, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas, United States of America, 4 Center for Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of
America, 5 Department of Neurology, Ohio State University, Columbus, Ohio, United States of America, 6 Department of Neurology, Heinrich Heine University, Düsseldorf,
Germany, 7 Neurology Section, VA North Texas Health Care System, Medical Service, Dallas, Texas, United States of America

Abstract
Background: Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS).
Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental
autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated
with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent
treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE.

Findings: Performing in vitro time kinetic studies of PEG minocycline-liposomes in human peripheral blood mononuclear
cells (PBMCs), we determined that PEG minocycline-liposome preparations stabilized with CaCl2 are effective in diminishing
MMP-9 activity. Intravenous injections of PEG minocycline-liposomes every five days were as effective in ameliorating
clinical EAE as daily intraperitoneal injections of minocycline. Treatment of animals with PEG minocycline-liposomes
significantly reduced the number of CNS-infiltrating leukocytes, and the overall expression of MMP-9 in the CNS. There was
also a significant suppression of MMP-9 expression and proteolytic activity in splenocytes of treated animals, but not in CNS-
infiltrating leukocytes. Thus, leukocytes gaining access to the brain and spinal cord require the same absolute amount of
MMP-9 in all treatment groups, but minocycline decreases the absolute cell number.

Conclusions: Our data indicate that less frequent injections of PEG minocycline-liposomes are an effective alternative
pharmacotherapy to daily minocycline injections for the treatment of CNS autoimmune diseases. Also, inhibition of MMP-9
remains a promising treatment target in EAE and patients with MS.

Citation: Hu W, Metselaar J, Ben L-H, Cravens PD, Singh MP, et al. (2009) PEG Minocycline-Liposomes Ameliorate CNS Autoimmune Disease. PLoS ONE 4(1):
e4151. doi:10.1371/journal.pone.0004151
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received May 26, 2008; Accepted November 21, 2008; Published January 7, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: OS was supported by a Start-up Grant from the Dallas VA Research Corporation, a New Investigator Award from VISN 17, Department of Veterans
Affairs, a Merit Review Award from the Department of Veterans Affairs, Research Grants from National Multiple Sclerosis Society (NMSS; RG3427A8/T, and
RG2969B7/T), and a grant from the Viragh Family Foundation. The funding agencies had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bernd.kieseier@uni-duesseldorf.de (BCK); olaf.stuve@va.gov (OS)
. These authors contributed equally to this work.

Introduction Minocycline is an oral tetracycline derivative with good

bioavailability within the CNS. It was shown previously that
Experimental autoimmune encephalomyelitis (EAE) is an minocycline attenuates neuroinflammation, neuropathological
antigen-specific T cell-mediated autoimmune diseases of the changes, and clinical disease severity in EAE [7–9]. The biological
central nervous system (CNS), which has long served as an effects of minocycline in EAE appear to be at least partially
animal model for the human demyelinating disorder multiple mediated through its effect on the expression and biological
sclerosis (MS) [1]. A pathological hallmark of EAE is the activity of MMP-9 [8]. Clinical trials of minocycline in patients
presence of perivascular mononuclear cell infiltrates in the brain with MS are ongoing. Systemic administration of minocycline has
and spinal cord [1]. In order to egress from the peripheral blood been associated with numerous, sometimes serious adverse effects.
into peripheral tissues, leukocytes have to transverse endothelial Liposomes are spherical vesicles that consist of one or more lipid
barriers, the basement membrane (basal lamina), and parenchy- bilayers that surround an aqueous space. Liposomes were
mal extracellular matrix (ECM). Matrix metalloproteinases developed as drug carriers because of their capability to enclose
(MMPs) are proteolytic enzymes that mediate leukocyte biological materials, and to deliver them to specific tissues. Long-
migration across the blood-brain barrier (BBB), and through circulating polyethylene glycol (PEG) liposomes have two
ECM [2–6]. interesting pharmacological properties: (1) Following administra-

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 Minocycline Liposomes in EAE

Figure 1. Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on the human peripheral blood mononuclear
cells (PBMC) in vitro. Quantitative analysis of the zones of gelatinolysis detected the reduced the proteolytic activity of matrix metalloproteinase
(MMP)-9 in the human PBMC sample treated with minocycline (3.0 mg/ml) compared with findings in controls (PBS). A more significant decrease of
MMP-9 activity was detected in cells treated with minocycline for 6 hours compared to cells that were treated for 1 hour (A). At 1 hour, there was no
significant MMP-9 activity difference among samples treated by the different PEG liposome preparation either with glucose, CaCl2 or MgCl2 (A). Time
kinetic studies of PEG minocycline-liposomes in vitro revealed that the MMP-9 gelatinolytic activity is significantly reduced at 6 hours after incubation
samples with PEG liposome+minocycline (B). Moreover, PEG minocycline-liposome+CaCl2 treatment inhibited the MMP-9 activity more strikingly than
the PEG minocycline-liposome+MgCl2 preparation (B). Gelatinolytic activity was detectable by gelatin-zymography at molecular weights of 92 kDa,
indicative of MMP-9, in the supernatants from all human PBMC samples studied. Incubation with PEG minocycline-liposomes with CaCl2 resulted in
remarkably decreased sizes of the bands at 92 kDa, pointing to decreased activation of MMP-9 in comparison with PEG minocycline-liposomes with
MgCl2 or glucose, respectively (C).
doi:10.1371/journal.pone.0004151.g001

tion, PEG liposomes remain intact in the blood compartment for The principal goal of this study was to test the treatment efficacy
extended periods of time; (2) PEG liposomes have a high affinity of PEG minocycline-liposomes in EAE, and to study their effect on
to, and accumulate predominantly within sites of inflammation MMP-9 expression by leukocytes in peripheral lymphoid organs
[10]. and the CNS.

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 Minocycline Liposomes in EAE

Figure 2. Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on the clinical course of experimental
autoimmune encephalomyelitis (EAE). A single intravenous (i.v.) injection of PEG minocycline-liposomes given after disease onset at day 15
post-immunization resulted in significant amelioration of clinical disease for eight days compared to a single injection of PBS when given shortly after
onset of clinical disease (A). This effect was not sustained (A). In subsequent experiments, PEG-liposomes were administered every five days.
Treatment with i.v. PEG minocycline-liposomes initiated after disease onset at day 15 post-immunization and administered every five days was as
effective in ameliorating clinical EAE as treatment with daily intraperitoneal (i.p.) injections of minocycline (B). In contrast, i.v. injections of empty PEG-
liposome every five days, or minocycline i.p. injections every five days had no detectable effect on the clinical course of EAE (B). The time of treatment
initiation is indicated by a red arrow.
doi:10.1371/journal.pone.0004151.g002

Results by gelatin-zymography, as previously described [5,11]. Densito-

metric analysis of the zones of gelatinolysis revealed a reduced
In vitro effects of PEG minocycline-liposomes on MMP-9 proteolytic activity of MMP-9 in those supernatants derived from
expression by human PBMCs PBMCs treated with minocycline (3.0 mg/ml) compared to
Proteolytic activity of MMP-9 was assessed in supernatants of control (PBS). Specifically, the reduction of MMP-9 proteolytic
human PBMCs after 24 hrs of stimulation with IL-2 and assessed activity following PEG minocycline-liposomes treatment was

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 Minocycline Liposomes in EAE

Table 1. Summary of the EAE disease coursea.

Treatment No. of Mice Incidenceb (%) Mortalityc (%) Average Day of Onset Average Cumulative Disease Scored

No Txe 5 5/5 (100) 0 17 3266

Minof (q5d) 5 5/5 (100) 0 17 3364
Lipog (q5d) 5 5/5 (100) 1 (20) 17 3269
Minoh (daily) 5 5/5 (100) 0 15 2161
Mino-Lipoi (q5d) 5 5/5 (100) 0 15 1862

a
Graded disease score as described in Materials and Methods.
b
Represents the percentage of mice that developed a clinical score of at least one.
c
Represents the percentage of mice that died or were sacrificed for humane purposes.
d
The cumulative disease score was calculated by adding the disease score from the day of onset to day 30. The values shown are the mean6SE of all the mice with
disease in each group.
e
No Tx = No Treatment.
f
Mino = Minocycline.
g
Lipo = Empty PEG-liposomes.
h
Mino = Minocycline.
i
Mino-Lipo = PEG minocycline-liposomes.
doi:10.1371/journal.pone.0004151.t001

more substantial when cells were harvested after 6 hours than Effects of PEG minocycline-liposomes on CNS
after 1 hour, both with PEG minocycline-liposome preparations inflammation
containing CaCl2 or MgCl2 (Fig. 1A). When time kinetic studies A decreased number of inflammatory foci in the brain of EAE
of PEG minocycline-liposomes were performed in vitro, we mice treated with PEG minocycline-liposomes was observed five
detected that PEG minocycline-liposomes with CaCl2 inhibited days after treatment initiation (Fig. 3A). Specifically, the number of
the proteolytic activity of MMP-9 more strikingly than PEG CD11b+ macrophages, CD3+ T lymphocytes, and the overall
minocycline-liposomes with MgCl2 or glucose (Fig. 1B&C). expression of MMP-9 within CNS tissue was decreased by PEG
Incubation with PEG minocycline-liposomes with CaCl2 resulted minocycline-liposomes treatment (Fig. 3A–B). There was no
in a substantial decrease of MMP-9 proteolytic activity difference with regard to the numbers of immunoreactive cells
compared with PEG minocycline-liposomes with MgCl2 or between mice that were treated daily with i.p. minocycline, and
glucose, respectively (Fig. 1C). Consequently, PEG minocycline- animals treated with i.v. PEG minocycline-liposomes (Fig. 3B).
liposome preparations containing CaCl2 were used in all EAE
experiments.
Effects of PEG minocycline-liposomes on the expression
of MMP-9 in splenocytes and CNS-infiltrating leukocytes
Effects of PEG minocycline-liposomes on the clinical The expression of MMP-9 by splenocytes was significantly
course of EAE reduced in mice treated daily with i.p. injections of minocycline
To test our hypothesis that less frequent intravenous (i.v.) and i.v. injections of PEG minocycline-liposomes every five days
injections of a liposome formulation have similar therapeutic compare to splenocytes from animals that received no treatment, i.p.
efficacy as daily injections of regular formulation minocycline, we injections of minocycline every five days, and i.v. injections of empty
employed treatment paradigms that did not favor our hypothesis. PEG-liposomes every five days, as shown by ELISA (Fig. 4A). As
Other investigators had previously shown that a daily intraperi- expected, MMP-9 proteolytic activity measured by zymography was
toneal (i.p.) treatment paradigm in itself may significantly lower also significantly diminished in mice treated daily with i.p. injections
clinical disease in mice with EAE, possibly through the release of of minocycline and i.v. injections of PEG minocycline-liposomes
anti-inflammatory mediators [9]. In initial experiments, a single every five days (Fig. 4B). The protein expression of MMP-9 by CNS-
dose of PEG minocycline-liposomes was administered after EAE infiltrating leukocytes was similar in all treatment groups (Fig. 4C).
disease onset at day 15 post-immunization to determine the There was no significant difference with regard to the number of
duration of efficacy of this preparation. A single i.v. injection of CD11b+ macrophages, CD3+ T lymphocytes, and the expression of
PEG minocycline-liposomes resulted in significant amelioration of MMP-9 in the CNS between animals that had received no
clinical disease for eight days compared to a single injection of PBS treatment, daily i.p. PBS injection, and empty PEG-liposomes
(Fig. 2A). This beneficial effect was not sustained (Fig. 2A). administered i.v. every five days (data not shown).
Consequently, it was decided to administer PEG-liposomes every
five days. In clinical practice, the earliest possible time of treatment
Discussion
initiation in patients with MS is after the first clinical attack [12].
Thus, therapy was started at day 15 post immunization, after Minocycline, a semisynthetic tetracycline, is used for the
experimental animals had developed clinical disease. Treatment treatment of pneumonia, rheumatoid arthritis (RA), acne, and
with i.v. PEG minocycline-liposomes every five days was as other infectious diseases. Furthermore, it has been successfully
effective in ameliorating clinical EAE as treatment with daily i.p. tested in animal models of neurodegeneration, CNS inflammation,
injections of minocycline (Fig. 2B, Table 1). Both treatment and traumatic brain injury [13]. It is currently thought that the
paradigms resulted in sustained clinical benefit. In contrast, i.v. clinical benefits achieved by minocycline are the result of caspase-
injections of empty PEG-liposomes every five days, or minocycline activated apoptosis, and its modulation of peripheral immuno-
i.p. injections every five days had no detectable effect on the competent cells and microglia with regard to their release of
clinical course of EAE (Fig. 2B, Table 1). cytokines, suppression of free radical production, and inhibition of

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 Minocycline Liposomes in EAE

Figure 3. Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on inflammation within the central nervous
system (CNS). A decreased number of inflammatory foci in the brain of mice with experimental autoimmune encephalomyelitis (EAE) treated with
PEG minocycline-liposomes at disease onset was detected (A). Specifically, the number of CD11b+ macrophages, CD3+ T lymphocytes, and the overall
expression of matrix metalloproteinase (MMP)-9 were decreased by PEG minocycline-liposomes treatment (A,B). There was no difference with regard
to the numbers of immunoreactive cells between mice that were treated daily with intraperitoneal (i.p.) minocycline, and animals that had received
intravenous (i.v.) PEG minocycline-liposomes (B). There was no difference with regard to the number of CD11b+ macrophages, CD3+ T lymphocytes,
and the expression of MMP-9 in the CNS between animals that had received no treatment, daily i.v. PBS injection, and empty PEG-liposomes
administered i.p every five days (data not shown).
doi:10.1371/journal.pone.0004151.g003

MMPs [13]. More recent studies suggest that minocycline all treatment groups, indicating that there is an absolute
treatment may worsen some diseases of the CNS [13] and the requirement for a certain level of MMP-9 expression by leukocytes
peripheral nerve [14]. Thus, minocycline might have therapeutic to migrate across the blood-brain barrier, and to gain access to
potential in neuroinflammatory disorders. However, long-term areas of inflammation. The absolute number of infiltrating cells
safety and efficacy need to be determined critically when was decreased.
considering its application in clinical neurology. Our present data emphasize the potential role of PEG liposomes
Minocycline has an acceptable side-effect profile and tolerability as a drug delivery system for pharmaceuticals for CNS
when utilized for short-term antibiosis. Potential adverse effects autoimmune diseases. Our data also indicate that inhibition of
associated with long-term daily minocycline therapy for patients MMP-9 remains a promising treatment target in patients with MS.
with MS are concerning. These potential side effects have perhaps
most frequently been observed in patients with RA. In a Materials and Methods
randomized, double-blind study of minocycline in patients with
active RA, patients received a maximal oral daily dose of 200 mg Preparation of long-circulating PEG–liposomes
for 26 weeks [15]. Six out of 40 patients stopped medication A lipid solution was prepared in ethanol, containing 100 mM
because of adverse events in the minocycline treatment group. dipalmitoyl phosphatidylcholine (DPPC), 8.1 mM PEG 2000
Specifically, gastrointestinal adverse effects and dizziness were distearyl phosphatidylethanolamine (DSPE) (Lipoid GmbH,
among the adverse events significantly increased in the minocy- Ludwigshafen, Germany) and 54 mM cholesterol (Sigma, Poole,
cline group compared to those patients receiving placebo. Other UK) in a molar ratio of 1.85:0.15:1.0. The lipid solution was
adverse events that were more frequent in the minocycline transferred to a round-bottom flask, and a lipid film was created
treatment group included rash and headaches. Long-term by rotary evaporation. The film was hydrated with a solution of
exposure to minocycline has also been associated with a variety 120 mM calcium chloride and 120 mM sodium acetate (MP
of clinical and serological autoimmune aberrations, including Biomedicals, Inc., Eschwege, Germany). The resulting lipid
serum sickness [16], drug-induced lupus [17], autoimmune dispersion was sized by multiple extrusions through polycar-
hepatitis [18], and vasculitis [19]. bonate filter membranes. Unencapsulated calcium chloride and
Some of the observed adverse events are not entirely surprising. sodium acetate was removed by dialysis. Mean particle size was
Minocycline is a small (495 kDa), highly lipophilic molecule capable determined by dynamic light scattering with a Malvern 4700
of crossing the blood–brain barrier (BBB). Because of the large system (Malvern, Worcestershire, UK). The diameter of the
volume of distribution of minocycline, the drug accumulates in liposomes was determined to be 100–110 nm with a polydis-
tissues other than the CNS, including the eye and prostate, and it is persity index below 0.2. To 8 ml liposomal formulation, 80 mg
detectable in high concentration in tears, saliva and breast milk [20]. minocycline hydrochloride (MP Biomedicals Inc.) was added.
The pharmacological half-life of minocycline is approximately This mixture was incubated at 50uC for 15 minutes. Phospho-
18 hours. Thus, high and frequent dosing is required. lipid content was determined by phosphate assay [22] in the
Preliminary data on the efficacy of minocycline in patients with organic phase after extraction of liposomal preparations with
MS are promising. In an ongoing clinical trial of minocycline in a chloroform. The aqueous phase after extraction was used for
relatively small number of patients with relapsing-remitting MS, reversed-phase HPLC-determination of the minocycline con-
study participants received oral minocycline 100 mg twice daily tent.
for 6 months after a 3-month run-in period [21]. A 30-month
treatment extension is ongoing. The preliminary results of this trial
demonstrated that minocycline therapy reduces gadolinium- In vitro assessment of mononuclear cells
enhancing lesions on magnetic resonance imaging (MRI) [21]. Peripheral blood was aseptically collected from healthy
Fortunately, no serious adverse effects of minocycline in MS have volunteers by standard venipuncture into vacuum tubes containing
been reported thus far. sodium heparin as anticoagulant. PBMCs were isolated by Ficoll-
As our study in EAE animals shows, pharmacotherapy with Hypaque (Pharmacia, Karlsruhe, Germany) density gradient
long-circulating PEG minocycline-liposomes would substantially centrifugation and resuspended (26106/ml) in complete culture
lower the total amount of minocycline administered to patients, media consisting of RPMI 1640 medium supplemented with 10%
yet provide similar clinical effectiveness. Thus, the risk of potential human serum, 2 mM glutamine (Sigma-Aldrich, Munich, Ger-
side effects of minocycline could be minimized. In our experi- many), 100 U/ml penicillin, and 100 IE/ml streptomycin (Invi-
ments, PEG minocycline-liposomes provided a sustained clinical trogen, Karlsruhe, Germany). PBMCs were cultured for 24 h at
benefit when given every five days, but not when given less 37uC and 5% CO2 in the presence of IL-2 (10 ng/ml; Sigma-
frequently. The total expression of MMP-9 within the CNS and by Aldrich). Afterwards, PEG-liposomes were added and superna-
splenocytes, a robust biomarker of minocycline activity, was also tants were obtained 1 and 6 h later. Informed written consent was
equally effected by daily minocycline treatment and injections with obtained from all healthy volunteers, and all study procedures
PEG minocycline-liposomes every five days. Interestingly, the were approved by the Institutional Review Board at the Heinrich
expression of MMP-9 by CNS-infiltrating leukocytes was similar in Heine University.

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 Minocycline Liposomes in EAE

Figure 4. Effects of PEG minocycline-liposomes on the expression of matrix metalloproteinase (MMP)-9 in splenocytes and CNS-
infiltrating leukocytes. The expression levels of MMP-9 were significantly down-regulated on day 20 after active induction of experimental
autoimmune encephalomyelitis (EAE) in splenocytes of mice following treatment with daily intraperitoneal (i.p.) injections of minocycline, and after
intravenous (i.v.) injections of PEG minocycline-liposomes every 5 days compared to experimental animals that received no treatment, that were
treated with i.v. injections of empty PEG-liposome every five days, or with minocycline i.p. injections every five days, as shown by ELISA (A). The
proteolytic activity of MMP-9 measured by zymography was also significantly diminished in mice treated daily with i.p. injections of minocycline and
i.v. injections of PEG minocycline-liposomes every five days (B). MMP-9 protein expression in CNS mononuclear cells was not found to be significantly
different between experimental groups (C).
doi:10.1371/journal.pone.0004151.g004

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 Minocycline Liposomes in EAE

Gelatin zymography dosing experiment, a single dose of i.v. injection of PEG

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis minocycline-liposomes or PBS (control) was given at disease onset.
(SDS/PAGE) zymography was performed for determination of
gelatinase activity as described before [5,11]. Supernatants were Histopathological evaluation
mixed 1:1 with Tris/glycine SDS sample buffer (Novex, CA), Inflammatory infiltrates in the brain were quantified by
and samples were applied to a 10% (w/v) polyacrylamide haematoxylin and eosin (H&E) staining, as well as immunohisto-
resolving gel containing 0.1% SDS and 0.1% gelatin type A from chemistry using antibodies against CD3, CD11b, and MMP-9 on
porcine skin (Sigma-Aldrich). Stacking gels were 5% (w/v) days 20 after active immunization according to published methods
polyacrylamide. After electrophoresis gels were washed in [11,24]. Selected brain, thoracic and lumbar spinal cord sections
renaturing buffer (Novex) containing Triton X-100 to remove were evaluated by an examiner blinded to the treatment status of
any SDS and incubated in developing buffer (Novex) for the experimental animals. Three mice per treatment group were
18 hours at 37uC. Gels were stained for 6 hours in 30% examined. The mice selected for histopathological evaluation had
methanol / 10% acetic acid containing 0.5% (w/v) Coomassie the mean disease score of their respective treatment group.
Brilliant Blue G-250 and destained in the same buffer without
dye. Gelatinase activity was detected as unstained bands on a Preparation of CNS mononuclear cells
blue background representing areas of gelatin digestion. As a Mononuclear cells were isolated from brains and spinal cords as
negative control, gels were incubated with 10 mol/L N- described [25]. CNS tissue was passed through a cell strainer
ethylenediaminetetraacetic acid (EDTA) prior to the activation (70 mm). After centrifugation, cells were resuspended in 37%
of gelatinases in parallel. Images of gels were captured by Percoll (GE Healthcare, WI), centrifuged at 2,118 g for 15 min,
scanning on a flatbed scanner. A standard curve was obtained and washed. To prepare cells for experiments, pooled CNS
for densitometric quantitation of MMP activity using purified samples were washed twice with 37% Percoll, resuspended in 30%
MMP-2 and -9 (Chemicon, CA). In each sample, MMP-9 Percoll, and layered over 70% Percoll. Cells harvested from the
proteolytic activity was calculated using an electrophoretic gel gradient interface were washed and resuspended in culture
lane calculation software after image inversion. medium consisted of RPMI 1640 (Invitrogen, CA) supplemented
with L-glutamine (2 mM) (Mediatech Inc. VA), sodium pyruvate
Mice (1 mM) (Mediatech), non-essential amino acids (0.1 mM) (Media-
Female C57BL/6 (B6) mice were purchased from the Jackson tech), penicillin (100 U ml21) (Mediatech), streptomycin
Laboratory (Bar Harbor, ME). The use of mice in these (0.1 mg ml21) (Mediatech), 2-mercaptoethanol (561025 M)
experiments was approved by the Institutional Animal Care and (Mediatech) and 10% (v/v) fetal bovine serum (Mediatech) for
Research Advisory Committee at the University of Texas further analysis.
Southwestern Medical Center at Dallas.
Enzyme immunoassay
Experimental autoimmune encephalomyelitis Supernatants from splenocytes and CNS mononuclear cells
EAE was induced in groups of five 6–10 week old female cultured with MOGp35–55 (2 ug/ml) were used for analysis. The
C57BL/6 mice by immunization with 200 mg of myelin concentration of total MMP-9 (pro-MMP-9, active MMP-9, and
oligodencrocyte glycoprotein (MOG) petide (p) 35–55 (BioSource tissue inhibitor of matrix metalloproteases [TIMP]-1-complexed
International, CA) in an emulsion with Complete Freund adjuvant MMP-9) in cell culture supernatants was determined by
(CFA) containing 4 mg ml21 of heat-inactivated Mycobacterium Quantikine Mouse MMP-9 (total) Immunoassay according to
tuberculosis H37Ra (Difco Laboratories, MI). On the day of the manufacturer’s instructions (R&D Systems, MN). The results
immunization and 48 hours later, C57BL/6 mice were injected of ELISA experiments are expressed as an average of triplicate
with 200 ng pertussis toxin (PTx, List Biological Laboratories, Inc., wells 6standard deviation (SD). A SOFTmax ELISA plate reader
CA) in phosphate buffered saline (PBS) i.p. Mice were examined and software was used for data analysis (Molecular Devices, CA).
daily for clinical signs of EAE and scored as follows: 0: No
paralysis; 1: Loss of tail tone; 2: Hindlimb weakness; 3: Hindlimb Statistical analysis
paralysis; 4: Hindlimb and forelimb paralysis; 5: Moribund or The means of two normally distributed samples were compared
death [23]. All experiments were repeated twice. by Student t-test. All other statistical comparisons between groups
were examined using one-way multiple range ANOVA test for
Pharmacotherapy multiple comparison. P-values,0.05 were considered significant.
For EAE treatment, the mice were treated with i.v. injections of Data are shown with SD of the mean.
PEG minocycline-liposomes (50 mg/g body weight [BW] in 100 ml
of PBS) every five days. Empty PEG-liposomes (50 mg/g BW in Author Contributions
100 ml of PBS), i.p injections of minocycline (50 mg/g BW in Conceived and designed the experiments: WH LHB TNE BCK OS.
100 ml of PBS) every five days, and not pharmacological treatment Performed the experiments: WH LHB MPS BCK OS. Analyzed the data:
constituted negative controls. Daily i.p. injections of minocycline WH LHB BCK OS. Contributed reagents/materials/analysis tools: WH
(50 mg/g BW in 100 ml of PBS) was the positive control. In one JM BCK OS. Wrote the paper: WH PCC EMF TNE MKR BCK OS.

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PLoS ONE | www.plosone.org 9 January 2009 | Volume 4 | Issue 1 | e4151