Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo.
ABSTRACT We exploited leukocyte-endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium in vitro and in vivo. Leukocyte-endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under in vitro flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium in vivo, and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium in vivo due to the addition of a targeting ligand. The leukocyte-inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.
- SourceAvailable from: Gerben A Koning[show abstract] [hide abstract]
ABSTRACT: In chronic inflammatory diseases, the endothelium is an attractive target for pharmacological intervention because it plays an important role in leukocyte recruitment. Hence, inhibition of endothelial cell activation and consequent leukocyte infiltration may improve therapeutic outcome in these diseases. We report on a drug targeting strategy for the selective delivery of the anti-inflammatory drug dexamethasone to activated endothelial cells, using an E-selectin-directed drug-Ab conjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalization of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-alpha-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.The Journal of Immunology 02/2002; 168(2):883-9. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: P selectin, expressed on surfaces of activated endothelial cells and platelets, is an adhesion receptor for leukocytes. We report that P selectin-deficient mice, generated by gene targeting in embryonic stem cells, exhibit a number of defects in leukocyte behavior, including elevated numbers of circulating neutrophils, virtually total absence of leukocyte rolling in mesenteric venules, and delayed recruitment of neutrophils to the peritoneal cavity upon experimentally induced inflammation. These results clearly demonstrate a role for P selectin in leukocyte interactions with the vessel wall and in the early steps of leukocyte recruitment at sites of inflammation. These mutant mice should prove useful in deciphering the contributions of P selectin in various inflammatory responses as well as in platelet functions.Cell 09/1993; 74(3):541-54. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)-treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18-deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP-stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18-independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.Blood 02/1990; 75(1):227-37. · 9.06 Impact Factor
Leukocyte-inspired biodegradable particles that
selectively and avidly adhere to inflamed
endothelium in vitro and in vivo
Harshad S. Sakhalkar*, Milind K. Dalal*, Aliasger K. Salem†, Ramin Ansari‡, Jie Fu§, Mohammad F. Kiani‡,
David T. Kurjiaka¶, Justin Hanes§, Kevin M. Shakesheff†, and Douglas J. Goetz*?
Departments of *Chemical Engineering and¶Biological Sciences, Ohio University, Athens, OH 45701;†School of Pharmaceutical Sciences, University of
Nottingham, Nottingham NG7 2RD, United Kingdom;‡Department of Biomedical Engineering, University of Tennessee Health Science Center,
Memphis, TN 38163; and§Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved October 29, 2003 (received for review March 12, 2003)
We exploited leukocyte–endothelial cell adhesion chemistry to
generate biodegradable particles that exhibit highly selective ac-
cumulation on inflamed endothelium in vitro and in vivo. Leuko-
adhesion to inflamed endothelium, relative to noninflamed endo-
thelium, under in vitro flow conditions similar to that present in
blood vessels, a 6-fold higher adhesion to cytokine inflamed
endothelium relative to non-cytokine-treated endothelium in vivo,
and a 10-fold enhancement in adhesion to trauma-induced in-
flamed endothelium in vivo due to the addition of a targeting
ligand. The leukocyte–inspired particles have adhesion efficiencies
similar to that of leukocytes and were shown to target each of the
major inducible endothelial cell adhesion molecules (E-selectin,
P-selectin, vascular cell adhesion molecule 1, and intercellular
adhesion molecule 1) that are up-regulated at sites of pathological
inflammation. The potential for targeted drug delivery to inflamed
endothelium has significant implications for the improved treat-
ment of an array of pathologies, including cardiovascular disease,
arthritis, inflammatory bowel disease, and cancer.
[i.e., vascular cell adhesion molecule (VCAM)-1, E-selectin,
P-selectin, and intercellular adhesion molecule (ICAM)-1] is
increased at sites of pathological inflammation. For example,
VCAM-1 is present in a localized fashion on aortic endothelium
that overlies early foam cell lesions (1) and is increased on
endothelium in models of colitis (2). P-selectin and E-selectin
are up-regulated in a variety of pathological settings, including
ischemia-reperfusion injury (3), arthritis (4), and colitis (5).
ICAM-1 expression is increased in ischemia-reperfusion injury
(3) and at sites of radiation-induced inflammation (6, 7). Addi-
tionally, the expression of these ECAMs may be increased on
endothelium within tumors (e.g., ref. 8).
These observations have led to a strong interest in the
development of drug delivery strategies that exploit the in-
creased expression of ECAMs to achieve selective delivery to
sites of diseased tissue (9–12). Drug carriers made from biode-
gradable polymers [e.g., poly(lactic acid), PLA] are easily pre-
pared, have a long shelf life, can carry several orders of magni-
tude more drug than a mAb, and can be designed to have well
defined drug-release rates (13–15). Because of these attributes,
it is well accepted that biodegradable drug carriers complement
and expand the possibilities of targeted drug delivery afforded
by other carriers (e.g., liposomes and mAbs) (14).
Recent studies have attempted to develop biodegradable
particles that exhibit selective adhesion to ECAM-expressing
endothelium. Previously, our group passively adsorbed a mAb to
E- and P-selectin onto poly(?-caprolactone) (PCL) particles and
found that the resulting mAb-coated PCL particles exhibited
selective adhesion to cells expressing E- and P-selectin (16).
However, the adhesion was low (?0.17% that of leukocytes) and
he expression of endothelial cell adhesion molecules
(ECAMs) known to play a role in leukocyte recruitment
occurred only at shear stresses ?0.3 dynes?cm2, a level of shear
that is not considered physiologically relevant (16). The limited
adhesion appeared to be due to a low level of mAb adsorbed to
the particles. Another group has conjugated SLex(a carbohy-
drate ligand for selectins) to biodegradable particles and found
that the SLexparticles roll on polystyrene surfaces coated with
purified P-selectin (17). However, the adhesion of the particles
to endothelial cells in vitro and the interaction of the particles
with the vasculature in vivo were not determined. It should also
be noted that neither of the polymers used previously (16, 17)
had incorporated stealth chemistry [e.g., poly(ethylene glycol)
(PEG)] that would presumably be needed to achieve reasonable
circulation times in vivo (18, 19). Thus, to date the goal of
developing biodegradable particles that avidly and selectively
adhere to inflamed endothelium has remained elusive.
In this paper, we describe the development of targeted,
similar to that of leukocytes. The targeted particles exhibit
(i) significant (up to 15-fold) selective adhesion to inflamed
endothelium, relative to noninflamed endothelium, under phys-
iologically relevant in vitro flow conditions, (ii) significant (6-
fold) selective adhesion for cytokine inflamed endothelium,
relative to non-cytokine-treated endothelium, in vivo, and (iii)
significant (up to 10-fold) enhancement in adhesion to trauma-
induced inflamed endothelium, in vivo, due to the addition of a
targeting ligand. We demonstrate that the particles can be made
to target all of the major inducible ECAMs (E-selectin, P-
selectin, VCAM-1, and ICAM-1) that have been shown to be
up-regulated in a host of disease settings (1–7). The design of
these particles and this targeting approach in general, is inspired
by the leukocyte–endothelial cell biochemistry that mediates the
selective recruitment of leukocytes to a site of inflammation.
Thus, by bridging the fields of drug delivery and vascular
cell–cell adhesion, we have successfully developed PEGylated
biodegradable polymeric particles that exhibit selective adhesion
to inflamed endothelium at levels similar to that of leukocytes.
Materials and Methods
Materials. PBS and Hanks’ balanced salt solution (HBSS) with
Ca2?and Mg2?(HBSS?) were from BioWhittaker (Walkers-
ville, MD). BSA was from Sigma and added to PBS to make the
blocking buffer (PBS, 1% BSA). FBS was added to HBSS?, 1%
BSA to make the assay buffer (HBSS?, 1% BSA, 2% FBS).
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: LEAP, leukocyte–endothelial cell adhesive particle; ECAM, endothelial cell
adhesion molecule; TNF-?, tumor necrosis factor ?; HUVEC, human umbilical vein endo-
thelial cells; PEG, poly(ethylene glycol); PLA, poly(lactic acid); FACS, fluorescence-activated
cell sorting; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion
?To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
December 23, 2003 ?
vol. 100 ?
no. 26 ?
Neutravidins were from Molecular Probes. Recombinant mouse
tumor necrosis factor ? (TNF-?) was from Calbiochem. All
reagents for culturing and activating human umbilical vein
endothelial cells (HUVEC) were as described (20).
Biotin–caproyl–protein A was from Accurate Chemical and
Scientific (Westbury, NY). The 19.ek.Fc PSGL-1 construct is a
chimera consisting of the first 19 aa of mature PSGL-1 linked to
an enterokinase cleavage site, which, in turn, is linked to the Fc
region of human IgG1. Fc liberated by enterokinase (ek.Fc)
served as a negative control. The 19.ek.Fc, ek.Fc, and murine
mAb HPDG2?3 (anti-human P-selectin; IgG1) were provided by
Raymond T. Camphausen (Wyeth Research, Cambridge, MA)
and have been described (21, 22). Murine mAb KPL-1 (anti-
human PSGL-1; IgG1), murine biotinylated mAb 68–5H11
(anti-human E-selectin; IgG1), rat biotinylated mAb 10E9.6
(anti-murine E-selectin; IgG2a), biotinylated rat IgG2a, and
biotinylated mouse IgG1 were from Pharmingen. Murine bio-
tinylated mAb 15.2 (anti-human ICAM-1; IgG1) and biotin-
ylated mAb 1.G11B1 (anti-human VCAM-1; IgG1) were from
HUVEC Culture. HUVEC were purchased from Clonetics (San
Diego, CA) and maintained in culture as described (21).
HUVEC were activated by 4-h treatment with 0.25 ng?ml IL-1?
or 25 ng?ml TNF-?. Note that, in preliminary experiments, we
characterized the adhesion molecule profile on unactivated, 4-h
IL-1?-, and 4-h TNF-?-activated HUVEC by using ELISA. The
adhesion molecule profiles on our HUVEC, described in Results
and Discussion, are similar to that reported by others (23).
PLA–PEG–Biotin Synthesis. We generated a PLA–PEG–biotin
polymer for the present study as described (24, 25). In brief, 1 g
of ?-amine-?-hydroxy PEG (Shearwater Polymers, average mo-
lecular mass, 3.4 kDa) was stirred with 0.250 g of N-
hydroxysuccinimide–biotin (Sigma) and 80 ?l of triethylamine
in 1 ml of dichloromethane and 2 ml of acetonitrile at room
temperature under argon overnight. Biotinylated PEG was
isolated by dissolving in hot isopropanol and then cooling. The
resulting precipitate was collected by vacuum filtration and dried
from toluene azeotrope. Second, 2.0 g of D,L-lactide (Purac,
Lincolnshire, IL) was polymerized with 0.35 g of ?-hydroxy
PEG-biotin by using 0.1 g of stannous 2-ethyl hexanoate (Sigma)
as the initiator by reflux in silanized glassware containing 25 ml
of anhydrous toluene to yield PLA–PEG–biotin. The final
polymeric material was recovered by dissolution in 10 ml of
dichloromethane and precipitation in 200 ml of cold ether.
PLA–PEG–Biotin Particle Synthesis. Particles were produced by
using a single emulsion technique in which 10 ml of a 25 mg?ml
solution of the polymer in dichloromethane was homogenized
for 2 min in 250 ml of a 0.1% (wt?vol) aqueous poly(vinyl
alcohol) (PVA) solution (PVA 88% hydrolyzed, PolySciences,
Warrington, PA). In certain cases, rhodamine was added to the
organic phase to produce rhodamine-loaded PLA–PEG parti-
cles. The resulting emulsion was stirred for 4 h at room tem-
perature in a chemical fume hood to allow the dichloromethane
to evaporate. Particles were collected by centrifugation, washed
performed with a Coulter Multisizer II (Beckman Coulter).
Batches with number average diameters between 1.0 and 2.5 ?m
were used in this study.
Ligand Conjugation to PLA–PEG–Biotin Particles. PLA–PEG–biotin
particles were incubated (7.5 ? 107particles per ml, 37°C for 20
min) in PBS containing 50 ?g?ml neutravidin. Neutravidin
Texas Red was used when preparing PLA–PEG particles for the
in vitro adhesion assays, and neutravidin Oregon Green was used
when preparing PLA–PEG particles for the 19.ek.Fc in vivo
assays. All other preparations were done with unconjugated
neutravidin. PLA–PEG–biotin particles with incorporated rho-
damine were used in the TNF-? in vivo experiments. Neutravi-
din-conjugated PLA–PEG particles were washed and incubated
(2 ? 107particles per ml, 24°C, 30 min) in PBS containing a
biotinylated mAb (various concentrations), biotinylated mouse
or rat IgG (30 ?g?ml; 60 ?g?ml), or biotinylated protein A (50
the resulting mAb or IgG-conjugated PLA–PEG particles were
washed with blocking buffer and held in blocking buffer at room
temperature (?4 h) until used in the fluorescence-activated cell
sorting (FACS) or adhesion assays. Washed protein A PLA–
PEG particles were incubated (4 ? 107particles per ml, 24°C,
1 h) in blocking buffer containing 17 ?g?ml of the 19.ek.Fc or
ek.Fc construct. The resulting suspension was stored overnight
at 4°C before performing the in vivo assays. For the FACS assays,
the 19.ek.Fc and ek.Fc particles were washed in blocking buffer
and immediately used.
Flow Cytometric Analysis. mAb conjugated PLA–PEG particles
were incubated (24°C, 20 min) with a FITC-conjugated goat
F(ab?)2 polyclonal anti-mouse (Caltag, Burlingame, CA) or
anti-rat antibody (Jackson ImmunoResearch), washed and fixed
in HBSS? containing 1% formaldehyde. 19.ek.Fc and ek.Fc
PLA–PEG particles were incubated (24°C, 20 min) with mAb
KPL-1 or HPDG2?3, washed, incubated (24°C, 20 min) with an
FITC polyclonal antibody to mouse IgG, washed and fixed in
HBSS? containing 1% formaldehyde. Fluorescence of PLA–
PEG particles was determined by using a FACSort flow cytom-
eter (Beckon Dickinson). To gain insight into the number of
mAbs bound to the PLA–PEG particles, Quantum Simply
Cellular beads (Bangs, Fishers, IN), which have a known number
of binding sites for mouse IgG, were used to calibrate the flow
cytometer as per the manufacturer’s instructions.
In Vitro Adhesion Assay.Aparallelplateflowchamber(Glycotech,
Rockville, MD), similar to that described by Smith and col-
leagues (26), was used in this study. Our particular set up has
been described (20). An image intensifier was used to enhance
detection of the PLA–PEG particles. A suspension of PLA–PEG
particles (6 ? 105in assay buffer) was drawn over the HUVEC
at 1.5 dynes?cm2. After 2.5 min of flow, the number of PLA–
different fields of view under fluorescence illumination. These
numbers were averaged and normalized to the area of the field
of view to give an n of 1. The results of n replicate experiments
were averaged to give the data presented.
In Vivo Adhesion Assay. We used intravital microscopy as de-
scribed (27). Briefly, animals were intubated, catheterized, and
placed on a surgical board where the right cremaster muscle was
pinned as a flat sheet. In certain cases, the mice were given an
intrascrotal injection of TNF-? (500 ng in saline) 2 h before the
surgery. Approximately 20 min after surgery, the PLA–PEG
particles were injected (femoral artery for the trauma model and
jugular vein for the TNF-? model). The number of adherent
(either rolling or firmly adherent) PLA–PEG particles was
determined by observing postcapillary venules through an in-
travital microscope by using fluorescent illumination and nor-
malized to the length of the venule, for firm adhesion, and the
length of the venule and the time of observation, for rolling
adhesion. Data were collected from n ? 3 separate mice for each
condition. These values were averaged to obtain the data
Statistical Analysis. Student’s t test was used to analyze the
difference between two means. Multiple comparisons against a
single control were evaluated by using ANOVA and, subse-
www.pnas.org?cgi?doi?10.1073?pnas.2631433100 Sakhalkar et al.
quently, a Bonferroni test was used to determine statistical
significance. In all instances, comparisons with P values ?0.05
were considered statistically significant. All error bars represent
Results and Discussion
PLA–PEG Particles Conjugated with mAbs to Inducible ECAMs Exhibit
Significant Selective Adhesion to Inflamed HUVEC Under Physiologi-
cally Relevant in Vitro Flow Conditions. We sought to determine
whether biodegradable particles conjugated with ligands to
inducible ECAMs could exhibit selective adhesion to inflamed
endothelium in vitro. We used HUVEC as our model endothelial
cell because HUVEC are of human origin, well characterized,
widely used to investigate vascular events in vitro (e.g., articles
citing ref. 28), and can be treated with cytokines to generate an
in vitro model of inflamed endothelium (23). The adhesion
molecule profile on the HUVEC used in this study, as deter-
mined by ELISA (data not shown), is similar to that reported by
others (23). Specifically, HUVEC cultured in the absence of
activating agents (e.g., IL-1? or TNF-?), have little, if any,
surface expression of E-selectin, VCAM-1, or P-selectin, but do
express ICAM-1. A 4-h treatment of HUVEC with the proin-
flammatory cytokines TNF-? or IL-1? induces E-selectin ex-
pression and increases ICAM-1 expression. A 4-h treatment of
HUVEC with TNF-? induces significant and reproducible
VCAM-1 expression. We did not detect P-selectin surface
expression on 4-h IL-1?- or TNF-?-treated HUVEC.
a biodegradable block copolymer consisting of biotinylated PEG
and PLA blocks. The phase separation of PEG and PLA upon
particle preparation using emulsion methods (13) ensures that
the particle surface is rich in biotinylated PEG, allowing facile
linkage of targeting moieties, via a neutravidin bridge, to the
particles at high densities. We conjugated mAbs to E-selectin,
determined by FACS (Fig. 1), the level of conjugated mAb
reached very high densities (in most cases ?200,000 mAbs per
particle) and was a function of the concentration of mAb used
during the coupling procedure. Thus, ECAM ligands are easily
coupled to the PLA–PEG particles and, by simply varying the
concentration of ligand in the coupling procedure, the ligand
density on the PLA–PEG particles can be controlled.
We next tested the adhesion of the mAb-conjugated PLA–
PEG particles to HUVEC in vitro. Studies focused on under-
standing leukocyte adhesion to the endothelium, a process
somewhat analogous to drug carrier adhesion to the endothe-
lium, have clearly revealed that the local fluid dynamics, in
particular the shear stress at the leukocyte–endothelial cell
interface, can greatly influence adhesion. Indeed, adhesion
events that are operative under low fluid shear conditions may
not occur under physiologically relevant fluid shear conditions
(30). Thus, we investigated the adhesion of the PLA–PEG
particles to HUVEC under in vitro flow conditions that mimic
fluid dynamic conditions present in vivo. Specifically, we used an
in vitro flow chamber that is routinely used to study leukocyte
adhesion to the endothelium under flow (26, 31).
In separate assays, we perfused the anti-E-selectin (?-E),
anti-VCAM-1 (?-V), and the anti-ICAM-1 (?-I) PLA–PEG
particles over inflamed HUVEC at physiologically relevant
levels of fluid shear. As shown in Fig. 2, each of the leukocyte–
endothelial cell adhesive particles (LEAPs) exhibited significant
levels of adhesion to inflamed HUVEC. The nature of the
adhesion was biphasic, wherein particles traveling at the free
stream hydrodynamic velocity abruptly attached to the inflamed
HUVEC and subsequently remained firmly adherent with no
rolling observed. This behavior is in contrast to leukocytes that
typically exhibit a rolling behavior before firm adhesion (30, 32).
The adhesion of the mAb PLA–PEG particles, or LEAPs, was
strikingly more avid than what was achieved previously with
passive adsorption of mAbs to biodegradable particles (16).
Specifically, the LEAPs exhibited significant adhesion to in-
flamed HUVEC at a physiologically relevant level of fluid shear
(1.5 dynes?cm2). With the previous system (16), adhesion only
occurred at ?0.3 dynes?cm2, which is significantly less than
physiologically relevant (i.e., ?1.0 dyne?cm2).
Neutrophils have exquisite biochemical and biophysical fea-
tures that impart the ability to efficiently attach (i.e., tether) to
inflamed endothelium under flow. Thus, neutrophil adhesion to
inflamed HUVEC provides a standard for characterizing the
level of adhesion of the LEAPs. We use our previous estimate
of neutrophil primary attachment to inflamed endothelium (16)
to calculate that the LEAPs achieve a level of attachment
between 16% and 38% that of neutrophils. Although this
represents a significant advance (?100-fold increase) relative to
LEAPs are as efficient as neutrophils at attaching to inflamed
HUVEC under flow. To make the detailed comparison, it is
important to realize that the primary mechanism by which
particles are delivered to within molecular distance of the
HUVEC monolayer in a flow chamber is the settling of the par-
ticles in response to the acceleration of gravity (33). The settling
velocity scales with D2, where D is the diameter of the particle
(34). Thus, all else being equal, more neutrophils (8-?m diam-
eter) will be delivered to within molecular distance of the
HUVEC monolayer and have an opportunity to attach relative
to the 2.5-?m-diameter LEAPs. Accounting for this difference
in transport by using an established mathematical model (33)
reveals that the efficiency of attachment of the LEAPs is equal
number of LEAPs and neutrophils are delivered to within
molecular distance of inflamed HUVEC, the number of LEAPs
that will attach to the HUVEC is equal to or exceeds the number
of neutrophils that will attach. Thus, the LEAPs appear to be as
efficient as neutrophils in regards to their ability to attach to
inflamed HUVEC under physiologically relevant levels of fluid
shear. Here, we have demonstrated biodegradable polymeric
particles. Separate aliquots of PLA–PEG particles, precoupled with neutravi-
respectively). Subsequently, the particles were treated with a FITC labeled
antibody and analyzed with FACS. PLA–PEG particles not treated with the
FITC-labeled antibody gave results similar to the top histogram (data not
shown). The 0.5 ?g?ml concentration was only used for anti-ICAM-1. Results
typical of n ? 5 experiments. Calibration with Quantum Simply Cellular beads
indicated that the 30 ?g?ml anti-VCAM-1, anti-ICAM-1, and anti-E-selectin
PLA–PEG particles had ?200,000 bound ligands.
mAbs to E-selectin, VCAM-1, and ICAM-1 can be coupled to PLA–PEG
Sakhalkar et al.
December 23, 2003 ?
vol. 100 ?
no. 26 ?
particles that have adhesion efficiencies to inflamed HUVEC on
par with leukocytes at physiologically relevant levels of fluid
The adhesion of the LEAPs to inflamed HUVEC was signif-
icantly higher than (i) the adhesion of the LEAPs to nonin-
flamed HUVEC (Fig. 2) and (ii) the adhesion of IgG (a negative
control) PLA–PEG particles to inflamed HUVEC (Fig. 2).
There are two key parameters that can be used to characterize
the effectiveness of the targeting. One parameter, the selectivity,
is the ratio of the number of particles delivered to the target,
inflamed endothelium (for the in vitro model, 4-h cytokine-
activated HUVEC) relative to the number of particles delivered
to the noninflamed endothelium (for the in vitro model,
HUVEC not treated with proinflammatory cytokines). The
other parameter, the ligand efficiency, is the ratio of the number
endothelium relative to the number of nontargeted particles
(e.g., IgG PLA–PEG particles) delivered to the inflamed endo-
thelium. We plotted the selectivity and ligand efficiency as a
function of the concentration of mAb used in preparing the mAb
PLA–PEG particles (Fig. 3).
The selectivity of LEAPs for inflamed endothelium was the
greatest when VCAM-1 or E-selectin was targeted, yielding a
maximal selectivity between 12 and 15 (Fig. 3A). Targeting
ICAM-1 resulted in a rather modest selectivity of ?2 (Fig. 3A).
This modest selectivity with ?-I-LEAPs reflects the fact that
noninflamed HUVEC express a basal level of ICAM-1. The
selectivity of ?-V-LEAPs and ?-E-LEAPs was a function of the
concentration of mAb used in the conjugation (Fig. 3A). In
contrast, the selectivity of ?-I-LEAPs was independent of the
concentration of anti-ICAM-1 used in the conjugation (Fig. 3A).
The ligand efficiency plot (Fig. 3B) revealed that, for all of the
LEAPs, (i) the addition of the mAb to the PLA–PEG particles
had a dramatic effect on the adhesion to inflamed HUVEC with
the ligand efficiency reaching maximal values between 27 and 33
and (ii) the ligand efficiency was a function of the mAb con-
centration used during the conjugation.
Combined, the results described above clearly demonstrate
that (i) the efficiency of adhesion of LEAPs to inflamed
HUVEC is on par with leukocytes, (ii) LEAPs exhibit significant
selective adhesion (up to 15-fold) for inflamed HUVEC relative
to noninflamed HUVEC, and (iii) addition of a targeting ligand
significantly increases (up to 33-fold) the efficiency of adhesion
of PLA–PEG particles for inflamed HUVEC. Importantly, the
LEAPs exhibited this adhesive behavior under physiologically
relevant fluid shear conditions.
PLA–PEG Particles Conjugated with a Recombinant PSGL-1 Construct
in Vivo. Although the in vitro flow chamber model captures
several of the characteristics of the in vivo environment, it is
(negative control), were perfused over HUVEC in an in vitro flow chamber. The number of LEAPs adherent to the HUVEC was determined after 2.5 min of flow.
Black bars indicate adhesion to 4 h cytokine (IL-1? in A and C or TNF-? in B) activated HUVEC. Gray bars indicate adhesion to unactivated HUVEC; ?g?ml is the
concentration of ligand (either a mAb or mouse IgG) used during the conjugation procedure. Ligand indicates which molecule was coupled to the PLA–PEG
of the LEAPs to cytokine activated HUVEC was a function of the concentration of mAb used in the coupling procedure. *, P ? 0.05 compared to LEAPs over
unactivated HUVEC (gray bars); #, P ? 0.05 compared to 30 ?g?ml mouse IgG PLA–PEG particles over 4 h IL-1? or TNF-?-activated HUVEC.
LEAPs exhibit selective adhesion to cytokine activated HUVEC. Separate sets of PLA–PEG particles, conjugated with a mAb to an ECAM or mouse IgG
The selectivity, defined as the ratio of the number of LEAPs that adhere to
inflamed HUVEC relative to the number of LEAPs that adhere to noninflamed
for ICAM-1. The selectivity for VCAM-1 and E-selectin was a function of the
concentration of anti-VCAM-1 and anti-E-selectin used in the conjugation,
whereas the selectivity for ICAM-1 appears to be independent of concentra-
adhere to inflamed HUVEC relative to the number of IgG PLA–PEG particles
used in the conjugation. The ligand efficiency was a function of the concen-
tration of mAb and reached a maximum value between 27 and 33. Open
The selectivity and ligand efficiency of LEAP adhesion to HUVEC. (A)
www.pnas.org?cgi?doi?10.1073?pnas.2631433100 Sakhalkar et al.
clearly an approximation. Thus, we used intravital microscopy,
a technique commonly used to study leukocyte adhesion to the
endothelium in vivo, to determine whether the LEAPs could
exhibit adhesion to inflamed endothelium in vivo. Exteriorizing
internal tissues (e.g., cremaster muscle) is known to lead to
P-selectin expression in the postcapillary vessels and subsequent
leukocyte rolling that is almost exclusively mediated by P-
selectin within the first hour after exteriorization of tissue (35,
36). The primary leukocyte ligand for P-selectin is PSGL-1 (22,
37). We have previously established that a recombinant PSGL-1
construct, termed 19.ek.Fc (consisting of the first 19 aa of
PSGL-1 linked to an enterokinase cleavage site that is, in turn,
liked to human Fc), binds to P-selectin in vitro and in vivo (21,
27, 38). The PSGL-1 portion of the 19.ek.Fc construct can be
cleaved by enterokinase, leaving the Fc region (21). The
the 19.ek.Fc construct (21, 27). We used these constructs and the
trauma-activated model of inflammation to determine whether
the LEAPs could exhibit significant adhesion in vivo.
FACS revealed that the 19.ek.Fc construct was coupled to the
PLA–PEG particles (Fig. 4A). Murine cremaster muscle was
exteriorized and prepared for visualization via intravital micros-
copy. Suspensions of 19.ek.Fc-LEAPs or ek.Fc PLA–PEG par-
ticles (negative control) were injected directly into the blood
stream of groups of mice. The interaction of the particles with
the postcapillary venules was observed via intravital microscopy.
Approximately 10-fold more 19.ek.Fc-LEAPs exhibited an ad-
hesive interaction with the vessel wall compared to the negative
control (i.e., ek.Fc) PLA–PEG particles (Fig. 4B). The majority
of the adhesive 19.ek.Fc-LEAPs exhibited a rolling adhesive
behavior (Fig. 4C) that was characterized by a slow nonconstant
velocity translation in the direction of flow (39). Thus, addition
of the PSGL-1 peptide to the PLA–PEG particles resulted in a
?10-fold increase in the adhesion of the PLA–PEG particles to
the inflamed endothelium, with the majority of the adhesive
19.ek.Fc-LEAPs exhibiting a rolling adhesion. Here, we have
to inflamed endothelium in vivo and demonstrated rolling
biodegradable particles in vivo.
PLA–PEG Particles Conjugated with a mAb to E-Selectin Exhibit
Significant Selective Firm Adhesion to TNF-?-Inflamed Endothelium in
Vivo. It is difficult to compare LEAP adhesion to inflamed
endothelium and LEAP adhesion to noninflamed endothelium
by using 19.ek.Fc and the trauma model described above. To
make this comparison, we investigated the adhesion of ?-E-
LEAPs in a TNF-?-induced model of inflammation (40).
Groups of mice were given an intrascrotal injection of TNF-?,
which elicits E-selectin expression (40), or no treatment. Two
hours later, the cremaster muscle was prepared for observation
and suspensions of ?-E-LEAPs or IgG PLA–PEG particles
(negative control) were injected. The number of adherent par-
ticles in the postcapillary venules was determined 10 min after
As shown in Fig. 5, 6-fold more ?-E-LEAPs were adherent in
TNF-?-treated mice compared to the number adherent in mice
not treated with TNF-?, and 4.7-fold more ?-E-LEAPs were
adherent in TNF-?-treated mice compared to the number of IgG
PLA–PEG particles adherent in TNF-?-treated mice. Thus, the
selectivity was 6 and the ligand efficiency was 4.7. In contrast to
the rolling adhesive behavior observed in the previous experi-
ments (Fig. 4), the ?-E-LEAPs exhibited a biphasic adhesive
behavior, wherein particles traveling at the free-stream hydro-
dynamic velocity abruptly adhered and subsequently remained
firmly adherent with no rolling observed. Once firmly adherent,
the ?-E-LEAPs remained adherent for the entire observation
period (as long as 25 min). Note that the LEAPs we used in the
present study are probably not endocytosed by the endothelium
because of their size (41).
Combined, the data presented in Figs. 4 and 5 clearly dem-
onstrate in vivo targeting to inflamed endothelium with selec-
tivity as high as 6 and ligand efficiency as high as 10. In addition,
the results demonstrate that different types of adhesion can be
achieved (i.e., rolling and firm adhesion) and suggest that the
nature of the adhesion can be controlled by particle design.
microvascular endothelium in vivo. (A) PLA–PEG particles, precoupled with
either 19.ek.Fc (a recombinant PSGL-1 construct) or ek.Fc (negative control),
were treated with a mAb to human PSGL-1 (KPL-1) or human P-selectin
(HPDG2?3), washed, treated with an FITC-labeled polyclonal antibody, and
(Middle) 19.ek.Fc-LEAPs treated with a mAb to P-selectin (isotype-matched
control mAb). (Bottom) ek.Fc PLA–PEG particles treated with a mAb to PSGL-1
(KPL-1). Results shown are typical of n ? 2 separate experiments. (B) 19.ek.Fc
of rolling particles was determined. A significantly greater number of rolling
(C and D) A segment of a postcapillary venule from a typical experiment is
shown. (C) Three images were taken 1 s apart and superimposed to generate
the composite image. The white sphere (marked by the arrows) is a 19.ek.Fc-
LEAP rolling along the wall of the venule. (D) Two images were taken 1?30th
of a second apart and superimposed to generate the composite image. The
white blur is a PLA–PEG particle(s) not interacting with the vessel wall.
PSGL-1-conjugated PLA–PEG particles adhere to trauma-activated
in vivo. Mice were given an intrascrotal injection of TNF-? or no injection.
Approximately 2 h later, ?-E-LEAPs or rat IgG PLA–PEG particles (negative
control) were injected into the mice (5 ? 106per mouse), and the number of
adherent particles was observed in the postcapillary venules of the cremaster
muscle. A significantly greater number of ?-E-LEAPs were adherent in TNF-?-
of ?-E-LEAPs were adherent in TNF-?-pretreated mice compared to rat IgG
PLA–PEG particles. All adherent particles were firmly adherent (i.e., not roll-
ing). Ligand indicates which molecule was coupled to the PLA–PEG particles,
of mice with TNF-? 2 h before the experiment (?) or no pretreatment (?);
*, P ? 0.05 compared to right bars. FACS revealed that the ?-E-mAb can be
conjugated to PLA–PEG particles (data not shown).
?-E-LEAPs exhibit selective adhesion to TNF-? inflamed endothelium
Sakhalkar et al.
December 23, 2003 ?
vol. 100 ?
no. 26 ?
Leukocyte–endothelial cell adhesion chemistry and techniques
proven indispensable for unraveling the mechanisms of leuko-
cyte recruitment to sites of inflammation were applied to guide
our design and testing of novel leukocyte-inspired biodegradable
particles. This approach resulted in the generation of PEGylated
biodegradable particles that exhibit highly selective accumula-
tion on inflamed endothelium in vitro and in vivo. The LEAPs
exhibit (i) in vitro attachment efficiencies to inflamed endothe-
lium on par with that of leukocytes, (ii) up to 15-fold selective
adhesion to inflamed endothelium under physiologically rele-
vant in vitro flow conditions, (iii) a 6-fold selective adhesion for
cytokine inflamed endothelium, relative to non-cytokine-
treated endothelium, in vivo, and (iv) up to 10-fold enhancement
in adhesion to trauma-induced inflamed endothelium in vivo due
to the addition of a targeting ligand.
These results bode well for this targeting approach. However,
it is important to point out other issues involved in drug delivery.
For example, although the first step was to demonstrate selective
adhesion to inflamed versus noninflamed endothelium and to
characterize the adhesion, another key issue is to optimize
selectivity for inflamed endothelium versus uptake by the re-
ticuloendothelial system. Additionally, the possibility of un-
wanted effects of the particles (e.g., whether the LEAPs elicit an
inflammatory response) needs to be addressed. Nevertheless,
this work is clearly a significant step forward. The approach
presented here and the reagents generated will foster the
rational and efficient development of drug delivery schemes that
seek to target drugs to diseased tissues via the heterogeneous
expression of endothelial surface moieties. As the endothelium
continues to be mapped, it is anticipated that opportunities for
targeted drug delivery via the endothelium will further increase.
A concerted and vigorous effort between investigators from a
variety of disciplines, as has occurred in the field of leukocyte
adhesion, will be integral to the full exploitation of these
We thank Dr. Takahiro Morita for assistance with particle preparation
and Dr. Sriram Neelamegham and Yi Zhang (Department of Chemical
Engineering, State University of New York, Buffalo) for useful discus-
sions and calculations of particle transport within the flow chamber. This
work was supported by grants from The Whitaker Foundation (to
D.J.G.), National Science Foundation Grants BES 9733542?0096303 (to
D.J.G.) and BES0090009 (to M.F.K. and D.J.G.), the American Heart
Association (M.F.K.), and a Merck Junior Faculty Development Award
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www.pnas.org?cgi?doi?10.1073?pnas.2631433100Sakhalkar et al.