| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of
*
Pharmacokinetics and Drug Delivery and
Pathology and Laboratory Medicine, Medical Biology Section, Groningen University Institute for Drug Exploration, Groningen, The Netherlands;
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands; and
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-stimulated
endothelial cells and not to resting endothelial cells. After binding,
the conjugate was internalized and routed to multivesicular bodies,
which is a lysosome-related cellular compartment. After intracellular
degradation, pharmacologically active dexamethasone was released, as
shown in endothelial cells that were transfected with a
glucocorticoid-responsive reporter gene. Furthermore, intracellularly
delivered dexamethasone was able to down-regulate the proinflammatory
gene IL-8. In conclusion, this study demonstrates the possibility to
selectively deliver the anti-inflammatory drug dexamethasone into
activated endothelial cells, using an anti-E-selectin Ab as a
carrier molecule. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in arthritis, psoriasis, and Crohn’s disease
(5, 6, 7). The endothelium is an attractive target for pharmacological intervention in chronic inflammation, because it plays an important role in leukocyte recruitment and infiltration (8). Once activated, endothelial cells express adhesion molecules on their surface and secrete a broad variety of cytokines and chemokines, thereby directing the multistep adhesion cascade of leukocytes into inflamed tissue (9).
To reduce leukocyte recruitment, Abs that block endothelial adhesion molecules have been therapeutically applied (10, 11, 12). These strategies are not always successful, possibly due to redundant leukocyte recruitment mechanisms in the body. Alternatively, selective inhibitors of cellular activation pathways have been developed, which, for example, reduce adhesion molecule and cytokine expression in endothelial cells (13, 14). However, the in vivo use of these inhibitors is limited due to the importance of these activation pathways in other cells and processes in the body.
In the strategy described in this report, a so-called drug targeting approach is applied in which an Ab to E-selectin (CD62E) is used to deliver the covalently coupled anti-inflammatory drug dexamethasone into activated endothelial cells. The ultimate goal of this drug targeting approach is to selectively block endothelial cell activation at the site of inflammation. It is anticipated that the low drug concentrations in other cell types in the body will simultaneously diminish drug-associated side effects (15).
In the present study, binding, uptake, and intracellular routing of an immunoconjugate consisting of the glucocorticoid dexamethasone covalently attached to an Ab recognizing E-selectin was investigated in HUVECs. Additionally, the pharmacological effect of the intracellularly delivered dexamethasone was determined using glucocorticoid reporter gene transfected endothelium as well as IL-8 Northern blotting. In this way, we investigated whether the immunoconjugate can be used for selective delivery of anti-inflammatory drugs into activated endothelial cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The H18/7 (mouse anti-human E-selectin) mAb-producing hybridoma (AbhEsel)3 was kindly provided by Dr. M. Gimbrone, Jr. (Boston, MA). AbhEsel was purified from the culture medium by protein A affinity chromatography (protein A-Sepharose fast flow; Pharmacia, Roosendaal, The Netherlands) followed by dialysis against PBS. The 10E9 (rat anti-mouse E-selectin) mAb-producing hybridoma (AbmEsel) was kindly provided by Dr. D. Vestweber (Institute of Cell Biology, ZMBE, University of Münster, Münster, Germany). AbmEsel was purified from the culture medium by protein G affinity chromatography (protein G-Sepharose fast flow; Pharmacia) followed by dialysis against PBS. An irrelevant control Ab (MOC31, mouse anti-human EGP-2 (AbCtrl)) was kindly provided by IQProducts (Groningen, The Netherlands).
The anti-dexamethasone polyclonal Ab was prepared in our
laboratory (16) and was purified by absorption to
polyvinylidene difluoride membranes coated with
-tocopherol-succinate-BSA (prepared similarly as dexamethasone
(dexa)-AbhEsel (described below), using 10
equivalents of -tocopherol-succinate and one equivalent BSA). This
procedure was performed to remove Abs recognizing the succinate linker
in the conjugate. As a result, only Abs recognizing conjugated
dexamethasone remained in the antiserum.
Human E-selectin/IgG fusion protein was kindly provided by Dr. J. van Dijk (Vrije Universiteit, Amsterdam, The Netherlands); human P-selectin/IgG fusion protein was kindly provided by Dr. B. Appelmelk (Vrije Universiteit).
Preparation of dexa-Ab conjugates
All chemicals used for preparation of the conjugates were of
analytical reagent grade or higher. Dexamethasone (Bufa, Hilversum, The
Netherlands) was conjugated via a succinate linker to the Ab according
to Melgert et al. (16) (Fig. 1
A). For this,
dexamethasone-21-hemisuccinate (dexa-suc) was prepared by reacting
dexamethasone with succinic anhydride according to McLeod
(17) with minor modifications. Briefly, dexamethasone (2.5
mmol), excess succinic anhydride (42 mmol), and 4-dimethylaminopyridine
(2.6 mmol) were dissolved in 80 ml of anhydrous acetone and allowed to
react for 24 h at room temperature. After removal of the solvent
by evaporation under reduced pressure, the crude product was
recrystallized from ethanol:water (3:7). The identity of the
synthesized dexa-suc was confirmed by mass spectrometry (molecular
mass, 492.5 Da; molecular mass found, 492.1 Da).
|
Characterization of dexa-Ab conjugates
Dexa-Ab conjugates were analyzed for protein content according to Lowry (18). Dexamethasone content was analyzed by HPLC using a chromatography system consisting of a Waters 600 controller, 717plus autosampler and 486 tunable absorbance detector, operated at 240 nm, in combination with a µBondapak C18 guard-pak precolumn and a µBondapak C18 column (300 x 3.9 mm) (all from Waters, Milford, MA). Elutions were performed with a water/acetonitrile/trifluoroacetic acid mixture (60/40/0.05) at a flow rate of 1.5 ml/min. Dexamethasone and dexa-suc eluted at 5.8 and 9.3 min, respectively.
The amount of coupled dexamethasone was analyzed after alkaline hydrolysis of the succinate linker during 1 h, followed by neutralization of the pH and direct analysis of released dexamethasone. For this, 20 µl of the dexa-Ab solution was treated with 50 µl of 0.1 N NaOH, mixed and incubated at room temperature for 1 h. After addition of 50 µl of 0.1 N HCl and mixing, 30 µl of the solution was injected into the HPLC.
SDS-PAGE analyses were performed on a mini-PROTEAN II system (Bio-Rad, Veenendaal, The Netherlands) using 7.5% polyacrylamide Tris-HCl gels. Gels were either stained for protein (Coomassie brilliant blue staining) or blotted on polyvinylidene difluoride membrane, followed by immunostaining for conjugated dexamethasone.
Binding of dexa-AbhEsel to E-selectin in a surface plasmon resonance study
A BIAcore 2000 instrument (Biacore, Uppsala, Sweden) was used to analyze the interaction between dexa-AbhEsel and human E-selectin. All experiments were performed at 20°C using PBS containing 0.005% Tween 20. IgG fusion proteins (E-selectin, P-selectin) were immobilized on a CM5 sensor chip according to the manufacturer’s instructions.
Binding interaction of AbhEsel or dexa-Abhsel with human E-selectin was determined by passing a cycle of five injections over the immobilized E-selectin chimeric protein at a flow rate of 30 µl/min: 1) running buffer, 2) solution containing the primary ligand (either dexa-AbhEsel or AbhEsel, both at a concentration of 500 nM in running buffer), 3) running buffer, 4) solution containing the secondary ligand (anti-dexamethasone polyclonal antiserum diluted in running buffer), and 5) running buffer. At the end of each cycle, the sensor chips were regenerated with 0.1 M HCl and equilibrated with running buffer. Sensorgrams were corrected for background by baseline subtraction using an empty flow channel. Data were analyzed using BIAevaluation software (V2.1, Pharmacia Biosensor, Uppsala, Sweden).
Endothelial cells
HUVECs were isolated and cultured as previously described (19) and obtained from the Endothelial Cell Facility Groningen University/Academic Hospital Groningen (Groningen, The Netherlands). Primary isolates were cultured on 1% gelatin-precoated tissue culture flasks (Costar, Cambridge, MA) at 37°C under 5% CO2/95% air. The culture medium consisted of RPMI 1640 supplemented with 20% heat-inactivated FCS, 2 mM L-glutamine, 5 U/ml heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml endothelial cell growth factor supplement extracted from bovine brain. The H5V mouse endothelioma cell line was kindly provided by Dr. A. Vecchi (Istituto Ricerche Farmacologische Mario Negri, Milan, Italy). These cells were grown in tissue culture flasks at 37°C under 5% CO2/95% air. The culture medium consisted of DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 300 µg/ml gentamicin.
Both cell types, upon confluence, were detached from the surface by trypsin/EDTA (0.5/0.2 mg/ml in PBS) and split at a 1:3 ratio. For the experiments described, HUVEC were used up to passage three.
Analysis of binding of dexa-Ab conjugates to endothelial cells
Binding of dexa-Ab conjugates to endothelial cells was
investigated using both immunohistochemistry and flow cytometry. HUVECs
were grown to confluence on gelatin-precoated six-well-plates and
stimulated with 100 ng/ml recombinant human (rh)TNF- (Boehringer
Mannheim, Mannheim, Germany) for 4 h. After three washes
with cold PBS, cells were trypsinized and for immunohistochemical
analysis spun down on slides using the Shandon Cytospin 3 Cell
Preparation System (Life Sciences International, Veldhoven, The
Netherlands). Cytospots were acetone-fixed, preincubated with 10%
normal goat serum, and subsequently incubated with 10 µg/ml dexa-Ab
for 1 h at room temperature in a humid chamber. After extensive
washing with PBS, Ab was detected by incubation with HRP-conjugated
goat anti-mouse IgG (DAKO, Glostrup, Denmark), whereas
conjugated dexamethasone was detected by rabbit anti-dexamethasone
antiserum, followed by HRP-conjugated goat anti-rabbit IgG (DAKO).
After color development counterstaining was performed with Mayers’
hematoxylin and slides were mounted with glycerin.
For flow cytometry, trypsinized cells were incubated with 10 µg/ml dexa-AbhEsel or unconjugated AbhEsel in 5% FCS/PBS for 1 h at 4°C. Ab was subsequently detected using FITC-labeled rabbit anti-mouse F(ab')2 (DAKO, Glostrup, Denmark) and samples were analyzed using a Coulter Epics-Elite flow cytometer (Coulter Electronics, Hialeah, FL).
Data were analyzed using WinList (version 3D; Verity Software House, Topsham, ME) and WinMDI (version 2.8; The Scripps Research Institute, La Jolla, CA) software.
Internalization of dexa-Ab conjugates by endothelial cells
Internalization of dexa-Ab conjugates by endothelial cells was studied using confocal laser scanning microscopy (CLSM) and immuno-transmission electron microscopy (immuno-TEM).
HUVECs were grown to confluence on fibronectin-coated chamber slides
(Lab-Tek; Nalge Nunc International, Naperville, IL) or
gelatin-coated flat-bottom 48-well plates for CLSM or immuno-TEM
experiments, respectively. Cells were stimulated with 100 ng/ml
rhTNF- for 4 h. After 4 h, dexa-Ab was added to the medium
to a final concentration of 10 µg/ml. After various incubation
periods, cells were washed with cold PBS and fixed with 2%
paraformaldehyde/PBS overnight at 4°C.
CLSM
For detection of the localization of the conjugate by CLSM, cells were preincubated with 10% normal goat serum followed by permeabilization of the cells with 0.1% saponin in PBS. After permeabilization, Ab and conjugated dexamethasone were detected by, respectively, incubation with tetramethyl rhodamine-labeled goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL), and rabbit anti-dexamethasone antiserum followed by incubation with cyanine dye 5-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
Slides were embedded in antifading medium consisting of 0.25% Dabco (1,4-diazabicyclo[2.2.2]octane; Merck, Darmstadt, Germany) in glycerol/PBS (9/1 v/v). Slides were examined using a confocal laser-scanning microscope equipped with a 488-nm argon, 568-nm krypton, and 647-nm HeNe laser (Leica TCS-SP; Leica Microsystems, Rijswijk, The Netherlands). Images were analyzed using Leica TCS-SP Power Scan software.
Immuno-TEM
For detection of Ab, cells were incubated with goat anti-mouse IgG labeled with 6 nm of colloidal gold (Aurion, Wageningen, The Netherlands). For localization of conjugated dexamethasone, cells were incubated with rabbit anti-dexamethasone followed by incubation with goat anti-rabbit IgG labeled with 6 nm of colloidal gold (Aurion). All Abs were dissolved in 1% BSA/PBS containing 0.05% Triton X-100. Cells and Abs were fixed with 2% glutaraldehyde/PBS for 15 min at room temperature. After several washes with water, silver enhancement was performed for 5 min to increase average gold particle size (R-gent; Aurion). Cells were washed with distilled water and fixed in 2% glutaraldehyde/PBS. Specimens were postfixed in 1% OsO4 for 2 h and dehydrated with increasing alcohol concentrations, followed by propylene oxide. Thereafter, cells were embedded in Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections (80 nm) were stained with uranylacetate and lead citrate and examined by use of a Philips 210 transmission electron microscope (Philips, Eindhoven, The Netherlands) at 60 kilovolts.
Reporter gene analysis of dexamethasone release from a dexa-AbmEsel conjugate
H5V mouse endothelioma cells were transfected with
lipoplexes containing the synthetic amphiphile Saint2 (Saint,
Groningen, The Netherlands) and a reporter gene construct encoding
glucocorticoid responsive element (GRE)-driven Firefly luciferase
(Clontech Laboratories, Palo Alto, CA). The cells were cotransfected
with pCMV-Luc encoding Renilla luciferase (Promega, Madison, WI) to
correct for transfection efficiency. The molar ratio Firefly:Renilla
DNA was 500:1 and the total amount of DNA used for transfection was 1
µg. Twenty-four hours after transfection, rhTNF- (250 ng/ml) was
added in either the absence or presence of dexamethasone (1, 10, or 100
nM) or dexa-AbmEsel (10 µg/ml). Cells were
harvested after 48 h and luciferase activity was measured using a
Dual Luciferase Assay kit (Promega).
Down-regulation of IL-8 expression by dexa-AbhEsel
HUVECs were grown to confluence on gelatin-precoated
25-cm2 flasks and stimulated with 100 ng/ml
rhTNF-. Where indicated, dexamethasone (100 nM) or
dexa-AbhEsel (10 µg/ml) was added to the cells
1 h before or simultaneous with start of TNF- stimulation,
respectively. After 6 h of TNF- stimulation, total RNA was
extracted using TRIzol reagent (Life Technologies, Gaithersburg,
MD). The amount of RNA extracted was measured by optical density at 260
nm. RNA (4 µg) was separated on a 1% agarose/formaldehyde gel and
blotted overnight onto Hybond N+ membrane
(Boehringer Mannheim) in 10x SSC (150 mM sodium chloride, 15 mM sodium
citrate, pH 7.0). 32P-labeled radioactive
random-primed IL-8 and GAPDH probes were synthesized from PCR products,
using the Strip-EZ DNA kit (Ambion, Austin, TX).
According to Church and Gilbert (20), the blot was hybridized with the IL-8 probe at 65°C and subsequently exposed to a phosphor imager screen. After removal of the radioactive probe from the membrane, according to the Strip-EZ protocol (Ambion), hybridization was repeated, this time using the random-primed GAPDH as a probe.
The obtained IL-8 and GAPDH signals were quantified using Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (Molecular Dynamics).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dexa-AbhEsel conjugates were prepared
by coupling dexa-suc to primary amino groups of the Ab (Fig. 1
A). SDS-PAGE followed by anti-dexamethasone Western
blotting demonstrated that dexamethasone indeed had been covalently
conjugated to the 150-kDa Ab (Fig. 1B). To assess the amount
of coupled dexamethasone molecules, the ester linkage between the drug
and protein was chemically degraded and the amount of dexamethasone was
determined by HPLC. The results of this analysis, in combination with
the protein content of the conjugates, yielded an average dexamethasone
content of 2.3 drug molecules per AbhEsel
molecule, i.e., 56% conjugation efficiency. Similar drug conjugation
efficiencies were found for AbCtrl- and
AbmEsel-based drug-Ab conjugates.
Binding of dexa-AbhEsel to E-selectin in a surface plasmon resonance study
The binding of dexa-AbhEsel conjugate to
immobilized human E-selectin/IgG chimeric protein was investigated
using surface plasmon binding technology. In theory, the coupling of
dexamethasone could disrupt the Ag recognition of
AbhEsel to human E-selectin. To discriminate
between the binding of unmodified AbhEsel and
dexa-AbhEsel, anti-dexamethasone was applied
as a secondary ligand to the (dexa-)AbhEsel
molecules bound on the chip. The sensorgrams clearly showed binding of
dexa-AbEsel to E-selectin, as reflected by the
secondary increase in the resonance signal upon incubation with the
anti-dexamethasone Ab (Fig. 2).
|
). Analysis of binding of dexa-Ab conjugates to endothelial cells
We next addressed the question of whether
dexa-AbhEsel was also able to selectively bind to
E-selectin expressed on the endothelial cell surface. The conjugate
specifically bound to rhTNF--stimulated HUVEC as shown by
immunohistochemical analysis of the presence of both the Ab and the
conjugated dexamethasone (Fig. 3, B and C). As controls,
dexa-AbhEsel did not bind to resting HUVEC (Fig. 3A), and dexa-AbCtrl did not bind to
activated endothelial cells (Fig. 3D).
|
F), similar to binding observed
with unconjugated AbhEsel (Fig. 3E). Internalization of dexa-Ab conjugates by endothelial cells
After binding, dexa-AbEsel needs to be
internalized by endothelial cells to be degraded, which is a
prerequisite for dexamethasone to be released and to have a
pharmacological effect. Using CLSM, we determined that
dexa-AbhEsel bound to and was internalized by
HUVECs stimulated for 4 h with rhTNF- (Fig. 4). At this time point HUVEC had maximal
expression of E-selectin on their surface (data not shown). After
1 h of incubation, a comparable staining pattern for
AbhEsel was found for both unmodified
AbhEsel and dexa-AbhEsel
(Fig. 4, A and D).
|
E), and this
staining pattern colocalized completely with the staining for
AbhEsel (Fig. 4F). This indicates that
(part of) the dexamethasone molecules were still conjugated to
AbhEsel upon transport into the endothelium.
Initial binding at the cell surface was already seen at 10 and 20 min
after conjugate incubation (Fig. 4, G and H).
Starting from 1 h after incubation, surface staining decreased and
an intracellular, punctuate, vesicle-like staining pattern could be
observed for both the dexamethasone and AbhEsel
part of the conjugate (Fig. 4, F and I–L).
Staining intensities decreased after 18–24 h of incubation (Fig. 4, K and L), most likely indicating intracellular
degradation of the conjugate. In control experiments,
dexa-AbhEsel was not internalized by resting
HUVEC, nor was dexa-AbCtrl internalized by
activated HUVEC, resulting in fully black images (data not shown).
Immuno-TEM studies were used to investigate the nature of the
vesicle-like structures as detected by the CLSM studies. After 1 h
of incubation, both the dexamethasone and AbhEsel
parts of the conjugate, although with a clear difference in quantity,
localized at the cell membrane, in small vesicles just below the plasma
membrane, as well as in multivesicular bodies (Fig. 5). In time, cell membrane localization
of the conjugate decreased, whereas accumulation in multivesicular
bodies increased (data not shown). Lysosomes (in the definition of
electron dense bodies), and hence the presence of the conjugate in
lysosomes, were hardly observed in the endothelial cells.
|
Reporter gene analysis of dexamethasone release from dexa-AbmEsel
After internalization and degradation, dexamethasone has to
diffuse into the cytoplasm and bind to its receptor to be able to exert
a pharmacological effect. In theory, upon binding of dexamethasone to
the glucocorticoid receptor, the complex translocates to the nucleus
where it binds to GREs in the DNA, as a result of which gene expression
is affected. We measured this effect by analyzing the expression of a
luciferase reporter gene that contains a GRE promoter. Because the
primary cell type HUVEC was difficult to transfect (only 
2%
transfection efficiency was obtained using the protocol described in
Materials and Methods), the mouse endothelioma cell line H5V
was transfected with the GRE-luciferase plasmid (20–25% transfection
efficiency). This cell line expresses E-selectin after TNF-
stimulation, which is a prerequisite for testing E-selectin-directed
conjugates.
After transfection, H5V cells were incubated with
dexa-AbmEsel (10 µg/ml, corresponding to 100
nM dexamethasone) and the GRE-mediated luciferase expression was
compared with the expression obtained after incubation with 1, 10, and
100 nM unconjugated dexamethasone, respectively. GRE-mediated
luciferase expression was measured in three independent
experiments.
The result of one representative experiment is shown in Fig. 6. Luciferase activity increased with
increasing concentration of unconjugated dexamethasone. In all three
experiments, the luciferase activity induced by
dexa-AbmEsel was comparable with the activity
induced by unconjugated dexamethasone at a concentration between 1 and
10 nM. From this, it was concluded that
dexa-AbmEsel is indeed capable of delivering
pharmacologically active dexamethasone into activated endothelial
cells, although the amount of delivered dexamethasone is less than what
enters the cell by passive diffusion of unconjugated dexamethasone.
|
In our cellular system with the primary endothelial cell type
HUVEC, no effects of freely administered dexamethasone on the
expression of adhesion molecules VCAM-1 or ICAM-1 could be
demonstrated. Therefore, to confirm pharmacological activity of
intracellularly delivered dexamethasone, down-regulation of the
proinflammatory gene encoding IL-8 was analyzed by Northern blotting.
Incubation of HUVEC with TNF- resulted in increased expression of
IL-8 mRNA, which was partly down-regulated by coincubation with either
free dexamethasone or dexa-AbhEsel conjugate.
Band intensities of IL-8, normalized to GAPDH mRNA expression levels,
quantitatively demonstrated 28 and 22% decreases in IL-8 mRNA
accumulation after treatment with 100 nM dexamethasone and 10 µg/ml
dexa-AbhEsel conjugate, respectively (Fig. 7). Similar decreases in IL-8 mRNA levels
were observed using other techniques, including competitive RT-PCR and
cDNA expression array analysis (S. A. Ásgeirsdóttir,
R. Kok, M. Everts, D. Meÿer, and G. Molema, manuscript in
preparation).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
E-selectin was chosen as a target molecule because this adhesion molecule is solely expressed on activated endothelial cells (21), thus enabling selective delivery of the drug into this cell type. Furthermore, it is an internalizing adhesion molecule, in contrast with, e.g., the adhesion molecules ICAM-1 or VCAM-1 (22). Internalization is an important quality of a target molecule because it is essential for intracellular degradation of the conjugate and hence release of the targeted drug.
Detailed studies on targeting drugs to endothelial cells are scarce and, until now, they have mainly focused on the delivery of cytotoxic drugs (23). However, in inflammation, killing of endothelial cells is not considered a rational strategy to achieve improved therapeutic outcome. Therefore, we aim at the down-regulation of endothelial activation by selective delivery of anti-inflammatory drugs. Dexamethasone was chosen as a model drug because of its broad spectrum of pharmacological effects. Furthermore, a polyclonal anti-dexamethasone antiserum was available to study the fate of the conjugated dexamethasone during intracellular handling of the conjugate by the endothelium.
The surface plasmon resonance studies and cellular binding demonstrated that the dexa-AbhEsel conjugate, with an average of two conjugated dexamethasone molecules per Ab, still recognized its target molecule. Higher dexamethasone loading ratios (up to 10 dexamethasone molecules per AbEsel molecule) reduced the solubility of the conjugate and diminished the E-selectin recognition (data not shown). Because the conjugation of high amounts of dexamethasone may also result in an increased distribution of the conjugate to the liver in vivo (16), the dexa-AbEsel conjugate with two molecules of dexamethasone per carrier molecule was used in the experiments described in this work.
Our CLSM and immuno-TEM studies indicate that the dexa-AbhEsel conjugate is intracellularly routed to vesicle-like structures, identified as multivesicular bodies. Because multivesicular bodies are lysosomal-related structures (24, 25), these data are in agreement with other studies on the routing of an unmodified anti-E-selectin Ab, where a final lysosomal pathway of the Ab was indicated (22, 26). Thus, the intracellular routing of the carrier AbhEsel was not affected by conjugation of the anti-inflammatory drug dexamethasone.
The presence of dexa-AbhEsel in multivesicular bodies and the absence of the conjugate in the nucleus is in contrast with the intracellular routing of a hydrocortisone-BSA conjugate (27). This steroid-protein conjugate localized in the nucleus after binding to its hormone receptor in the target cells. A likely explanation of this discrepancy is the difference in the number of drug molecules conjugated to the carrier protein. Whereas the hydrocortisone-BSA conjugate contained 21 molecules of hydrocortisone per BSA molecule, AbhEsel was modified with only two molecules of dexamethasone per Ab molecule.
Dexa-AbEsel conjugates were prepared in which the drug molecule was coupled via a succinate linker to primary amino groups of the Ab. This succinate linkage is sufficiently stable to deliver dexamethasone to the intracellular compartments of the target cells where the conjugate is degraded (16, 17). The pharmacologically active dexamethasone is subsequently released and can exert an effect, as shown by the reporter gene analysis in transfected endothelial cells. Despite the lower amount of drug delivered by dexa-AbmEsel when compared with intracellular accessibility of free dexamethasone, the conjugate has the advantage over unconjugated dexamethasone of being selective in delivering the drug into activated endothelial cells. Drug-associated side effects in other cell types will thus be diminished.
Because the artificial read-out system, consisting of the GRE-luciferase reporter gene, does not reflect the physiological effects of delivered dexamethasone, effects of dexamethasone on proinflammatory IL-8 mRNA levels were characterized by Northern blotting. Results demonstrated the ability of selectively delivered dexamethasone to decrease proinflammatory gene expression in activated endothelial cells. To further extend analysis of pharmacological effects of dexamethasone, gene expression profiling was performed, using a human cDNA cytokine/receptor array. The observed changes in mRNA levels of various cytokines and related molecules support our data presented in this report that dexamethasone is released in a pharmacologically active form, once intracellularly delivered by the dexa-AbhEsel conjugate (S.A. Ásgeirsdóttir, R. Kok, M. Everts, D. Meÿer, and G. Molema, manuscript in preparation).
In conclusion, the immunoconjugate dexa-AbEsel was internalized and intracellularly degraded by activated endothelial cells, allowing the release of pharmacologically active dexamethasone. Therefore, this study shows the possibility of using an anti-E-selectin immunoconjugate to selectively deliver anti-inflammatory drugs into activated endothelial cells. Further in vivo evaluation will elucidate the potential of this drug targeting strategy to selectively interfere with endothelial cell activation and hence leukocyte recruitment in chronic inflammatory diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Maaike Everts, Department of Pharmacokinetics and Drug Delivery, Groningen University, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail address: M.Everts{at}farm.rug.nl
3 Abbreviations used in this paper: AbhEsel, mouse anti-human E-selectin Ab (H18/7); AbmEsel, rat anti-mouse E-selectin Ab (10E9); AbCtrl, mouse anti-human EGP-2 Ab (MOC31); dexa, dexamethasone; dexa-suc, dexa-21-hemisuccinate; CLSM, confocal laser scanning microscopy; immuno-TEM, immuno-transmission electron microscopy; GRE, glucocorticoid responsive element; rh, recombinant human.
Received for publication August 30, 2001. Accepted for publication November 15, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
therapy of rheumatoid arthritis: what have we learned?. Annu. Rev. Immunol. 19:163.[Medline]
4 integrin monoclonal antibody. J. Clin. Invest. 92:372.
This article has been cited by other articles:
|
|
 |
|
  S. A. Asgeirsdottir, P. J. Zwiers, H. W. Morselt, H. E. Moorlag, H. I. Bakker, P. Heeringa, J. W. Kok, C. G. M. Kallenberg, G. Molema, and J. A. A. M. Kamps Inhibition of proinflammatory genes in anti-GBM glomerulonephritis by targeted dexamethasone-loaded AbEsel liposomes Am J Physiol Renal Physiol, March 1, 2008; 294(3): F554 - F561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Danielyan, B.-S. Ding, C. Gottstein, D. B. Cines, and V. R. Muzykantov Delivery of Anti-Platelet-Endothelial Cell Adhesion Molecule Single-Chain Variable Fragment-Urokinase Fusion Protein to the Cerebral Vasculature Lyses Arterial Clots and Attenuates Postischemic Brain Edema J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 947 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Muro, T. Dziubla, W. Qiu, J. Leferovich, X. Cui, E. Berk, and V. R. Muzykantov Endothelial Targeting of High-Affinity Multivalent Polymer Nanocarriers Directed to Intercellular Adhesion Molecule 1 J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1161 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-S. Ding, T. Dziubla, V. V. Shuvaev, S. Muro, and V. R. Muzykantov Advanced drug delivery systems that target the vascular endothelium. Mol. Interv., April 1, 2006; 6(2): 98 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Kuldo, J. Westra, S. A. Asgeirsdottir, R. J. Kok, K. Oosterhuis, M. G. Rots, J. P. Schouten, P. C. Limburg, and G. Molema Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1229 - C1239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Sakhalkar, M. K. Dalal, A. K. Salem, R. Ansari, J. Fu, M. F. Kiani, D. T. Kurjiaka, J. Hanes, K. M. Shakesheff, and D. J. Goetz Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo PNAS, December 23, 2003; 100(26): 15895 - 15900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bhaskar, D. A. Law, E. Ibsen, D. Breinberg, K. M. Cass, R. B. DuBridge, F. Evangelista, S. M. Henshall, P. Hevezi, J. C. Miller, et al. E-Selectin Up-Regulation Allows for Targeted Drug Delivery in Prostate Cancer Cancer Res., October 1, 2003; 63(19): 6387 - 6394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Dagia and D. J. Goetz A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1{beta}- and TNF-{alpha}-induced ECAM expression and adhesion Am J Physiol Cell Physiol, October 1, 2003; 285(4): C813 - C822. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and dexa-AbhEsel
in a concentration of 10 µg/ml (
). The increase in luciferase
activity indicates the successful delivery of pharmacologically active
drug in the endothelial cells. RLU, Relative luciferase units. Bars
represent mean values ± SD (n = 3); *,
p < 0.05; **, p < 0.01 vs
control (Dex, 0 nM), as determined by two-tailed Student’s
t test, assuming similar variances.