Shell cross-linked nanoparticles designed to target angiogenic blood vessels via αvβ3 receptor-ligand interactions

Article abstract of DOI:10.1021/ma048824e

The design, synthesis, and characterization of a novel polymeric nanostructured material bearing surface-attached integrin antagonists are demonstrated. The covalent coupling of a complex and biologically active small molecule to a well-defined nanostructured material combines the elegance of synthetic organic chemistry with the state-of-the-art polymer chemistry. This unique material offers potential in targeting to tumor neovasculature and delivery of diagnostic and therapeutic agents.

Full text of DOI:10.1021/ma048824e

Macromolecules 2004, 37, 7109-7115
7109
Shell Cross-Linked Nanoparticles Designed To Target Angiogenic Blood
Vessels via Rvâ3 Receptor-Ligand Interactions
Dip a n ja n P a n , J effr ey L. Tu r n er , a n d Ka r en L. Wooley*
Washington University in Saint Louis, Center for Materials Innovation and Department of Chemistry,
One Brookings Drive, CB 1134, Saint Louis, Missouri 63130-4899
Received J une 14, 2004; Revised Manuscript Received J uly 14, 2004
ABSTRACT: The design, synthesis, and characterization of a novel polymeric nanostructured material
bearing surface-attached integrin antagonists are demonstrated. The covalent coupling of a complex and
biologically active small molecule to a well-defined nanostructured material combines the elegance of
synthetic organic chemistry with the state-of-the-art polymer chemistry. This unique material offers
potential in targeting to tumor neovasculature and delivery of diagnostic and therapeutic agents.
inhibitor free, Aldrich) was dried over calcium hydride and
distilled from sodium/benzophenone. Isoprene was purified by
passing through a neutral alumina column to remove the
inhibitor. N,N-Dimethylformamide (DMF, 99.8%) and tert-
butyl acrylate (98%) were purchased from Aldrich Chemical
Co. and distilled over calcium hydride prior to use. Nitrogen
(UHP, 99.99%) was used for polymerization and storage of
materials. Flash column chromatography was performed using
32-63 D 60 Å silica gel from ICN SiliTech (ICN Bio-
medicals GmbH, Eschwege, Germany). Spectra/Por membrane
(MWCO: 12 000-14 000 Da) used for dialysis was obtained
from Spectrum Medical Industries, Inc. (Laguna Hills, CA).
In tr od u ction
For the general detection, diagnosis, and treatment
of tumors, targeting of the vasculature has received
considerable attention. Identification of cell surface
receptors that are overexpressed and ligands that can
interact selectively with those receptors1 is a key area
in which rapid developments are being assisted by
advances in proteomics and genomics and by the
development of analytical tools. From the many pos-
sibilities, the integrin Rvâ3, which is found in large
numbers upon the endothelial cells of many angiogenic
tumor-associated blood vessels and is known to play a
key role in cell matrix recognition and facilitate gene
transfer, has been the focus of several studies aimed at
the detection2 and treatment3 of cancers. To accomplish
therapeutic efficacy, Hood et al.4 demonstrated that the
attachment of a ligand that targets Rvâ3 to cationic
nanoparticles allowed for packaging, transport, and
delivery of antiangiogenic genes to Rvâ3-overexpressing
M21 melanoma cells in mice, resulting in tumor regres-
sion. Given the promising in vivo data that were
reported for this Rvâ3 targeting ligand, our interest has
been its implementation as a targeting moiety for well-
defined nanostructured materials, comprised of an
amphiphilic core-shell morphology, which can be uti-
lized for the sequestration of multiple and variable guest
molecules.5
To develop a novel, endothelial cell specific delivery
vehicle, the preparation of a shell cross-linked (SCK)
nanoparticle6,7 bearing an integrin Rvâ3-targeting ligand
(1) was undertaken. The SCK was also labeled fluores-
cently to allow for characterization and tracking of the
nanostructure. Intracellular pH plays an important role
in many cellular events including receptor-mediated
signal transduction, ion transport, endocytosis, cell
adhesion, and various other cellular processes.8 In
recent years, pH-sensitive fluorescent dyes are often
applied to monitor changes in intracellular pH.
Mea su r em en ts. 1H NMR (300 MHz) and 13C NMR (75
MHz) were recorded as solutions on a Varian Mercury 300
MHz spectrometer with the solvent signal as standard.
Hydrodynamic diameter distribution and distribution averages
for the SCKs in PBS solutions were determined by dynamic
light scattering. A Brookhaven Instruments Co. (Holtsville,
NY) DLS system equipped with a model BI-9000AT digital
correlator, a model EMI-9865 photomultiplier, and a model
95-2 Ar ion laser (Lexel Corp., Fremont, CA), operated at 514.5
nm, was used. Measurements were made at 20 ( 0.1 °C.
Nanoparticles were dialyzed into 50 mM PBS, pH 7.2, prior
to analysis. Buffered nanoparticle solutions were either cen-
trifuged in a model 5414 microfuge (Brinkman Instrument Co.,
Westbury, NY) for 4 min or filtered through 0.22 µm poly-
(vinylidene fluoride) and 0.1 µm poly(ether sulfone) membrane
filters (Pall Gelman, East Hills, NY) to remove dust particles.
Scattered light was collected at a fixed angle of 90°. The digital
correlator was operated with 522 ratio spaced channels, an
initial delay of 1.4 µs, a final delay of 10 ms, and a duration
time of 15 min. A photomultiplier aperture of 400 µm was used,
and the incident laser power was adjusted to obtain a photon
counting rate between 200 and 300 kcps. Only measurements
for which the measured and calculated baselines of the
intensity autocorrelation function agreed to within (0.1% was
used to calculate nanoparticle hydrodynamic diameter values.
All determinations were made in triplicate. The calculations
of the nanoparticle diameter distributions and distribution
averages were performed with the ISDA software package
(Brookhaven Instruments Co.), which employed single-expo-
nential fitting, cumulants analysis, and nonnegatively con-
strained least-squares particle size distribution analysis rou-
tines. Solution state hydrodynamic diameter determination of
the fluoresceinated SCK samples were performed by using
Brookhaven Instruments Co. 90 PLUS particle size analyzer
equipped with a solid-state laser operated at 658 nm. All the
UV-vis spectroscopy data were acquired on a Varian Cary 1E
UV-vis spectrophotometer. SCK solutions (∼0.3 mg/mL) and
a solution of Rvâ3-targeting ligand (0.2 mg/mL) were made in
deionized water prior to the absorbance measurements. All
Exp er im en ta l Section
Ma ter ia ls. Unless otherwise listed, all solvents and re-
agents were purchased from Aldrich Chemical Co. (St. Louis,
MO) and used as received. Tetrahydrofuran (HPLC grade,
* To whom corresponding should be addressed: e-mail:
klwooley@artsci.wustl.edu.
10.1021/ma048824e CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/27/2004

7110 Pan et al.
Macromolecules, Vol. 37, No. 19, 2004
the SCK concentrations have been determined by lypholization
of the aqueous sample solutions. All the fluorescence spec-
troscopy data were acquired on a Varian Cary Eclipse fluo-
rescence spectrophotometer. Each sample was prepared inde-
pendently from a stock solution of 7 at 0.25 mg/mL. Sample
solutions at pH values 5.9, 6.3, 6.9, 7.3, and 7.8 were excited
at λex ) 488 nm. Fluorescence emission spectra (λem ) 517 nm)
from 7 at different pH values in 50 mM sodium phosphate
buffered saline were recorded. Zeta potential (ú) values for the
SCKs were determined with a Brookhaven Instrument Co.
(Holtsville, NY) model Zeta Plus zeta potential analyzer.
Measurements were made following dialysis (MWCO 12-14
kDa dialysis tubing, Spectrum Laboratories, Rancho Domingu-
ez, CA) of SCK solutions into 1 mM KH2PO4, 1 mM KCl, pH
7.1 buffer. Data were acquired in the phase analysis light
scattering (PALS) mode following solution equilibration at 25
°C. Calculation of ú from the measured nanoparticle electro-
phoretic mobility (µ) employed the Smoluchowski equation: µ
) ꢀú/η, where ꢀ and η are the dielectric constant and the
absolute viscosity of the medium, respectively. Measurements
of ú were reproducible to within (2 mV of the mean value
given by 16 determinations of 10 data accumulations. Trans-
mission electron microscopy carbon grids were prepared by
oxygen plasma treatment to make the surface hydrophilic.
Particle samples were diluted 9:1 in water and further diluted
1:1 with a 1% phosphotungstic acid (PTA) stain. Micrographs
were collected at 100 000× magnification. The number-average
particle diameter values and standard deviations were gener-
ated from the analyses of a minimum of 100 particles from
three micrographs. Tapping-mode atomic force microscopy
measurements were conducted in air with a Nanoscope III
BioScope system (Digital Instruments, Santa Barbara, CA)
operated under ambient conditions with standard silicon tips
(type, OTESPA-70; L, 160 µm; normal spring constant, 50 N/m;
resonance frequency, 246-282 kHz). The samples were pre-
pared for AFM analysis by depositing a 2 µL drop of the
solution onto freshly cleaved mica and allowing it to dry freely
in air. The number-average particle height values and stan-
dard deviations were generated from the section analysis of a
minimum of 100 particles from at least five different analysis
regions. SEC was conducted on a Waters Chromatography Inc.
model 150-CV, equipped with a model 410 differential refrac-
tometer, a Precision Detectors Inc. model PD2040 dual-angle
(15° and 90°) light scattering detector, and a three-column
series of Polymer Laboratories Inc. PLgel 10 µm mixed B 300
× 7.5 mm columns. The system was equilibrated at 35 °C in
anhydrous THF, which served as the polymer solvent and
eluent (flow rate set to 1.00 mL/min, then determined gravi-
metrically). Data collection was performed with Precision
Detectors Inc. Precision Acquire software. Data analysis was
performed with Precision Detectors Inc. Discovery 32 software.
Interdetector delay volume and the light scattering detector
calibration constant were determined from a nearly monodis-
this solution was added alkoxyamine (2,2,5-trimethyl-3-(1-
phenylethoxy)-4-phenyl-3-azahexane) (401 mg, 1.23 mmol) and
nitroxide (2,2,5-trimethyl-4-phenyl-3-azahexane 3-nitroxide)
(13 mg, 0.062 mmol). The mixture was allowed to stir at room
temperature for 10 min to ensure homogeneity, prior to
performing three freeze-pump-thaw cycles. After the final
degassing, the tube was sealed, allowed to warm to room
temperature, and placed in an oil bath at 130 °C. After 47 h,
the tube was immersed in an ice bath to quench the polym-
erization. Excess tBA was removed via distillation, and 15 mL
of tetrahydrofuran was added to the tube and allowed to stir
until complete dissolution had occurred. The resultant polymer
was precipitated three times into a 50% methanol/water (1 L)
solution at 4 °C. The white polymer product was collected via
filtration and dried under vacuum overnight to give poly(tert-
NMR
butyl acrylate) (PtBA). Yield: 10.3 g (76%). Mn
Da, Mn
) 10 300
GPC
) 11 100 Da, Mw/Mn ) 1.14. Tg ) 33 °C. IR: 3437,
1
2972, 1723, 1443, 1357, 1252, 1163, 753 cm-1. H NMR (300
MHz, CDCl3, ppm): δ 1.4 (-COOC(CH3)3), 1.6-2.0 (-CH2-
CH(COOC(CH3)3)-), 2.0-2.4 (-CH2-CH(COOC(CH3)3)-), 7.0-
7.2 (aromatic H’s from initiator). 13C NMR (75 MHz, CDCl3,
ppm): δ 28.1, 36.0-39.2, 41.4-43.8, 81.4, 173.8.
P r ep a r a tion of P tBA-b-P I Diblock Cop olym er . A 100
mL Schlenk tube connected to a double manifold under N2,
fitted with a stir bar (oven-dried at 110 °C overnight), was
charged with PtBA macroinitiator (2.11 g, 0.205 mmol) and
placed under vacuum (<0.1 mm Hg) for 24 h. The tube was
backfilled with N2 and fitted with a rubber septum. Isoprene
(20 mL, 200 mmol, freshly distilled from CaH2) was transferred
to the Schlenk tube via cannula followed by the addition of
nitroxide via a gastight syringe. The mixture was allowed to
stir at room temperature for 10 min to ensure homogeneity,
prior to performing three freeze-pump-thaw cycles. After the
final degassing, the tube was sealed, allowed to warm to room
temperature, and placed in an oil bath at 143 °C. After 72 h,
the tube was immersed in an ice bath to quench the polym-
erization. Excess isoprene monomer was removed via distil-
lation, and 25 mL of tetrahydrofuran was added to the tube
and allowed to stir until complete dissolution had occurred.
The resultant polymer was precipitated three times into a 70%
methanol/water (1 L) solution at 4 °C. A white solid was
collected via filtration and dried under vacuum overnight to
NMR
give PtBA-b-PI diblock copolymer. Yield: 2.63 g (77%). Mn
) 16 700 Da, Mn
GPC
) 15 100 Da, Mw/Mn ) 1.11. (Tg)PI ) -74
°C, (Tg)PtBA ) 32 °C. IR: 3437, 3079, 2972, 1723, 2640, 2443,
1357, 1234, 1159, 860, 783, 750, 783 cm-1. 1H NMR (300 MHz,
CDCl3, ppm): δ 1.4 (-COOC(CH3)3), 0.9-2.1 (br, backbone H’s
and side-chain CH3’s), 2.1-2.3 (-CH2-CH(COOC(CH3)3)-),
4.4-5.0 (br, 1,2 -CHdCH2 and 3,4 -C(CH3)dCH2), 5.0-5.2
(1,4-, -CH2-C(CH3)dCH-CH2-), 5.7-5.9 (br, 1,2 -CHd
CH2), 7.0-7.2 (aromatic H’s from end groups). 13C NMR (75
MHz, CDCl3, ppm): δ 15.8, 23.2, 26.5-27.8, 30.7, 39.5, 41.7-
42.1, 80.1-80.8, 124.1-124.8, 127.2-128.2, 130.3, 134.8, 147.9,
173.7-174.
perse polystyrene calibrant (Pressure Chemical Co., Mp
)
90 000 g/mol, Mw/Mn < 1.04). The differential refractometer
was calibrated with standard polystyrene reference material
(SRM 706 NIST), of known specific refractive index increment
dn/dc (0.184 mL/g). The dn/dc values of the analyzed polymers
were then determined from the differential refractometer
response. Glass transition temperatures (Tg) were measured
by differential scanning calorimetry on either a Perkin-Elmer
DSC-4 (polymer samples) or Mettler Toledo DSC822e (SCK).
The Perkin-Elmer instrument was upgraded with an Instru-
ment Specialties (Antioch, IL) temperature program interface
PE, and data were acquired and analyzed with TA-PC software
(version 2.11, Instrument Specialists). Measurements obtained
using the Mettler Toledo DSC822e were analyzed using Mettler
Toledo Star SW 7.01 software. Heating rates for both instru-
ments were 10 °C/min, and the Tg values were taken at the
midpoint of the inflection tangent, upon the third heating scan.
P r ep a r a tion of P tBA Hom op olym er . A 100 mL Schlenk
tube connected to a double manifold under N2, fitted with a
stir bar (oven-dried at 110 °C overnight and flame-dried under
vacuum immediately prior to use), was covered with a rubber
septum and charged with 43 mL (38 g, 300 mmol) of tBA. To
P r ep a r a tion of P AA-b-P I Diblock Cop olym er 3. To a
250 mL two-necked round-bottom flask equipped with a stir
bar and West condenser was added PtBA-b-PI (2.48 g, 0.148
mmol (polymer), 11.5 mmol (ester)). The polymer was dissolved
into an 80:20 toluene:acetic acid (v:v) solution (250 mL) and
allowed to stir for 20 min to ensure homogeneity. To this
solution was added methanesulfonic acid (0.0519 mL, 0.800
mmol) neat via a syringe. The solution was allowed to stir at
reflux and monitored via 1H NMR. The reaction was stopped
after 1.5 h at reflux, and excess solvent was removed under
vacuum. The resultant solid was taken up into tetrahydrofuran
(100 mL), and 100 mL of water was added dropwise over a 30
min period. Purification was afforded through dialysis (MWCO
3500 Da) against water for 4 days. Lyophilization of the
resultant solution afforded pure PAA-b-PI as a colorless solid.
Yield: 1.89 g (100%). (Tg)PI ) -73 °C, (Tg)PAA ) 127 °C. IR:
3100-3600, 3079, 2972, 1680, 1640, 1601, 1443, 1347, 1243,
1212, 860, 783 cm-1. 1H NMR (300 MHz, THF-d8, ppm): δ 1.3-
2.1 (br, backbone H’s and side-chain CH3’s), 2.3-2.5 (-CH2-
CH(COOH)-), 4.5-5.0 (br, 1,2 -CHdCH2 and 3,4-, -C(CH3)d
CH2), 5.2 (1,4 -CH2-C(CH3)dCH -CH2-), 5.0-5.2 (1,4-,

Macromolecules, Vol. 37, No. 19, 2004
Shell Cross-Linked Nanoparticles 7111
Sch em e 1a
a
Reagents and conditions: (a) tetrahydrofuran, followed by addition of water and dialysis against water; (b) 2,2′-
(ethylenedioxy)bis(ethylamine), 1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide, rt, overnight, followed by dialysis
against water; (c) 5, 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimidemethiodide, rt, overnight, followed by dialysis against sodium
phosphate buffered saline at pH 7.2.
-CH2-C(CH3)dCH-CH2-), 5.7-5.9 (1,2-, -CHdCH2), 7.0-
7.2 (aromatic H’s from end groups). 13C NMR (75 MHz, THF-
d8, ppm): δ 15.9, 23.1, 26.7-27.1, 31.2, 35.5-36.2, 40.7-42.0,
80.2-80.8, 124.1-124.9, 127.3-128.8, 130.4, 134.8, 148.9,
175.2.
P r ep a r a tion of P AA-b-P I Micelle 4. Micellization of the
PAA-b-PI diblock copolymer was induced by dissolving the
polymer (950 mg) into tetrahydrofuran (475 mL), followed by
the addition of Nanopure water at a rate of ∼10 mL/h over 2
days. Afterward, a second 500 mL of water was added neat,
and the resulting solution was allowed to stir 2 h before being
transferred to dialysis bags (MWCO 3500 Da), and dialysis
was performed against water for 5 days. Final concentration
) 0.435 mg/mL. Dh (DLS) ) 52 ( 9 nm.
P r ep a r a tion of Sh ell Cr oss-Lin k ed (SCK) Na n op a r -
ticles 2. To a stirred solution of micelles (500 mL, 218 mg,
0.0177 mmol (polymer), 1.4 mmol (acid residues)) was added
a solution of 2,2′-(ethylenedioxy)bis(ethylamine) (0.0190 g,
0.152 mmol) in water (20 mL) dropwise over a 10 min period.
The resultant solution was allowed to stir for 1 h at ambient
temperature. To this solution was added a solution of 1-[3′-
(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (0.0903
g, 0.304 mmol) in water (20 mL) dropwise over 10 min. The
resultant solution was allowed to stir overnight under ambient
conditions. Removal of small molecule contaminants was
achieved through dialysis through a 10-12 kDa MWCO
membrane against pure water for 1 week. DSC (Tg) ) -70 °C
(an endotherm for the transition of the PI core only was
observed). IR (KBr): 3560-3200, 2930, 2440, 1651, 1556, 1450,
1387, 1339, 1155, 1075, 1036, 874 cm-1. 1H NMR [D2O(1):THF-
d8(1), overnight]: δ 0.98-3.3 (aliphatic protons of polymer
backbone), 7.2 ppm (aromatic end groups). DLS volume-
average Dh (λ ) 514.5 nm): 31 ( 1 nm; DLS volume-average
Dh (λ ) 658 nm): 33 ( 1 nm; Dah (TEM): 17 ( 3 nm; Hav
(AFM): 2.7 ( 1 nm; zeta (ú): -26 ( 5 mV.
residues available) in a round-bottom flask was added a
solution of 1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide
methiodide (0.4 mg, 0.0013 mmol) in deionized water (1 mL).
The solution was allowed to stir for 20 min at room temper-
ature. To this reaction mixture was added a solution of 5 (0.3
mg, 6.5 × 10-4 mmol), dissolved in deionized water (1 mL).
The reaction mixture was allowed to react overnight at room
temperature. The solution was then transferred to a presoaked
dialysis membrane tube (MWCO 12-14 kDa) and allowed to
dialyze for 3 days against 50 mM sodium phosphate, 50 mM
sodium chloride, pH 7.2. IR (KBr): 3430, 3070, 2980-2860,
2440, 2390, 2350, 2344, 2300, 1734, 1728, 1710, 1690, 1680,
1650, 1630, 1555, 1536, 1500, 1459, 1438, 1250, 1130, 1050,
990, 950, 860, 810-630 cm-1 1H NMR [D2O(2):THF-d8 (1),
.
overnight]: δ 0.98-3.4 (aliphatic protons along the backbones
of the core polymer chains). DLS volume-average Dh: (λ )
514.5 nm): 42 ( 5 nm; DLS volume-average Dh (λ ) 658 nm):
48 ( 7 nm; Dah (TEM): 21 ( 8 nm; Hav (AFM): 2.8 ( 1 nm;
zeta (ú): -20 ( 3 mV.
F lu or escen ce La belin g of th e rvâ3-Ta r getin g Liga n d
F u n ction a lized Sh ell Cr oss-Lin k ed Na n op a r ticles 7. To
a stirred solution of SCK-Rvâ3 1 (0.20 mg/mL, 0.0043 mmol of
acrylic acid residues available) in a round-bottom flask was
added a solution of 1-[3′-(dimethylamino)propyl]-3-ethylcar-
bodiimide methiodide (0.3 mg, 0.001 mmol) in deionized water
(1 mL). The solution was allowed to stir for 20 min at room
temperature. To this reaction mixture was added a solution
of 6 (0.2 mg, 3.9 × 10-4 mmol), dissolved in deionized water
(1 mL). The reaction mixture was allowed to react overnight
at room temperature. The solution was then transferred to a
presoaked dialysis membrane tube (MWCO 12-14 kDa) and
allowed to dialyze for 7 days against 50 mM sodium phosphate,
50 mM sodium chloride, pH 7.3. DLS volume-average Dh (λ )
658 nm): 46 ( 6 nm; Dah (TEM): 24 ( 8 nm; Hav (AFM): 2.6
( 2 nm; zeta (ú): -27 ( 2 mV.
P r ep a r a tion of th e rvâ3-Ta r getin g Liga n d F u n ction -
a lized Sh ell Cr oss-Lin k ed Na n op a r ticles 1. To a stirred
solution of SCK 2 (0.20 mg/mL, 0.0092 mmol of acrylic acid
Syn th esis of 5- a n d (6)-Ca r boxyflu or escein Su ccin im -
id yl Active Ester (CF -Osu ) 9. To a solution of 5- and (6)-
carboxyfluorescein (8) (0.25 g, 0.66 mmol) in anhydrous DMF

7112 Pan et al.
Macromolecules, Vol. 37, No. 19, 2004
Ta ble 1. Su m m a r y of Ch a r a cter iza tion Da ta for SCK 2,
SCK 1, La beled w ith th e rvâ3 Ta r getin g Liga n d , a n d SCK
7, La beled w ith th e rvâ3 Ta r getin g Liga n d a n d 5- a n d
(6-)Ca r boxyflu or escein
2,2′-(ethylenedioxy)bis(ethylamine) (10) (4.15 mL, 0.0275 mol)
in dioxane (20 mL) was added dropwise at ambient temper-
ature over 20 min a solution of di-tert-butyl dicarbonate (1.02
g, 0.0046 mol) in dioxane (5 mL). The mixture was allowed to
stir for 24 h and concentrated. 50 mL of water was added to
it, and it was rinsed with 50 mL of dichloromethane. The
combined organic layer was dried over MgSO4 and concen-
trated to give 0.44 g of the desired product. Yield: 37%. 1H
NMR (300 MHz, CDCl3, ppm): δ 1.4 (s, 9H, tBu), 1.5 (brs, 2H,
NH2), 2.8 (t, 2H), 3.3 (m, 2H), 3.5-3.6 (m, 4H), 3.7 (s, 4H), 5.2
(brs, 1H, NHBoc).
SCK-Rvâ3-
targeting
ligand (1)
SCK-Rvâ3-targeting
ligand-carboxy
fluorescein (7)
property
SCK (2)
Dh/DLSa
33 ( 6 nm
33 ( 1 nm
17 ( 3 nm
2.7 ( 1 nm
-26 ( 5 mV
42 ( 5 nm
48 ( 7 nm
21 ( 8 nm
2.8 ( 1 nm
-20 ( 3 mV
Dh/DLSb
46 ( 6 nm
24 ( 8 nm
2.6 ( 2 nm
-27 ( 2 nm
D
H
ah/TEM
av/AFM
ú/ZETA
Rea ction of 5- a n d (6)-Ca r boxyflu or escein Su ccin im -
id yl Ester (9) w ith 2-Am in o-2′-[(ter t-bu toxyca r bon yl)-
a m in o]eth ylen e Glycol Dieth yl Eth er (11) 12. To a stirred
solution of CF-Osu (9) (0.05 g, 0.11 mmol) in 4 mL of dry DMF,
a solution of mono-boc-protected diamine (11) was added (0.029
g, 0.12 mmol) in 2 mL of dry DMF. The reaction mixture was
allowed to stir for 3 h and 40 min. Reaction was monitored by
TLC. It was then poured in 10 mL of deionized water, mixed
well, and extracted with ethyl acetate. The organic layer was
washed with water (two times), dried (MgSO4), and evapora-
tion of solvent afforded the crude product. This was purified
by column chromatography (silica gel, chloroform and metha-
a
b
Using an Ar ion laser at λ ) 514.5 nm. Using a solid-state
laser at λ ) 658 nm.
(5 mL) was added EDAC (0.16 g, 0.8 mmol) followed by
N-hydroxysuccinimide (HOSu, 0.095 g, 0.8 mmol). The reaction
was covered with foil, stirred under N2. After 4.5 h, additional
EDAC (0.025 g) was added, and the reaction mixture was
allowed to stir for an additional 18.5 h. Reaction mixture was
rinsed into a separatory funnel with DMF (2-3 mL) and
diluted with acetone (10 mL). Buffer (0.05 M, pH 6.0 phosphate
buffer Na3PO4, 13 mL) was added, and the mixture was
extracted with ether/ethyl acetate (2:1) (50 mL). The combined
organic layer was washed with water three times, dried over
MgSO4, and solvent removed to furnish 0.1 g of crude 5- and
(6)-carboxyfluorescein succinimidyl esters. This compound was
immediately used for the next step without further purifica-
tion. Yield: 32%. 1H NMR (300 MHz, DMSO-d6, ppm): δ 2.88
(s) and 2.93 (s) (total 4H), 6.50-6.60 (m) (total 2H), 6.60-6.75
(m) (total 4H), 7.56 (d, J ) 8.2 Hz) and 8.20 (d, J ) 8.0 Hz)
(total 1H), 7.9 (s) and 8.54 (s) (total 1H), 8.30-8.45 (m) (total
2H), 10.20 (br s) (total 1H).
1
nol; 2:1). Yield: 0.04 g (66%). H NMR (300 MHz, DMSO-d6,
ppm): δ 1.3 (s, 9H), 2.94-3.60 (m, 12H), 6.50-6.75 (m, 4H),
7.35 (d, J ) 8.2 Hz) and 8.24 (d, J ) 8.0 Hz) (total 1H), 7.62
(s) and 8.42 (s) (total 1H), 8.05 (d, J ) 8.0 Hz) and 8.21 (d, J
) 8.0 Hz) (total 2H), 8.67 (m) and 8.80 (m) (total 1H), 10.20
(brs) (total 1H).
Dep r otection of th e ter t-Boc: P r ep a r a tion of Ca r -
boxyflu or escein a m in e (6). tert-Boc-protected fluorescein-
amine (12) (20 mg) was taken in a round-bottom flask, and to
it 3-4 mL of a 1:2 mixture of dichloromethane and trifluoro-
acetic acid (cold) was added and allowed to stir for 1 h at 0-5
°C and an additional 2.5 h at ambient temperature. Excess
P r ep a r a tion of 2-Am in o-2′-[(ter t-bu toxyca r bon yl)a m i-
n o]eth ylen e Glycol Dieth yl Eth er (11).9 To a solution of
F igu r e 1. (a, b, c) Transmission electron microscopy images of 2, 1, and 7, respectively. The samples were prepared by addition
of 1% phosphotungstic acid, followed by drop deposition from the aqueous solution upon a carbon-coated copper grid and allowing
to dry freely in air. (d, e, f) Tapping-mode AFM images of 2, 1, and 7, respectively. The samples were prepared by drop deposition
from aqueous solution onto freshly cleaved mica and allowing to dry freely in air.

Macromolecules, Vol. 37, No. 19, 2004
Shell Cross-Linked Nanoparticles 7113
Resu lts a n d Discu ssion
As is illustrated in Scheme 1, the SCK nanoparticles
(2) were prepared in two steps from the diblock copoly-
mer poly(acrylic acid-b-isoprene) (3, PAA78-b-PI97).10
Briefly, a diblock copolymer composed of polyisoprene
(1,4-isomer predominant) and poly(tert-butyl acrylate)
blocks was synthesized by sequential nitroxide-mediated
radical polymerization (NMRP). The nitroxide and
alkoxyamine initiator were prepared and utilized in the
polymerization of tert-butyl acrylate according to the
method of Hawker and co-workers.11 The resultant
alkoxyamine end-functionalized PtBA homopolymer was
then used as a macroinitiator for the polymerization of
isoprene to obtain the diblock copolymer (poly(tert-butyl
acrylate)-b-polyisoprene). The tert-butyl ester groups
were hydrolyzed by treatment of the polymer with
methanesulfonic acid in a mixture of acetic acid and
toluene at reflux to produce the amphiphilic diblock
copolymer 3.10b,d The hydrolysis reaction was continued
for 1.5 h and was monitored by the disappearance of
F igu r e 2. Overlaid UV-vis absorption spectra of (a) SCK 2
(0.3 mg/mL), (b) SCK 1, labeled with Rvâ3 targeting ligand (0.3
mg/mL), and (c) Rvâ3-targeting ligand (5) (0.2 mg/mL).
trifluoroacetic acid and dichloromethane were removed by
rotary evaporator. It was then dissolved in excess amount of
water and lyophilized. The crude product was then purified
by flash column chromatography (silica, chloroform: methanol
1:1). Yield: (0.01 g) 63%. 1H NMR (300 MHz, DMSO-d6,
ppm): δ 2.82-3.60 (m, 12H), 6.46-6.75 (m, 4H), 7.35 (d, J )
8.2 Hz) and 8.22 (d, J ) 8.0 Hz) (total 1H), 7.62 (s) and 8.42
(s) (total 1H), 8.70 (brs) (total 1H), 8.10 (d, J ) 8.0 Hz) and
8.20 (d, J ) 8.0 Hz) (total 2H), 8.67 (m) and 8.80 (m) (total
1H). MALDI-TOF (matrix: R-cyanocinnamic acid): (M + H)+
507.9 (observed) (calculated 507.3).
1
tert-butyl groups resonances in the H NMR spectros-
copy. An aqueous solution of micelles 4 composed of
PAA78-b-PI97 were formed by controlled addition of
deionized water to a solution of 3 in THF, followed by
dialysis against deionized water. The 20% cross-linking
of the total acrylic acid residues constituting the micel-
lar periphery was accomplished by condensation reac-
tions with 2,2′-(ethylenedioxy)bis(ethylamine) as a di-
amino cross-linker (0.22 mol equiv, amine-to-acid ratio),
Sch em e 2a
a
Reagents and conditions: (a) 6, 1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide, rt, overnight, followed by dialysis
against sodium phosphate buffered saline at pH 7.3, 7 days.

7114 Pan et al.
Macromolecules, Vol. 37, No. 19, 2004
lic acid groups of the SCKs with 1-[3′-(dimethylamino)-
propyl]-3-ethylcarbodiimide methiodide (0.14 equiv based
upon the remaining acrylic acid residues) was followed
by the addition of 5 (0.07 equiv amine-to-acid ratio), and
the reaction was allowed to proceed overnight at ambi-
ent temperature in aqueous solution. This resulted in
the attachment of nominally 530 ligands per SCK
nanoparticle. The SCKs (1), labeled with 5, were puri-
fied by exhaustive dialysis using a 12-14 kDa MWCO
membrane against sodium phosphate buffered saline at
pH 7.2.
Detailed characterization of these nanoparticles was
performed by multiple analytical techniques. Dynamic
light scattering (DLS) measurements were made to
obtain the hydrodynamic diameters (Dh) of the micelles
and SCKs in aqueous solution. The SCKs exhibited a
slight increase in Dh upon functionalization with the
Rvâ3 targeting ligand (Table 1). Transmission electron
microscopy (TEM) and atomic force microscopy (AFM)
gave smaller diameter and height values, respectively,
in comparison to the DLS Dh values, obtained in a
hydrated state.
These findings are consistent with the expected
reduction in SCK dimensions due to the dehydration of
the SCK shell and also to the effects of particle shape
distortion that occur as the fluidlike polyisoprene-filled
SCKs flatten upon adsorption onto the substrates
required for TEM and AFM imaging.10b,c
F igu r e 3. Fluorescence emission spectra (excitation wave-
length λex ) 488 nm) from 7 at different pH values in 50 mM
sodium phosphate buffered saline. Inset: plot of fluorescence
emission intensities from 7 (at λem ) 517 nm) against pH. Each
sample was prepared independently from a stock solution at
0.25 mg/mL.
facilitated by 1-[3′-(dimethylamino)propyl]-3-ethylcar-
bodiimide methiodide (0.22 mol equiv relative to the
carboxylic acids). The SCK nanoparticles were purified
by dialysis, using a 10 kDa molecular weight cutoff
(MWCO) cellulose membrane, against deionized water.
The peptidomimetic integrin antagonist, Rvâ3-target-
ing ligand, 5, was prepared starting from commercially
available taurine following reported procedures4,12 and
was then attached within the shell of the SCK (2).
Activation of a fraction of the available surface carboxy-
Zeta potential values (ú) of these nanoparticles were
also determined by electrophoretic light scattering
measurements (Table 1). In each case, the ú value was
Sch em e 3a
a
Reagents and conditions: (a) N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydrochloride, N-hydroxysuccinimide (HOSu),
anhy DMF, rt, 23 h; (b) 2-amino-2′-[(tert-butoxycarbonyl)amino]ethylene glycol diethyl ether (11), DMSO, rt, 3 h 40 min; (c)
trifluoroacetic acid (TFA):dichloromethane (2:1), rt, 3.5 h; (d) di-tert-butyl dicarbonate (tBoc2O), dioxane, rt, 24 h.

Macromolecules, Vol. 37, No. 19, 2004
Shell Cross-Linked Nanoparticles 7115
negative, owing to the presence of remaining carboxy-
late groups on the SCK surface. UV-vis spectroscopy
of the functionalized nanoparticles demonstrated the
successful covalent attachment of ligand 5 (Figure 2).
Even though the SCK 2 lacks significant concentra-
tions of UV chromophores, there is a shoulder in the
UV-vis spectrum that results from the presence of the
aromatic rings as polymer chain termini and/or scat-
tering of light from the nanoparticles. Upon reaction
with 5, the intensity of this shoulder (λmax ) 250)
increases, indicating the conjugation to produce 1.
Fluorescence labeling of the nanoparticles was achieved
by activating a portion of the available carboxylic acid
groups of 1 upon reaction with 1-[3′-(dimethylamino)-
propyl]-3-ethylcarbodiimide methiodide (0.23 mol equiv
based upon the remaining acrylic acid residues) and
then allowing reaction to occur with the 5- and (6-)-
carboxyfluorescein derivative (6) (0.1 mol equiv, amine-
to-acid ratio) overnight at ambient temperature. Fluo-
resceinated SCKs (7) were purified by exhaustive dialysis
through 12-14 kDa MWCO membrane against sodium
phosphate buffered saline at pH 7.3 for a prolonged
period of time (Scheme 2). This resulted in the attach-
ment of nominally 710 fluoresceins per SCK nanopar-
ticle.
gineering, for providing access to the red laser DLS
facility.
Refer en ces a n d Notes
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DLS studies showed that there is no significant
change in Dh after the fluorescence tagging (Table 1).
Fluorescence spectroscopy, performed as a function of
pH, illustrated the pH dependence of the emission
intensity from 7 (Figure 3). The carboxyfluorescein
derivative, 6, was also synthesized in a straightforward
way from the commercially available mixture of 5- and
(6-)carboxyfluorescein (8) (Scheme 3).
Con clu sion s
In summary, a peptidomimetic Rvâ3-targeting ligand
was synthesized, well-defined SCKs were prepared by
the cross-linking of supramolecular assemblies of am-
phiphilic block copolymers, and these two components
were then conjugated to produce a nanoscale vessel that
is decorated with biologically active ligands. With the
additional introduction of a pH-sensitive fluorophore,
which can act as an optical marker, these materials are
now undergoing advanced characterization studies to
probe their behavior when challenged under in vitro and
in vivo conditions. Studies are in progress to test the
efficacy of this integrin mediated polymeric drug deliv-
ery vehicle in targeting tumor vasculature.
Ack n ow led gm en t. This project has been funded in
whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health, under
Contract N01-CO-27103 and by a NIH Chemistry-
Biology Interface Pathway Training Grant Fellowship
for J .L.T. (5T32GM08785-03). Mass spectrometry was
provided by the Washington University Mass Spectrom-
etry Resource with support from the NIH National
Center for Research Resources (Grant P41RR0954). The
authors are also thankful to Mr. G. Michael Veith,
Washington University, Department of Biology, for
TEM imaging and Professor Shelly E. Sakiyama-Elbert,
Washington University, Department of Biomedical En-
(12) (a) Duggan, M. E.; Duong, L. T.; Fisher, J . E.; Hamil, T. G.;
Hoffman, W. F.; Huff, J . R.; Ihle, N. C.; Leu, C. T.; Nagy, R.
M.; Perkins, J . J .; Rodan, S. B.; Wesolowski, G.; Whitman,
D. B.; Zartman, A. E.; Rodn, G. A.; Hartman, G. D. J . Med.
Chem. 2000, 43, 3736. (c) Egbertson, M. S.; Homnick, C. F.;
Hartman, G. D. Synth. Commun. 1993, 23, 703.
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