Determination of the bioavailability of biotin conjugated onto shell cross-linked (SCK) nanoparticles

Article abstract of DOI:10.1021/ja039647k

Shell cross-linked nanoparticles (SCKs) presenting surface- and bioavailable biotin functional groups were synthesized via a mixed micelle methodology, whereby co-micellization of chain terminal biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) and nonbiotinylated PAA-b-PMA were cross-linked in an intramicellar fashion within the shell layer of the mixed micelles, between the carboxylic acid groups of PAA and the amine functionalities of 2,2′-(ethylenedioxy)diethylamine. The hydrodynamic diameters (D_h) of the micelles and the SCKs with different biotinylated block copolymer contents were determined by dynamic light scattering (DLS), and the dimensions of the SCKs were characterized with tapping-mode atomic force microscopy (AFM) and transmission electron microscopy (TEM). The amount of surface-available biotin was tuned by varying the stoichiometric ratio of the biotinylated PAA-b-PMA versus the nonbiotinylated PAA-b-PMA, as demonstrated with solution-state, binding interaction analyses, an avidin/HABA (avidin/4′-hydroxyazobenzene-2-carboxylic acid) competitive binding assay, and fluorescence correlation spectroscopy (FCS). The avidin/HABA assay found the amount of available biotin at the surface of the biotinylated SCK nanoparticles to increase with increasing biotin-terminated block copolymer incorporation, but to be less than 25% of the theoretical value. FCS measurements showed the same trend.

Full text of DOI:10.1021/ja039647k

Published on Web 05/07/2004
Determination of the Bioavailability of Biotin Conjugated onto
Shell Cross-Linked (SCK) Nanoparticles
†
‡
§
Kai Qi, Qinggao Ma, Edward E. Remsen, Christopher G. Clark, Jr., and
Karen L. Wooley*
Contribution from the Center for Materials InnoVation and Department of Chemistry,
Washington UniVersity, One Brookings DriVe, Saint Louis, Missouri 63130-4899
Received November 17, 2003; E-mail: klwooley@artsci.wustl.edu
Abstract: Shell cross-linked nanoparticles (SCKs) presenting surface- and bioavailable biotin functional
groups were synthesized via a mixed micelle methodology, whereby co-micellization of chain terminal
biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) and nonbiotinylated PAA-b-PMA were
cross-linked in an intramicellar fashion within the shell layer of the mixed micelles, between the carboxylic
acid groups of PAA and the amine functionalities of 2,2′-(ethylenedioxy)diethylamine. The hydrodynamic
h
diameters (D ) of the micelles and the SCKs with different biotinylated block copolymer contents were
determined by dynamic light scattering (DLS), and the dimensions of the SCKs were characterized with
tapping-mode atomic force microscopy (AFM) and transmission electron microscopy (TEM). The amount
of surface-available biotin was tuned by varying the stoichiometric ratio of the biotinylated PAA-b-PMA
versus the nonbiotinylated PAA-b-PMA, as demonstrated with solution-state, binding interaction analyses,
an avidin/HABA (avidin/4′-hydroxyazobenzene-2-carboxylic acid) competitive binding assay, and fluores-
cence correlation spectroscopy (FCS). The avidin/HABA assay found the amount of available biotin at the
surface of the biotinylated SCK nanoparticles to increase with increasing biotin-terminated block copolymer
incorporation, but to be less than 25% of the theoretical value. FCS measurements showed the same
trend.
Introduction
interest in preparing nanoparticles conjugated with biomol-
2
7,28
29-31
32-39
ecules
for biomimetics,
targeted delivery,
bio-
Functionalization of nanoparticles enables tuning of the
interactions among themselves as well as with their surrounding
environment, by which controlled formation of supramolecular
architectures can be achieved.1-10 These nanostructured materi-
als have potential applications for the construction of devices
with unique optical,
catalytic properties.2
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10.1021/ja039647k CCC: $27.50 © 2004 American Chemical Society
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9
6599

A R T I C L E S
Qi et al.
detection,4
0-44
diagnosis, and treatment purposes,33,45-49 each
confinement versus flexibility of the partially crosslinked
7
2,73
of which depends on the biomolecule nanoparticle conjugates
remaining bioactive and bioavailable after conjugation.
Shell cross-linked nanoparticles (SCKs),5
polymer chains constituting the SCK shell,
the mobility of
7
4,75
the entire nanostructure,
tional groups.
and the composition of the func-
0-58
a class of well-
The interaction of biotin and avidin as a ligand-receptor pair,
widely used in the field of biology and medicine for purification,
localization, and diagnostics, has served as a well-defined model
defined, polymeric, nanostructured materials with a hydrophobic
core domain and a hydrophilic shell layer, have received recent
attention as biocompatible59 and stable nanoscale scaffolds
60
76,77
6
1-64
system to probe bioavailability.
Applications utilizing protein
from which bioactive elements can be presented.
The
recognition of biotinylated species, including small molecules,
polymers, lipids, nucleic acids, proteins, and nanoparticles, have
been extended to fabricate novel nanoscopic assemblies, such
general methodology that has been developed for the preparation
of SCKs involves the supramolecular assembly of block
copolymers into polymer micelles,6
5-68
followed by covalent
78
as two-dimensional arrays of biotin-avidin conjugates, protein-
cross-linking reactions throughout the shell layer. Synthetic
strategies for surface functionalization of SCKs have also been
7
9
80
polymer amphiphiles, protein-lipid monolayers, protein
8
1
82
6
9
70,71
multilayers, protein-polymer multilayers, protein-DNA
established, based upon mixed micelle formation
or
19,83
84-87
postpreparation functionalization reactions.64 In each case, the
multilayers,
protein-nanoparticle composites,
and func-
among others. Biotinylated nanopar-
and microparticles96,97 with sizes ranging from
globular proteins to cells, have been utilized as model systems
8
8-92
tionalized surfaces,
ticles,
determination of the surface- and bioavailability of the functional
groups covalently linked within the hydrogel-like shell layer
of the SCKs remains challenging, due to the interplay of the
9
3-95
3
0,98-100
to study and mimic the multivalent interactions
that occur
in protein-cell recognition and cell-cell adhesion pro-
cesses.
Given the high application potential and fundamental sig-
nificance of biotinylated nanoparticles, the synthesis and study
of biotinylated SCKs were undertaken. Interest in these materials
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VOL. 126, NO. 21, 2004

Bioavailability of Biotin on SCK Nanoparticles
A R T I C L E S
Scheme 1
Figure 1. 1H NMR spectra of (a) biotin, 4, (b) biotinylated alcohol, 5, and
(c) biotinylated initiator, 3, in CD3OD.
Scheme 2
is based primarily upon their value in the determination of the
surface- and bioavailability of functional groups that are
incorporated into the SCK nanostructure via a mixed micelle
methodology. The mixed micelle methodology, nominally, is a
co-micellization process involving amphiphilic block copoly-
mers, at least one of which contains a biotin unit as the
hydrophilic chain terminus. It is expected that an advantage of
the mixed micelle strategy is the ability to control the degree
of surface coverage by the biotin functional groups, via control
over the stoichiometric ratio of the chain end functionalized
and nonfunctionalized, amphiphilic block copolymers. Although
the placement of the functionality is at the hydrophilic chain
end, the actual location of this functionality with respect to the
SCK’s surface will depend on many factors. These include
conformations of the polymer chains, which are dependent upon
the nature of the chain end functionality, the composition of
the block copolymers, the conditions employed for micelle
formation, and the reaction conditions during shell cross-
In addition, the hydrophilic block segment that carried the biotin
unit was lengthened to enhance its presentation on the surfaces
of the micelles and the corresponding SCKs. To further increase
the solubility of the biotin functional group in aqueous media
and to reduce interparticle aggregation, which could potentially
result from the functionalized chain end, an ethylene oxide linker
was used.107
Therefore, amphiphilic diblock copolymers, poly-
acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) with, 1,
(
and without, 2, a biotin chain terminus, were prepared for co-
micellization, followed by intramicellar cross-linking. Nano-
structures were prepared using 0%, 0.2%, 1%, 2%, 10%, 40%,
and 100% biotinylated block copolymer. These ratios were
selected to span from low numbers of biotin to full biotinylation.
The synthesis of block copolymer, 1, having a biotin unit at
the hydrophilic chain terminus, utilized a biotinylated initiator,
3, for atom transfer radical polymerization (ATRP) (Scheme
2). Biotin, 4, was first activated by 1,1′-carbonyldiimidazole,
followed by coupling to a hydrophilic linker 2-(2′-aminoethoxy)-
ethanol to form the biotinylated alcohol, 5. Esterification of 5
with 2-bromo-2-methyl propionic acid, mediated by 4-(N,N-
dimethylamino)pyridine (DMAP) and 1,3-dicyclohexylcarbo-
diimide (DCC), afforded 3 in 43% yield after purification by
1
02-104
linking.
It was hypothesized that at least a portion of the
functional groups, which are biotin in this particular case, will
be surface-exposed after both micellization and shell cross-
linking. The results of the present study support this hypothesis
based on detailed solution-state, binding interaction analyses,
employing an avidin/HABA (avidin/4′-hydroxyazobenzene-2-
carboxylic acid) competitive binding assay and fluorescence
correlation spectroscopy (FCS).
Results and Discussion
The preparation of biotinylated SCK nanoparticles via the
mixed micelle methodology involves a combination of co-
micellization and covalent stabilization within the shell layer
1
flash chromatography. The composite of H NMR spectra
(Figure 1) indicates the formation of 3, wherein the methylene
resonance for the protons labeled as Hj of 5 shifted downfield
to 4.2 ppm, overlapping with the protons He and Hf on the biotin
unit, upon formation of 3. The appearance of the singlet
resonating at 1.9 ppm confirmed the presence of the isobutyryl
methyl groups of 3. The urea protons on biotin were not
observed, due to the rapid proton exchange with the solvent,
CD3OD.
Preparation of the block copolymers, 9 and 10, was ac-
complished by sequential ATRP of tert-butyl acrylate and
methyl acrylate, initiated from 3 and 6, respectively (Scheme
(Scheme 1). The block copolymer precursors were designed to
be composed of similar hydrophobic and hydrophilic block
segment compositions and lengths to provide a uniform distribu-
105,106
tion of the mixed polymer chains throughout the micelles.
(
(
(
(
(
102) Ma, Q.; Remsen, E. E.; Clark, C. G., Jr.; Kowalewski, T.; Wooley, K. L.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5058-5063.
103) Ma, Q.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Am. Chem.
Soc. 2001, 123, 4627-4628.
104) Huang, H.; Kowalewski, T.; Remsen, E. E.; Gertzmann, R.; Wooley, K.
L. J. Am. Chem. Soc. 1997, 119, 11653-11659.
105) Tian, M.; Qin, A.; Ramireddy, C.; Webber, S. E.; Munk, P.; Tuzar, Z.;
Proch a´ zka, K. Langmuir 1993, 9, 1741-1748.
106) Qin, A.; Tian, M.; Ramireddy, C.; Webber, S. E.; Munk, P.; Tuzar, Z.
Macromolecules 1994, 27, 120-126.
(107) B u¨ t u¨ n, V.; Wang, X. S.; de Paz B a´ n˜ ez, M. V.; Robinson, K. L.; Billingham,
N. C.; Armes, S. P.; Tuzar, Z. Macromolecules 2000, 33, 1-3.
J. AM. CHEM. SOC.
9
VOL. 126, NO. 21, 2004 6601

A R T I C L E S
Qi et al.
Scheme 3
Figure 2. 1H NMR spectra of (a) biotinylated PtBA, 7, and (b) nonbio-
tinylated PtBA, 8, in CDCl3.
Table 1. Molecular Weights and Molecular Weight Distributions
for Biotinylated and Nonbiotinylated PtBAm Homopolymers and
1
PtBAm-b-PMAn Diblock Copolymers by H NMR and SEC
polymer
ma
na
M
n
(1H NMR)b
M
n
(SEC)
w n
M /M
7
8
9
110
102
110
102
0
17 200
12 500
25 200
15 400
14 400
13 200
20 100
18 100
1.27
1.22
1.01
1.09
0
66
57
10
Figure 3. 1H NMR of spectra (a) biotinylated PtBA-b-PMA, 9, and (b)
nonbiotinylated PtBA-b-PMA, 10, in CDCl3. A magnified view of proton
resonances from the biotin unit is also shown.
a
Number of tert-butyl acrylate repeating units and methyl acrylate
repeating units based on SEC characterization. b Molecular weights of
biotinylated polymers were determined by comparison of the integration
area of the average of the protons on the biotinylated initiator at the chain
end to that of the resonance of the methine groups on the polymer backbone
between 2 and 2.4 ppm, in the 1H NMR spectra. Molecular weights of
nonbiotinylated polymers were determined by comparison of the integration
area of the average of the methylene proton resonance of the ethyl ester
groups and the methine group at the chain ends to that of the resonance of
the methine groups on the polymer backbone between 2 and 2.4 ppm, in
3). Polymerization of tert-butyl acrylate initiated by 3, using
CuBr/N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA)
as the catalyst/ligand system, was allowed to proceed at 55 °C
to 76% conversion to give a 56% yield of biotinylated poly-
(tert-butyl acrylate) (PtBA) homopolymer, 7. A small amount
1
the H NMR spectra.
of N,N-dimethylformamide was added to solvate 3 and provide
for a homogeneous polymerization mixture. Growth of the
methyl acrylate chain segment from 7 was performed in bulk,
in the presence of CuBr/PMDETA at 50 °C, and was allowed
to proceed to <10% conversion, giving biotinylated poly(tert-
butyl acrylate)-b-poly(methyl acrylate) (PtBA-b-PMA) diblock
copolymer, 9, in 41% yield. The synthesis of the nonbiotinylated
PtBA homopolymer, 8, and the corresponding PtBA-b-PMA
diblock copolymer, 10, followed a similar route, using ethyl
block copolymers, 1 and 2, was then accomplished by lyoph-
ilization (Scheme 3). The selective cleavage of the tert-butyl
groups was demonstrated by the disappearance of the tert-butyl
1
proton resonance at 1.42 ppm in the H NMR spectra and by
the disappearance of the tert-butyl group stretching bands at
-
1
1393 and 1367 cm in the IR spectra. In addition, the
broadening of the carbonyl stretching band and the absorption
-
1
from 3500 to 2500 cm , characteristic of carboxylic acids,
indicated the formation of PAA from PtBA. Although a number
of solvents and solvent mixtures were employed (e.g., DMSO,
THF/D2O), upon cleavage of the tert-butyl groups of 9 to form
amphiphilic block copolymer, 1, the resonances for the protons
2
-bromopropionate, 6, as the ATRP initiator. The presence of
the biotin functional group at the chain terminus was confirmed
1
by H NMR spectroscopy for the biotinylated homopolymer,
7
, and the biotinylated diblock copolymer, 9, which are shown
1
in Figures 2 and 3, in comparison to their nonbiotinylated
of the biotin unit were no longer visible by H NMR spectros-
analogues 8 and 10, respectively. In Figure 3, a magnified view
of a region of the H NMR spectrum for the biotinylated diblock
copy. The lack of observation of the chain end unit is consistent
with a solubility behavior for a polymer having very different
chain segment compositions. The primary ester linkage between
the biotin unit and the polymer is stable under the TFA reaction
conditions, which was confirmed by model studies whereby the
biotinylated initiator treated under the same conditions for the
cleavage of biotinylated PAA-b-PMA, 9, was found to undergo
1
copolymer is provided to illustrate the resonances for Hc, Hd,
He, and Hf of the biotin unit. The molecular weights and
molecular weight distributions of polymers, 7-10, were deter-
mined by size exclusion chromatography (SEC), equipped with
multiangle laser light scattering and refractive index detection.
1
1
The molecular weights were also determined via H NMR end
no cleavage, as observed by H NMR spectroscopy. Further-
group analysis. The results are summarized in Table 1.
The tert-butyl ester groups on 9 and 10 were cleaved
selectively by reaction with trifluoroacetic acid (TFA) in
dichloromethane for ca. 14 h at room temperature. After removal
of the solvent and excess TFA, the residue was dissolved in
THF, and the amphiphilic block copolymers were purified by
dialysis against deionized water (cellulose membrane dialysis
tubing, MWCO 6000-8000 Da). Isolation of the amphiphilic
more, confirmation of the persistence of the biotin chain end
unit was made through assays that identified its presence on
the surface of the micelles and SCK nanostructures, as described
below. Glass transition temperatures Tg of 1, 2, and 7-10 were
measured by differential scanning calorimetry (DSC) (Table 2).
The micellization process followed a two-step procedure.
Amphiphilic block copolymer PAA-b-PMA was dissolved in
THF (a solvent for the PMA and PAA segments), followed by
6602 J. AM. CHEM. SOC.
9
VOL. 126, NO. 21, 2004

Bioavailability of Biotin on SCK Nanoparticles
A R T I C L E S
Table 2. Glass Transition Temperatures for Biotinylated and
Nonbiotinylated Homopolymers and Diblock Copolymers
Characterized by DSC
T
g
(°C)a
T
g
(°C)a
T
g
(°C)a
polymer
(PtBA)
(PMA)
(PAA)
7
8
9
45
43
41
43
not observed
16
not observed
13
1
1
2
0
124
123
a
Measurements were performed with a heating rate of 10 °C/min under
N2 flow. Tg was taken as the midpoint of the inflection tangent upon the
third or subsequent heating scans.
Table 3. Mixed Micelle Formation and DLS Characterization for
the Mixed Micelles
biotinylated
THF
2
H O
micelle
h
D (nm)
block copolymer amount amount amount amount concentration (number-
a
micelle content (mol %) of 1 (mg) of 2 (mg) (mL)
(mL)
(mg/mL)
average)
11
12
13
14
15
0%
1%
2%
10%
40%
0
60.0
58.6
58.0
62.0
18.6
60
60
60
64
30
60
60
60
64
30
0.261
0.253
0.245
0.271
0.267
26 ( 5
27 ( 2
28 ( 1
29 ( 3
32 ( 4
0.7
1.3
6.8
12.0
a
Biotinylated block copolymer content was calculated on the basis of
the molar percentage of the biotinylated PAA-b-PMA block copolymer to
the nonbiotinylated PAA-b-PMA block copolymer.
Figure 4. Intensity autocorrelation functions for the 2% biotinylated SCK,
18 (dots), and the CONTIN fit (line) at 30° scattering angle, and the residual
plots for the corresponding CONTIN fit are also shown.
the gradual addition of deionized water (a nonsolvent for PMA)
into the polymer THF solution. Normally, equal volumes of
water and THF were added to obtain spherical micelles with
good reproducibility. The micelle stability was further estab-
lished upon dialysis of the mixed H2O/THF solution against
deionized water to remove the THF solvent. The calculated
concentration of the micelle solution was determined by
measuring the final volume of the micelle obtained together
with the initial weight of the polymer precursors used. By
controlling the initial molar ratio of the biotinylated PAA-b-
PMA to the nonbiotinylated PAA-b-PMA, a series of micelles
and mixed polymer micelles, 11-15, were formed, with a
theoretical biotinylated chain incorporation of 0%, 1%, 2%,
excellent agreement between CONTIN-computed autocorrela-
tion functions and experimental autocorrelation functions as
exemplified by the representative results shown for the 2%
biotinylated SCK, 18, in Figure 4. Small residuals found for
the CONTIN analyses and depicted graphically in Figure 4
-4
produced RMS errors on the order of 10 for all SCKs studied.
The intensity-average diameter distribution provided by
CONTIN was geometrically transformed, assuming a spherical
shape for the SCKs, into a volume-average diameter distribution.
This was accomplished by dividing the volume fraction of
nanoparticles, V(D), with diameter, D, in the intensity-average
diameter distribution by the product of the angular Mie scattering
coefficient, P(θ), for the nanoparticle and the cube of its
10%, and 40%, respectively. The hydrodynamic diameters (Dh)
3
of the micelles, 11-15, were determined by dynamic light
scattering (DLS) (Table 3).
diameter, D . Under the assumption that all particles in the
volume-average distribution have the same density, the volume-
average distribution is equivalent to the weight-average diameter
distribution.
The resulting volume-average diameter distribution was
transformed into a number-average diameter distribution by
Intramicellar cross-linking of the polymer micelles was
achieved by intermolecular bond formation between the car-
boxylic acid groups in the polymer micelle shell layers, by
activation with 1-[3′-(dimethylamino)propyl]-3-ethylcarbodi-
imide methiodide and reaction with 2,2′-(ethylenedioxy)dieth-
ylamine. Each of the micelles was cross-linked at a calculated
mean-cross-linking density of 60%, based on the stoichiometry
of amine functional groups of the diamino cross-linker to the
carboxylic acid groups from the PAA-b-PMA block copolymers.
The SCKs, 16-20, having varying degrees of biotinylated block
copolymer content, were purified by dialysis against deionized
water (cellulose membrane tubing, MWCO 6000-8000 Da) to
remove the unreacted cross-linker and byproducts. The amide
bond formation was confirmed by IR spectroscopy with the
3
dividing V(D) for volume-average diameter distribution by D .
The use of the volume- and number-average hydrodynamic
diameter distributions afforded mean volume and number
hydrodynamic diameters, Dh,volume and Dh,number, respectively.
The ratio, Dh,volume/Dh,number, served as a measure of polydis-
persity for the DLS-derived hydrodynamic diameter distribution
that was not skewed by the minute fraction of aggregates present
in aqueous solutions of the SCKs. As shown in Table 4, Dh,volume/
Dh,number ranged from 1.11 to 1.24 across the set of SCKs,
indicating that the distributions of hydrodynamic diameter were
narrow as shown in Figure 5. Measurements of hydrodynamic
diameter distributions conducted at two additional angles, 30°
and 125°, confirmed the finding of narrow hydrodynamic
diameter distributions for all SCKs studied. In the case of
micelles and SCKs prepared from 100% biotinylated PAA-b-
-1
introduction of amide I and II bands at ca. 1640 and 1560 cm .
DLS characterization of SCKs in aqueous solution provided
intensity autocorrelation functions that were deconvoluted into
intensity-average hydrodynamic diameter distributions using
CONTIN. The reliability of the analysis was confirmed by the
J. AM. CHEM. SOC.
9
VOL. 126, NO. 21, 2004 6603

A R T I C L E S
Qi et al.
Table 4. DLS Characterization for the Biotinylated and the
Table 5. Size Characterization Data for the SCKs
Nonbiotinylated SCKs
biotinylated
biotinylated
SCK
D
h
(nm)
D
h
(nm)
D
h
(nm)
block copolymer
content (mol %)
Dh
a
(nm)
Hav
b
(nm)
Dav
b
(nm)
Dav (nm)
c
block copolymer concentration (intensity- (volume- (number-
SCKa content (mol %)
D
h,volume
/
SCK
(DLS)
(AFM)
(AFM)
(TEM)
(mg/mL)
average) average) average)
D
h,number
16
0%
1%
2%
10%
40%
25 ( 2
25 ( 1
28 ( 1
25 ( 2
26 ( 1
2.6 ( 0.6
1.2 ( 0.5
1.3 ( 0.4
0.5 ( 0.2
1.0 ( 0.4
68 ( 8
102 ( 8
84 ( 11
65 ( 12
71 ( 8
30 ( 5
27 ( 5
24 ( 4
36 ( 7
26 ( 4
1
1
1
1
2
6
7
8
9
0
0%
1%
2%
10%
40%
0.260
0.249
0.244
0.263
0.253
73 ( 2 31 ( 1 25 ( 2
33 ( 3 29 ( 2 25 ( 1
36 ( 7 31 ( 1 28 ( 1
46 ( 1 30 ( 2 25 ( 2
38 ( 1 30 ( 1 26 ( 1
1.24
1.16
1.11
1.20
1.15
17
18
19
20
a
Number-average hydrodynamic diameters of SCKs in aqueous solution
a
were characterized by dynamic light scattering. b Average heights and
average diameters of SCKs were measured by tapping-mode AFM, averaged
from the diameters of ca. 150 particles. c Average diameters of SCKs were
measured by TEM, averaged from the diameters of ca. 150 particles.
SCKs were prepared with 60% average cross-link density, as based on
the ratio of amine functional groups from the diamino cross-linker to the
carboxylic acid groups from the PAA-b-PMA block copolymer.
hydrophobic carbon surface, the substrate for TEM characteriza-
7
5
tion. The characterization data for the SCK samples are
summarized in Table 5.
Avidin/HABA Binding Assay. The bioavailability of biotin
presented from the SCKs to its protein receptor was evaluated
by a competitive binding assay (avidin/HABA) and fluorescence
correlation spectroscopy (FCS) studies. The amount of surface-
available biotin on each functionalized SCK sample was
1
08-111
quantified by the avidin/HABA assay.
Figure 5. DLS characterization of 2% biotinylated SCK, 18, in aqueous
solution at 20 °C. Histograms are averaged by intensity, volume, and
number, respectively (CONTIN fit).
The results of these analyses are shown in the overlaid UV-
vis spectra (Figure 8a). Upon addition of SCK nanoparticles
with different biotinylated block copolymer content, the change
of absorbance at 500 nm increased with an increase in
biotinylated block copolymer content, suggesting the avidin/
HABA complex is displaced by biotin presenting on the SCK
surface. The amount of biotin in each of the biotinylated SCK
sample solutions was calculated, and the data are summarized
in Table 6. The amount of available biotin for each of the
biotinylated SCK nanoparticles corresponded to nanomoles of
biotin per milliliter of SCK sample, which increased with an
increase in biotinylated block copolymer content, controlled by
the initial stoichiometry of 1:2 during the process of mixed
micelle formation.
To compare the relative amount of surface-available biotin
among SCK samples, the relative amounts of biotin on the SCK
surfaces were calculated by correction for concentration dif-
ferences and normalization with the amount of surface-available
biotin using the 1% biotinylated SCK, 17, as the normalization
standard. The normalized amount of biotin, available for
interaction with avidin, for each sample is plotted against the
percentage of biotinylated block copolymer content in Figure
8c. The relative numbers of biotin units exposed on the SCK
surface and available for binding with avidin increased as the
theoretical numbers increased.
Figure 6. Tapping-mode AFM images of SCKs: (a) 16, (b) 17, (c) 18,
(d) 19, (e) 20, and (f) 100% biotinylated SCK. Samples were prepared by
drop deposition onto freshly cleaved mica and allowed to dry in air.
PMA copolymers, species characterized by sizes greater than
100 nm and by nonspherical, irregular shapes were found by
DLS and AFM measurements, respectively. Thus, low percent-
ages of biotinylated block copolymer contents were chosen,
ranging from 0% to 40% as mentioned above, for the study of
their interactions with avidin.
To estimate the functional group availability obtained via the
mixed micelle methodology, the amount of measured biotin
based on the avidin/HABA assay was compared against the
The dimensions of the SCKs were characterized by tapping-
mode atomic force microscopy (AFM) and transmission electron
microscopy (TEM). The diameter values obtained by AFM
(
(
(
108) Green, N. M. Biochem. J. 1965, 94, 23c-24c.
(Figure 6) and TEM (Figure 7) were substantially larger than
109) Green, N. M. Methods Enzymol. 1970, 18, 418-424.
110) Savage, M. D. A Laboratory Guide to Biotin-Labeling in Biomolecule
Analysis; Meier, T., Fahrenholz, F., Eds.; Birkh a¨ user Verlag: Basel,
Boston, Berlin, 1996; pp 1-28.
were the heights measured by AFM. These discrepancies are
the result of the low Tg of the methyl acrylate core domain
allowing for the particles to deform upon adsorption onto the
(
111) HABA is a dye that binds to avidin in the same binding pocket used to
bind biotin. HABA has a maximum UV absorbance at 350 nm, and once
an avidin/HABA complex has formed, the maximum UV absorbance shifts
to 500 nm. As compared with the strong affinity for biotin exhibited by
7
4,75
solid substrates employed for AFM and TEM imaging.
In
addition, the larger diameter values obtained from AFM
measurements in comparison to those from TEM indicate greater
deformation of the particles on the hydrophilic mica surface,
the substrate for AFM characterization, as opposed to the
-
15
avidin (K
d
) 10
M), avidin’s affinity for HABA is much weaker (K
d
-
6
) 10 M). Thus, when biotin is added to a solution of avidin/HABA
complex, HABA is displaced quantitatively by biotin, and this displace-
ment can be quantitatively monitored by the decrease in UV absorbance
at 500 nm.
6604 J. AM. CHEM. SOC.
9
VOL. 126, NO. 21, 2004

Bioavailability of Biotin on SCK Nanoparticles
A R T I C L E S
Figure 7. TEM images of SCKs: (a) 16, (b) 17, (c) 18, (d) 19, and (e) 20. Samples were stained with phosphotungstic acid and drop deposited onto a
carbon-coated copper grid.
effects is unknown. The most likely cause of reduced biotin
availability was trapping of biotinylated chain ends below the
nanoparticle surface during micelle formation. Other potential
sources of inhibition for biotin binding include SCK cross-
linking density, surface rigidity, chain dynamics, and polydis-
persity.
Fluorescence Correlation Spectroscopy (FCS). The mul-
tivalent binding event expected for biotinylated-SCK:protein
receptor interactions was also studied using FCS. The measure-
ment of the translational diffusion coefficient for a fluorescent
species within a small focal volume is provided by FCS.1
Because the diffusion of spherical molecules is inversely
proportional to the cube root of the molecular weight, any
association or binding interaction that leads to the increase of
molecular weight will result in a decrease of the diffusion
coefficient, which can be identified and quantified by the
correlation time from an autocorrelation analysis of fluorescence
intensity fluctuations.
13-119
120
The FCS instrument was home-built, based on the principle
1
21-124
of a solid immersion microscope,
and the schematic
drawing of the instrumentation setup was shown in Figure 9a.
Alexa Fluor 488 labeled avidin was analyzed by FCS, and a
hydrodynamic diameter of 9.2 nm was found. This diameter,
as expected, is small as compared with values obtained for
nonfluorescence SCK nanoparticles using DLS (Dh ca. 30 nm).
As a result, SCK:protein binding due to biotin-avidin recogni-
tion was expected to decrease the diffusion coefficient of the
fluorescently labeled avidin. In addition, as more avidin was
bound to SCK nanoparticles, a larger fraction of the slower
diffusing SCK:protein complex was anticipated.
Figure 8. (a) UV-vis spectra of the avidin-HABA complex and the
avidin-HABA complex added with 16-20, respectively, in 50 mM PBS
buffer, 50 mM NaCl, pH 7.1. The zoomed region at 500 nm is shown in
the subset. (b) Calibration curve for avidin/HABA assay. (c) Normalized
amount of surface-available biotin versus the biotinylated block copolymer
content (16-20 corresponding to 0%, 1%, 2%, 10%, and 40%, respectively).
FCS measurements of Alexa Fluor 488 labeled avidin and
SCKs, 16-20, with different biotinylated block copolymer
contents were performed in 10 mM of HEPES buffer with 1
mM EDTA, 1 M NaCl, and at pH 7.1. A high salt concentration
was used to eliminate the nonspecific binding interactions.1
theoretical values, which were calculated on the basis of the
initial stoichiometry of 1 and 2 being used in the preparation
of the mixed micelles (Table 6). The surface accessibilities of
the biotin units for the micelles and SCKs were similar, as
determined for the samples having 10% biotinylated block
copolymer incorporation, 19, 21, and 22.1 It is uncertain
whether the degree of shell cross-linking of the SCKs and
rigidity of the particles play a role in the bioavailability of the
surface-accessible groups. Within the experimental error, the
percentage of biotin units available in comparison to those
incorporated in the formulation remained consistently at 10-
25
12
(
(
(
113) Elson, E. L.; Magde, D. Biopolymers 1974, 13, 1-27.
114) Magde, D.; Elson, E. L.; Webb, W. W. Biopolymers 1974, 13, 29-61.
115) Schwille, P.; Oehlenschl a¨ ger, F.; Walter, N. G. Biochemistry 1996, 35,
10182-10193.
(116) Wohland, T.; Friedrich, K.; Hovius, R.; Vogel, H. Biochemistry 1999,
38, 8671-8681.
(117) Sch u¨ ler, J.; Frank, J.; Trier, U.; Sch a¨ fer-Korting, M.; Saenger, W.
Biochemistry 1999, 38, 8402-8408.
(
118) Medina, M. AÄ .; Schwille, P. BioEssays 2002, 24, 758-764.
119) Haustein, E.; Schwille, P. Methods 2003, 29, 153-166.
25%. The less than complete surface accessibility and bioavail-
(
ability can be explained by several effects, although the
magnitude of the contributions from each of these potential
(120) Clark, C. G., Jr.; Remsen, E. E.; Wooley, K. L., manuscript in preparation.
(121) Mansfield, S. M.; Kino, G. S. Appl. Phys. Lett. 1990, 57, 2615-2616.
(122) Ghislain, L. P.; Elings, V. B. Appl. Phys. Lett. 1998, 72, 2779-2781.
(123) Ghislain, L. P.; Elings, V. B.; Crozier, K. B.; Manalis, S. R.; Minne, S.
C.; Wilder, K.; Kino, G. S.; Quate, C. F. Appl. Phys. Lett. 1999, 74, 501-
503.
(
112) Sample 21 is a 10% biotinylated micelle, prepared by mixing 1 with
SEC
nonbiotinylated PAA93-b-PMA76, M
n
w n
) 18 600 g/mol, M /M ) 1.12,
which is slightly different from the nonbiotinylated block copolymer, 2,
used to prepare 19, and sample 22 is the corresponding SCK prepared
from 21, with 60% cross-linking density.
(124) Koyama, K.; Yoshita, M.; Baba, M.; Suemoto, T.; Akiyama, H. Appl.
Phys. Lett. 1999, 75, 1667-1669.
(125) Swamy, M. J.; Marsh, D. Biochemistry 2001, 40, 14869-14877.
J. AM. CHEM. SOC.
9
VOL. 126, NO. 21, 2004 6605

A R T I C L E S
Qi et al.
Table 6. Results of the Avidin/HABA Assay for the Micelle and SCK Samples
available biotin
per milliliter of
sample solutiond
(nmol/mL)
available biotin
per gram of
polymere
biotinylated
block copolymer
content (mol %)
sample
concentrationa
(mg/mL)
theoretical value of
available biotinb
(nmol/g)
(∆A500/34)
× 1000c
fraction of
surface-available
biotinf
sample
(nmol/mL)
(nmol/g)
1
1
1
1
2
2
2
1
6
7
8
9
1
2
0
5
0%
1%
2%
10%
10%
10%
40%
40%
2.53
2.59
1.39
2.93
2.63
2.51
2.45
1.38
0
900
1680
7560
7520
7520
30 000
30 000
0
0
0
99
410
n/a
11%
25%
11%
22%
19%
11%
12%
0.3
0.7
2.7
4.3
3.7
8.2
5.0
0.3
0.6
2.5
4.1
3.4
8.1
4.8
850
1600
1400
3300
3500
a
The micelle and SCK samples were concentrated using a stirred ultrafiltration cell equipped with an ultrafiltration membrane filter disk (NMWL 100 000
Da). b Theoretical values of the surface-available biotins based on the initial molar ratios of the biotinylated PAA-b-PMA to the nonbiotinylated PAA-b-
c
PMA during the preparation; the data are represented in units of nanomoles of biotin per gram of polymer precursor mixture. (∆A500/34) × 1000 was used
d
to calculate the amount of biotin in the sample solution being analyzed in nmol/mL. Surface-available biotin in the SCK or micelle solution by comparison
e
of the absorbance change with that of the calibration curve. Surface-available biotin per gram of polymer precursor mixture based on the calculated concentration
of the sample solutions used in each assay. f Fraction of the surface-available biotin is the molar percentage of available biotin to the theoretical value.
Table 7. Data Summary for the FCS Measurement
× 10-
(s)
5
D
T
× 10-11
(m2 s-1
τ
d
c
d
e
N
)
D
h
(nm)
D
h
(nm)
X
bound
OGa
8.2
6.4 ( 0.7 27 ( 3
1.5 ( 0.2
9.2 ( 1.4
avidina
0.67 41.7 ( 0.5 4.1 ( 0.6
1
1
1
2
2
6
8
9
0
0
0.72 50.5 ( 0.6 3.4 ( 0.5 11.2 ( 1.7 31 ( 1 0.14
0.76 62.1 ( 0.9 2.8 ( 0.4 13.8 ( 2.2 31 ( 1 0.32
0.87 65.1 ( 1.0 2.6 ( 0.4 14.4 ( 2.2 30 ( 2 0.42
0.79 73.0 ( 1.0 2.4 ( 0.4 16.2 ( 2.6 30 ( 1 0.41
b
17
78.4 ( 0.6 2.2 ( 0.3 17.4 ( 2.7 30 ( 1 0.47
a
OG is Oregon Green 488; avidin is Alexa Fluor 488 labeled avidin.
b
The same 40% biotinylated SCK sample, with a 10-fold increase of the
avidin concentration. c Hydrodynamic diameters of the fluorescent species
calculated from correlation time τd. Volume-average hydrodynamic
d
diameters of SCKs from DLS measurements, which were used to calculate
τbound for the fitting into the two-component model, eq 5 in the Supporting
Information. e Fraction of avidin-SCK complex, calculated by fitting with
eq 5 (Supporting Information).
Oregon Green 488, whose diffusion coefficient was determined
via calibration with Rhodamine 6G having a known diffusion
-
10
2
-1
126
coefficient of 2.80 × 10
m s (at 22 °C). Correlation
times and diffusion coefficients are summarized in Table 7. The
correlation time τd increased as the biotinylated block copolymer
content in each SCK sample increased, indicating more labeled
avidin was bound to SCK per unit concentration of avidin in
the solution, which additionally suggested that more biotin was
available to its protein receptors. The averaged hydrodynamic
diameters of the fluorescent species in each solution were
calculated using eqs 3 and 4 (Supporting Information), by
employing Oregon Green 488 as a calibrant for the calculation
of DT. The fluorescent species in solution were also analyzed
as either free labeled avidin or labeled avidin-bound SCKs using
a two-component model, eq 5 (Supporting Information). The
calculation of diffusion coefficients and the averaged hydro-
dynamic diameters of the fluorescent species using Oregon
Green 488 as calibrant have an experimental error up to 16%
in each determination, which mainly arise from the determina-
tion of the correlation time of Oregon Green 488. However,
the correlation time determination for each sample is indepen-
dent of the calibrant, so the trend observed in the determination
is valid. The calculations for the bound fraction of the
fluorescent species using the two-component model are also not
affected.
Figure 9. (a) Schematic drawing of FCS instrumentation setup; (b)
normalized autocorrelation function of Oregon Green 488, Alexa Fluor 488
conjugate, and mixture of Alexa Fluor 488 conjugate with 16-20, in 10
mM HEPES buffer with 1 mM EDTA and 1 M NaCl, pH 7.1, respectively.
A zoomed region of the autocorrelation function is shown in the subset.
The same concentration of the labeled avidin used to characterize
the diffusion coefficient for a solution of pure labeled protein
was employed for each SCK:protein solution mixture. Normal-
ized fluorescence intensity autocorrelation functions for SCK:
protein mixtures, the pure labeled protein, and an Oregon Green
488 calibration standard are shown in Figure 9b.
The correlation time τd for each measurement was calculated
by fitting the autocorrelation function. To calculate the diffusion
coefficient of the samples, Oregon Green 488 was used to
characterize the focal volume. The correlation time was
converted to diffusion coefficient by reference to the τd of
(
126) Rigler, R.; Mets, U¨ .; Widengren, J.; Kask, P. Eur. Biophys. J. 1993, 22,
169-175.
6606 J. AM. CHEM. SOC.
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VOL. 126, NO. 21, 2004

Bioavailability of Biotin on SCK Nanoparticles
A R T I C L E S
In agreement with the avidin/HABA assay, these FCS
measurements found that the amount of available biotin at the
surface of the biotinylated SCK nanoparticles increased with
increasing biotin-terminated block copolymer incorporation, as
evidenced by the increasing correlation times, and the fraction
of biotin that underwent binding with avidin was ca. constant.
To confirm there was no significant amount of available biotin
that remained unbound, the 40% biotinylated SCK sample was
also analyzed with an avidin concentration increased by 10-
fold. Under conditions of excess avidin, the lack of a significant
change of the hydrodynamic diameter of the avidin-SCK
complex and the presence of free avidin indicated a saturation
of the biotinylated SCKs with labeled avidin.
Conclusions
A novel biotinylated initiator was synthesized and utilized
to initiate, via ATRP, homo- and diblock copolymer bearing a
single biotin moiety at the chain terminus. The chain terminal
biotinylated amphiphilic diblock copolymer PAA-b-PMA was
mixed in solution with its nonbiotinylated analogue to form
mixed micelles. The mixed micelle approach offers control over
the functional group incorporation within the nanoparticles by
varying the stoichiometric ratio of the functionalized to non-
functionalized micelle precursors. Intramicellar cross-linking
within the PAA shell layer converted the supramolecular
assemblies to robust SCK nanoparticles presenting bioactive
biotin.
Figure 10. Schematic illustration of 60 avidins (with a diameter of 9.2
nm) packing at the surface of SCK (with a diameter of 30 nm) based on
“
hard sphere” surface contacts.
(
Figure 10). Accurate values of the SCK aggregation numbers
are yet to be determined, but they can be estimated to be between
00 and 800 when comprised from the diblock copolymers of
4
ca. 13 000 Da, which suggests that there will be significant steric
effects that will prevent the binding of avidins to all of the biotin
units present.
These early findings are instructive, indicating that binding
of biological macromolecules to the SCKs functionalized with
bioactive moieties is a complex process that can be controlled,
but which also requires substantial further investigation to be
understood. These initial results demonstrate a mechanism for
the preparation of well-defined nanostructured materials that
serve as a model for the study of molecular ligands, immobilized
on a nanoscopic surface, and recognized by their protein
receptors, a pervasive phenomenon in biological systems.
Further studies are in progress, utilizing the control in the
preparation of biotinylated SCK nanoparticles, and in conjunc-
tion with the compositional versatility and structural stability
of SCK nanoparticles as robust nanoscopic building blocks to
form supramolecular assemblies, namely fabrication of 2-D and
The surface- and bioavailability of the biotinylated SCK
nanoparticles, comprised of different percentages of biotinylated
block copolymer, were evaluated using an avidin/HABA
competitive binding assay and FCS. Data from these studies
support the trend that greater numbers of biotin were available
to bind with the protein receptors with an increase in the
biotinylated block copolymer incorporation. It is important to
note that the shell cross-linking of the nanostructure presenting
the biotin did not diminish the bioavailability for binding with
the biological macromolecules. The avidin/HABA assay quanti-
fied the amount of available biotin to be less than 25% of the
theoretical value, likely due to the loss of functional group
availability in the process of micellization and shell cross-
linking. However, kinetic studies that remain in progress are
revealing that the binding interactions are significantly more
complex than is considered here. These preliminary studies
indicate that there are kinetic differences and differences in the
ultimate level of available biotin units observed between the
samples; the rates of binding increase and the percentage of
available biotin units decrease with increasing biotinylation. FCS
results indicated the same trend for the binding capacity of 10%
and 40% biotinylated SCKs. With the enhanced sensitivity of
3-D nanostructured materials with biotinylated SCKs employing
biotin-avidin recognition.
Acknowledgment. This material is based upon work sup-
ported by the National Science Foundation under Grant No.
DMR-9974457 and 0210247. We gratefully acknowledge the
constructive comments and insights provided by Drs. B. Weiner
and G. Williams (Brookhaven Inst. Co.) into the algorithms
employed by the ISDA package (Brookhaven Inst. Co.) for
particle size distribution analysis. We thank Mr. G. Michael
Veith for assistance with TEM measurements and Mr. Jeffrey
L. Turner for schematic drawings of SCK nanoparticles. We
also acknowledge Mr. J. Todd Bartlett and Dr. Trevor Havard
1
18
fluorescence cross-correlation spectroscopy,
the binding
isotherm of the biotinylated SCKs of different biotin surface
coverage might be determined accurately, potentially advancing
the understanding of the thermodynamics and kinetics of this
multivalent model system. Geometric considerations, involving
hard sphere surface contacts, indicate that approximately 60
(Precision Detectors, Inc.) for assistance with the FCS data
acquisitions. Washington University Mass Spectrometry Re-
source, an NIH Research Resource (grant number P41RR0954),
Dr. Mei Zhu, and Mr. Xinping Liu are acknowledged for the
mass spectrometry characterization.
1
27
avidins
can be accommodated through binding upon the
surface of an SCK having a hydrodynamic diameter of 30 nm
Supporting Information Available: Full experimental details
and characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(
127) Using the angle (27.15°) between by two adjacent circles of radius 4.6
on the surface of a circle radius 15, the maximum number of smaller
spheres that can be packed on the surface of the larger sphere is 60. Sloane,
N. J. A.; Hardin, R. H.; Smith, W. D., and others. Spherical Codes. http://
www.research.att.com/∼njas/packings/index.html (accessed October 2003).
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