Immuno-polymeric nanoparticles by Diels-Alder chemistry

Article abstract of DOI:10.1002/anie.200701032

(Figure Presented) Hitting the target: Self-assembled polymeric nanoparticles from an amphiphilic copolymer were prepared with furan groups on the outer polyethylene glycol corona that were accessible for Diels-Alder reactions with maleimide-functionalize

Full text of DOI:10.1002/anie.200701032

Communications
DOI: 10.1002/anie.200701032
Bioorganic Chemistry
Immuno-Polymeric Nanoparticles by Diels–Alder Chemistry**
Meng Shi, Jordan H. Wosnick, Karyn Ho, Armand Keating, and Molly S. Shoichet*
Immuno-nanoparticles hold great promise for targeted deliv-
ery of drugs, therapeutics, and diagnostics as numerous
possible target receptors have been identified on the surface
hydrophobic small molecules primarily. The interest in
micellar self-assembling polymeric nanoparticles as delivery
vehicles is growing because they can both stabilize hydro-
phobic molecules at high drug loading and have their
[
1–4]
of cells in pathological conditions.
Under in vivo condi-
[
3,14–22]
tions, immuno-nanoparticles can deliver these agents to
specific sites by both passive targeting, where a hydrophilic
polymer outer layer such as polyethylene glycol (PEG) leads
composition tuned to allow functionalization.
Although
various polymeric micellar systems have been developed as
delivery vehicles, only a few cases have been designed to
[
1,3–5]
[3]
to longer circulation in the blood stream,
and active
incorporate antibodies for immunotargeting.
targeting mechanisms, where the “passive” nanoparticles
have antibody molecules or fragments covalently coupled to
their surface to achieve localized delivery to target cells that
To couple antibodies or antigen-binding fragments, mild
reaction conditions are required to achieve high efficiency
without significant loss of binding activity. While a number of
techniques for antibody conjugation have been estab-
[
1,3,4]
express the corresponding antigen receptors.
The design
[
3,6–13]
of immuno-polymeric nanoparticles requires not only the
creation of well-defined structures, but also the optimization
lished,
activation
these conventional methods often require either
or coupling reagents, thereby increasing the
[
6,8]
[10]
[
3,4,6–13]
of antibody conjugation chemistry.
Here we report the
complexity of the reaction protocol and the possibility of side
[
6–8,12,13]
design of a novel polymeric nanoparticle system using new
biodegradable graft copolymers of poly(2-methyl-2-carboxy-
trimethylene carbonate-co-d,l-lactide)-graft-poly(ethylene
glycol)-furan (poly(TMCC-co-LA)-g-PEG-furan; Scheme 1)
that self-assemble into micellar nanoparticles in aqueous
solution and provide reactive functional groups for coupling
of antibodies through Diels–Alder reactions.
reactions. Moreover, the highly reactive nature
and
[
11]
instability of the functional groups limit the efficiency of
these methods.
Diels–Alder reactions, which involve a highly selective
[4+2] cycloaddition reaction between a diene and a dieno-
[
23–25]
phile,
provide a means to overcome the limitations
associated with coupling chemically sensitive antibody mol-
ecules, particularly in aqueous environments where both the
rate and stereoselectivity of Diels–Alder reactions are
[
14–22]
Polymeric amphiphiles
self-assemble upon contact
with aqueous environments; the hydrophobic regions of the
copolymers spontaneously aggregate together to minimize
contact with the aqueous environment while the hydrophilic
segments are exposed to the aqueous environment and form
the outer corona. The inner hydrophobic domain, surrounded
by a hydrophilic outer shell, has been used to encapsulate
[
24,25]
dramatically increased.
Alder reactions in biotechnology applications has been
recognized,
The great potential of Diels–
[
26–30]
and here we demonstrate its applicability to
conjugate antibodies to nanoparticles for targeted delivery.
This simple, one-step reaction without by-products does not
require initiators, catalysts, or coupling reagents, is highly
efficient and selective under mild conditions, and results in
stable products. Our nanoparticles were designed with furan
groups (diene) on the outer PEG corona which are accessible
for reaction with antibodies functionalized with maleimide
(dienophile) groups in aqueous solution (Scheme 2). With a
view toward targeted delivery in breast cancer, we demon-
strate the synthesis of furan-functionalized micellar nano-
particles derivatized with maleimide-modified anti-HER2 (a
therapeutic antibody used to treat breast cancer) through
Diels–Alder reactions and the specific and efficient binding
with breast cancer HER2-over-expressing cell lines in vitro.
This nanoparticle system provides a novel means for the
delivery of combination immunotherapy/chemotherapy to
more effectively treat certain malignancies.
The copolymer, poly(TMCC-co-LA)-g-PEG-furan, was
designed specifically for the creation of immuno-nanoparti-
cles by combining strategies of polymeric self-assembly and
Diels–Alder reactions of bioconjugation in one process. The
copolymer is biodegradable and biocompatible for in vivo
applications; amphiphilic with the ability to self-assemble into
ordered structures; and capable of undergoing Diels–Alder
reactions because of the presence of furan groups at the PEG
[
*] M. Shi, Dr. J. H. Wosnick, K. Ho, Dr. M. S. Shoichet
Department of Chemical Engineering and Applied Chemistry
Department of Chemistry
Institute for Biomaterials and Biomedical Engineering
Terrence Donnelly Centre for Cellular and Biomolecular Research
University of Toronto
160 College Street, Toronto, ON M5S3E1 (Canada)
Fax: (+1)416-978-4317
E-mail: molly@chem-eng.utoronto.ca
Homepage: http://www.ecf.utoronto.ca/~molly
Dr. A. Keating
Cell Therapy Program
Princess Margaret Hospital/Ontario Cancer Institute
University Health Network, 620 University Avenue
Toronto, ON (Canada)
[
**] We thank Yumin Yuan for initial studies on the poly(TMCC-co-LA)
synthesis, Professor Mitchell Winnik for helpful discussions, and Dr.
Neil Coombs for TEM imaging. We are grateful to the Natural
Sciences and Engineering Research Council and the Canadian
Institutes for Health Research for funding through the Collaborative
Health Research Program.
Supporting information (including characterization data) for this
article is available on the WWW under http://www.angewandte.org
or from the author.
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termini. Scheme 1 shows the synthesis of the amphiphilic
terminal furan groups are easily accessible to maleimide-
copolymer. The resulting amphiphilic copolymer of
functionalized molecules in the aqueous solution, thus
enabling cycloaddition at the PEG termini of self-assembled
nanoparticles (Scheme 2).
À1
1
9.2 kgmol
contains
a
hydrophobic backbone of
To demonstrate the applicability of
Diels–Alder reactions in this system, a
maleimide-containing fluorescent probe
Alexa Fluor 488 C -Maleimide was cou-
5
pled with the furan-functionalized nano-
particles under aqueous conditions (in b-
morpholinoethanesulfonic acid (MES)
buffer, pH 5.5, 378C). As shown in
Figure 1, micellar nanoparticles incubated
with an excess of the fluorescent probe
showed strong fluorescent signals, which
indicated that the fluorescent probe was
coupled to the nanoparticles. There was
little to no evidence of fluorescence on the
nanoparticles in controls I and II, where
the maleimide groups were quenched
prior to reaction with the nanoparticles,
thus demonstrating the selectivity of the
Diels–Alder reaction. Interestingly, allow-
ing the furan-functionalized nanoparticles
and excess maleimide-containing fluores-
cent probes to react for 24 h resulted in
near-quantitative reaction of the accessi-
ble furan groups on the surface of the
micellar nanoparticles, as judged by the
amount of fluorescent probe coupled:
1 mg of nanoparticles had the capacity to
couple 3.6 nmol of maleimide-containing
molecules.
Scheme 1. Synthesis of poly(TMCC-co-LA)-g-PEG-furan amphiphilic graftcopolymer: The
copolymer backbone of poly(TMCC-co-LA) (4) was synthesized by ring-opening polymerization
of the cyclic carbonate monomer 2 (benzyl-protected TMCC), and d,l-lactide (LA) to yield
various copolymer compositions; the random copolymer with 13 mol% TMCC and 87 mol%
LA are described in detail herein. Poly(TMCC-co-LA) was then modified with a bifunctional
furan-poly(ethylene glycol)-amine (furan-PEG-NH ) (5) by coupling amine-terminated PEG to
2
the pendant carboxylic acid groups on the backbone polymer. The resulting amphiphilic,
À1
1
9.2 kgmol copolymer contains a hydrophobic backbone of poly(TMCC-co-LA) and an
average of one hydrophilic PEG chain grafted per backbone. (see Section S1 and Table S1 in
the Supporting Information for polymer characterization). Bn=benzyl.
To demonstrate the utility of
poly(TMCC-co-LA)-g-PEG
micellar
poly(TMCC-co-LA) and hydrophilic grafts of PEG with
furan groups located at the PEG termini. The amphiphilic
copolymer was dissolved in a mixture of dimethylformamide
nanoparticles as targeting vehicles, we constructed immuno-
nanoparticles by derivatizing preformed micellar nanoparti-
cles with herceptin (anti-HER2), a therapeutic monoclonal
antibody used to treat breast cancer. Herceptin is known to
bind to human epidermal growth factor receptor-2 (HER2),
which is over-expressed on 20–30% of breast and ovarian
(
DMF) and borate buffer and dialyzed against distilled water
to drive self-assembly. Scheme 2 shows a schematic represen-
tation of the self-assembly of micellar nanoparticles. The
hydrophobic segments of the poly(TMCC-co-LA) backbone
form the dense inner core while the hydrophilic PEG chains
orient toward the aqueous solution to form the outer corona.
Well-formed spherical nanoparticles were observed by trans-
mission electron microscopy (TEM; see Figure S1 in the
Supporting Information ) and have a hydrodynamic diameter
of 84 nm measured by dynamic light scattering. The formation
of hydrophobic microdomains in the micellar nanoparticles
was confirmed by fluorescence measurements using pyrene.
According to this method, the apparent critical aggregation
concentration (CACapp) of the copolymer was determined to
[
31]
cancer cell surfaces, thereby providing a basis for selective
immunotargeting in our system.
To couple herceptin antibodies (anti-HER2) to the
micellar nanoparticles by Diels–Alder reactions, an estab-
lished site-specific modification of the carbohydrate chains
within the Fc region of anti-HER2 was achieved. This enabled
the introduction of maleimide (Mal) groups while preserving
the intact Fab fragment for antigen binding and minimizing
[
32,33]
the loss of the bioactivity
(see Figure S3 in the Supporting
Information on modification of antibodies at the Fc region).
The average number of maleimide groups introduced per
antibody was approximately two. By using flow cytometry to
compare the binding capacity of maleimide-modified anti-
HER2 (anti-HER2-Mal) versus anti-HER2 to HER2-over-
expressing SKBR3 breast cancer cells, it was confirmed that
the modification of anti-HER2 with maleimide did not affect
its binding capacity (see Figure S4 in the Supporting Infor-
mation). Immuno-nanoparticles were prepared by incubating
À1
be approximately 3 mgmL (see Figure S2 in the Supporting
Information). In this system, the furan–maleimide Diels–
Alder reaction was designed to occur at the interface between
the PEG corona of the nanoparticles and the aqueous phase
in which the antibodies are dissolved. After the amphiphilic
copolymer self-assembles into micellar nanoparticles, the
hydrophilic segments orient outward to the water phase. The
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anti-HER2-Mal with nanoparticles in MES buffer at pH 5.5
and 378C (without any additional reagents). When the
nanoparticle/anti-HER2-Mal feed mass ratio was kept con-
stant (for example at 100:1), both the anti-HER2 density on
the immuno-nanoparticles and the coupling efficiency
(expressed as the percentage of the initial feed antibody
bound to the micellar nanoparticles) increased as a function
of reaction time (Figure 2a). Almost 100% of the initial anti-
HER2-Mal added was coupled within six hours of incubation,
À1
thus resulting in an antibody density of 64 pmolmg nano-
particle (approximated at 5 antibodies per nanoparticle. see
Section S4 in the Supporting Information). To better under-
stand the parameters that influence the yield of this Diels–
Alder reaction, the reaction time was fixed at 2 h and the
initial nanoparticle/anti-HER2-Mal mass ratio was increased.
As shown in Figure 2b, the coupling efficiency increased as
the nanoparticle/anti-HER2-Mal mass ratio increased; how-
ever, as might be expected, the anti-HER2 density on the
immuno-nanoparticle decreased. By using Diels–Alder reac-
Scheme 2. Schematic representation of the self-assembly of micellar
nanoparticles from poly(TMCC-co-LA)-g-PEG-furan and Diels–Alder
reactions between the nanoparticles and maleimide-modified antibod-
ies: the hydrophobic segments of poly(TMCC-co-LA) form the inner
core and the hydrophilic PEG chains orient toward the aqueous
solution to form the outer corona. Furan groups are located at PEG
termini which are easily accessible to maleimide-modified antibodies
for Diels–Alder reactions.
Figure 2. Immuno-nanoparticles prepared by Diels–Alder reactions.
a) Representative time-dependence of the coupling of anti-HER2-Mal
to nanoparticles. Reactions were carried out in pH 5.5 MES buffer at
378C. The nanoparticle/anti-HER2-Mal mass feed ratio was fixed at
Figure 1. Maleimide-containing Alexa Fluor 488 C -Maleimide (Fluor-
100:1. Anti-HER2-Mal was labeled with a fluorescent dye (Alexa
Fluor 430) for quantification. The coupling efficiency is expressed as
the percentage of the initial feed antibody bound to the micellar
nanoparticles. b) Representative nanoparticle/anti-HER2-Mal feed ratio
dependence of coupling of anti-HER2-Mal to nanoparticles. Reaction
time was fixed at 2 h. (see Section S4 in the Supporting Information:
the antibody density was converted into the average number of
antibody molecules per nanoparticle by estimating the aggregation
number of the micellar nanoparticles). Ab=antibody.
5
Mal) coupled to furan groups of self-assembled micellar nanoparticles
by Diels–Alder reactions. Control I: Maleimide groups on Fluor-Mal
fluorescent probes were quenched by incubating with excess cysteine
prior to reaction with furan-functionalized nanoparticles. Control II:
Maleimide groups on Fluor-Mal fluorescentprobes were quenched by
incubating with excess furfurylamine prior to reaction with furan-
functionalized nanoparticles (shown are the mean values of three
measurements Æstandard deviation).
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Chemie
tions, either prolonged reaction times or high nanoparticle/
anti-HER2-Mal feed ratios can be used to achieve maximum
coupling efficiency. The large excess of accessible nano-
particle furan groups relative to maleimide-modified anti-
bodies may account for the rapid reaction and the high
coupling efficiency achieved. To better understand the
coupling capacity of the furan-functionalized nanoparticles
in the Diels–Alder reaction, anti-HER2-coupled micellar
nanoparticles were further modified with maleimide-contain-
ing fluorescent probes. It was found that after coupling of the
antibody, the immuno-nanoparticle coupled a similar amount
of fluorescent probe as the unmodified (plain) nanoparticles.
When we compared the number of furan sites occupied by the
MB-468 (HER2 negative). Flow cytometry was employed to
measure the cell-associated fluorescence above background.
As shown in Figure 3a,b, the anti-HER2 immuno-nanopar-
ticles showed fourfold higher binding activity with SKBR3
than the IgG1K immuno-nanoparticle control (IgG1K acts as
a nonspecific antibody and shows little binding with SKBR3).
The binding of the immuno-nanoparticles was much lower
with low-HER2-expressing MCF7 cells than that observed
with SKBR3 cells. No binding was observed for the HER2-
negative MDA-MB-468 cells (Figure 3b). The binding of
immuno-nanoparticles correlates with the HER2 expression
[
31]
levels in these cell lines, which is well-documented and was
confirmed by incubation with herceptin under the same
conditions and by subsequent labeling with an FITC-anti-
À1
antibodies (11–64 pmolmg nanoparticle) with the total
number of accessible furan
groups for the Diels–Alder
À1
reaction
(3.6 nmolmg
nanoparticle, as determined
with small fluorescent probe
molecules), we found that
the bound antibodies occu-
pied only 1–2% of the
accessible furan groups.
This finding suggests that,
even after coupling of the
antibody, the majority of
furan groups are available
for further modification,
thus allowing the nanopar-
ticles to serve multiple func-
tions by sequential Diels–
Alder reactions, for exam-
ple, of targeting ligands,
imaging agents, and/or drug
molecules.
Importantly the hydro-
dynamic diameter of the
immuno-nanoparticles,
8
6.6 Æ 3.4 nm was similar to
that of unmodified (plain)
nanoparticles,
84.3 Æ 2.6
nm, (see Figure S5 in the
Supporting Information),
which suggests that neither
aggregation nor cross-link-
ing occurred during the
antibody-coupling proce-
dure.
Figure 3. Anti-HER2 immuno-nanoparticles binding with HER2-over-expressing cells. a) Flow cytometry
analysis of the specific binding of anti-HER2 immuno-nanoparticles with SKBR3 cells (HER2-over-expressing):
Top: Comparison of the binding of anti-HER2 immuno-nanoparticles and nonspecific IgG1K immuno-
nanoparticles. The antibody density on the anti-HER2 immuno-nanoparticles was 56 pmolmg nanoparticle.
IgG1K immuno-nanoparticles with the same antibody density were prepared under the same conditions as the
To demonstrate the
capability of anti-HER2
immuno-nanoparticles to
À1
bind
specifically
with anti-HER2 immuno-nanoparticles; bottom: Binding of anti-HER2 immuno-nanoparticles with SKBR3 cells
presaturated with anti-HER2. b) Mean fluorescent intensity measured for immuno-nanoparticles binding with
SKBR3 cells (HER2-over-expressing), MCF7 (HER2-low-expressing), and MDA-MB-468 (HER2-negative) cells,
HER2-over-expressing
cells, the immuno-nanopar-
ticles were incubated with
the following cell lines:
SKBR3
expressing), MCF7 (HER2-
determined by flow cytometry. c) Representative dose-dependence for the binding of anti-HER2 immuno-
nanoparticles with (
6
&
) SKBR3, (~) MCF7, and (*) MDA-MB-468 cells. The antibody density was fixed at
4 pmolmg nanoparticle. d) Representative dependence of the antibody density of the binding of anti-HER2
À1
(HER2-over-
immuno-nanoparticles with SKBR3, MCF7, and MDA-MB-468 cells. The concentration of the immuno-
À1
nanoparticles was fixed at 187.5 mgmL (shown are the mean values of three measurements Æstandard
low-expressing), and MDA- deviation).
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Communications
human antibody (see Figure S6 in the Supporting Informa-
The reaction mixture was stirred in an ice bath under N overnight,
2
tion). As further confirmation of the specificity of the
interaction of the immuno-nanoparticles with cells, presatu-
ration of HER2 antigens on SKBR3 cells with free herceptin
was found to inhibit the binding of immuno-nanoparticles
with SKBR3 cells by more than 70% (Figure 3b). The
binding capacity of the immuno-nanoparticles to SKBR3 cells
increased with increased concentrations of immuno-nano-
particles (Figure 3c). A significantly lower, but similar
observation was made with MCF7 cells, and there was no
measurable dose-dependent effect of immuno-nanoparticle
concentration on binding to HER2-negative MDA-MB-468
cells. The binding capacity of herceptin immuno-nanoparti-
cles to SKBR3 cells increased as the density of herceptin per
immuno-nanoparticle increased (Figure 3d); there was no
effect of an increased herceptin density on binding to either
MCF7 or MDA-MB-468 cell lines.
then filtered, and concentrated by rotary evaporation. The residue
was recrystallized twice from ethyl acetate and dried in a vacuum
oven overnight at RT to yield 2 as a white solid (13.5 g, 84%).
3
: Monomer 2 (2.5 g, 0.01 mol) was copolymerized with d,l-
lactide (LA; 6.5 g, 0.045 mol) in a bulk melt in the presence of one
drop of stannous 2-ethylhexanoate (Sn(oct) ) at 1208C for 24 h. The
2
resulting product was then dissolved in chloroform and precipitated
twice with hexane. After drying the product in a vacuum oven
overnight at RT, the copolymer 3 was collected (6.8 g, 76%).
4
: Copolymer 3 (6.0 g) was dissolved in EtOAc and the benzyl
protecting groups removed under hydrogen (H ) in the presence of
2
palladium on activated carbon (10 wt%) for 48 h at RT. The resulting
polymer was then filtered and precipitated twice with hexane. After
drying the product in a vacuum oven overnight at RT, the copolymer 4
was collected (4.4 g, 82%).
6
: Copolymer 4 (100 mg) was dissolved in DMF (5 mL) and MES
buffer (0.5 mL; 10 mm, pH 5.5). N-ethyl-N’-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDAC, 10 wt%) and N-hydroxy-
sulfosuccinimide (sulfo-NHS, 10 wt%) were added. The reaction
This proof-of-concept for cancer cell targeting with these
immuno-nanoparticles has demonstrated the continued bio-
activity, binding capacity, and specificity of the anti-HER2-
coupled nanoparticles. The success that we have achieved can
be attributed to the herceptin antibodybeing specifically
modified with maleimide groups in its Fc fragment prior to
nanoparticle coupling to avoid altering the binding character-
istics of the antibody. Moreover, the mild aqueous conditions
required for Diels–Alder cycloadditions of the antibodies to
the nanoparticles avoids both long reaction times and the use
of coupling or potentially denaturating reagents, thereby
preserving the selectivity and efficient binding capability of
the immobilized antibodies. Importantly, the antibodies are
coupled to the terminal groups of the PEG corona which are
easily accessible to antigen receptors on the cell surfaces.
In conclusion, we have synthesized new biodegradable,
amphiphilic, furan-functionalized copolymers that self-assem-
ble in aqueous conditions to form micellar nanoparticles. By
designing the nanoparticles with furan functional groups at
the termini of the PEG corona, anti-HER2 breast cancer
antibodies, modified with maleimide functional groups in
their Fc region, were covalently bound to the nanoparticles by
Diels–Alder cycloadditions. Anti-HER2 immuno-nanoparti-
cles specifically bound with HER2-over-expressing cells, thus
demonstrating the capacity of this technique to create
bioactive immuno-nanoparticles. The versatility of the nano-
particle system can be extended to create complex and
multiple functional delivery vehicles by taking advantage of
the Diels–Alder reaction to immobilize molecules appropri-
ate for imaging, diagnostics, or the targeted delivery of
therapeutic agents. Combining immunotherapy with locally
delivered bioactive agents or chemotherapy is under inves-
tigation.
solution was incubated at RT for 30 min. The furan-PEG-NH2
5
(50 mg; see Section S1 in the Supporting Information for synthesis
details) was dissolved in borate buffer (1 mL; 500 mm, pH 9.0). This
solution was then slowly added to the activated copolymer 4 solution
under stirring. The reaction mixture was incubated at RT for 24 h,
after which the reaction solution was dialyzed against distilled water
and then passed through a sepharose 4B column equilibrated with
distilled water to remove any remaining PEG. The collected fractions
containing particle suspensions were freeze-dried and copolymer 6
was collected as a white solid (75 mg, 50%).
Nanoparticle preparation by the dialysis method: Poly(TMCC-
co-LA)-g-PEG-furan 6 was dissolved in DMF/borate buffer (500 mm,
À1
pH 9.0) at a concentration of 10 mgmL . The solution was dialyzed
against distilled water using a dialysis membrane with a molecular-
À1
weight cut off (MWCO) of 12–14 kgmol at RT for 24 h (see
Section S2 in the Supporting Information for detailed methods).
Immuno-nanoparticle preparation by Diels–Alder reactions:
À1
Nanoparticle solution (1 mL; 4.0 mgmL in distilled water) was
À1
added to a solution of the maleimide-modified antibody (1 mgmL
in 100 mm MES buffer of pH 5.5). The reaction solution was
incubated at 378C under gentle agitation for various time periods.
The immuno-nanoparticles were purified by passing through a
sephacryl S-300HR column in 10 mm phosphate-buffered saline
(PBS) at pH 7.4 (see Sections S3 and S4 in the Supporting
Information for detailed methods).
Immuno-nanoparticle binding with cells: The antibody-coupled
immuno-nanoparticles were labeled with Alexa Fluor 488 C -Mal-
5
eimide by the same Diels–Alder reaction as used for the flow
5
cytometry analysis. 2 ” 10 cells were suspended in 0.05 mL PBS
buffer of pH 7.4 with 4% fetal bovine serum (FBS) and then added to
a solution of the immuno-nanoparticles (0.15 mL, various concen-
trations in 10 mm PBS, pH 7.4). After incubation of the mixture at
4
8C for 30 min, PBS buffer (1 mL) with 1% FBS and 2 mm
ethylenediamine tetraacetic acid (EDTA) was added to rinse the
cells and the mixture was then centrifuged. The supernatant was
removed. The harvested cells were resuspended in PBS buffer
À1
(
0.5 mL) with 1% FBS and 6 mgmL propidium iodide and then
Experimental Section
4
analyzed by flow cytometry. The first 10 events were recorded for
subsequent analysis of the gated live-cell population. For controls
where HER2 receptors on the breast cancer cell lines were
presaturated prior to interaction with immuno-nanoparticles, cells
2
(benzyl-protected TMCC): Compound 1 (12.7 g, 0.08 mol), derived
by nucleophilic substitution of the sodium salt of 2,2-bis(hydroxyme-
thyl)propionic acid with benzyl bromide, and ethyl chloroformate
(19.1 g, 0.176 mol) were dissolved in THF (250 mL). The reaction
À1
solution was cooled in an ice bath under nitrogen (N ). Triethylamine
were incubated with excess free herceptin (10 mgmL ) at 378C for
2
(19.4 g, 0.192 mol) was added dropwise to the rapidly stirring solution.
30 min prior to their incubation with the immuno-nanoparticles. (see
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