Synthesis of fluorine-18 functionalized nanoparticles for use as in vivo molecular imaging agents

Article abstract of DOI:10.1021/ja802010d

Nanoparticles containing fluorine-18 were prepared from block copolymers made by ring opening metathesis polymerization (ROMP). Using the fast initiating ruthenium metathesis catalyst (H₂IMes)(pyr)₂(Cl)₂Ru=CHPh, low polydispersity amphiphilic block copolymers were prepared from a cinnamoyl-containing hydrophobic norbornene monomer and a mesyl-terminated PEG-containing hydrophilic norbornene monomer. Self-assembly into micelles and subsequent cross-linking of the micelle cores by light-activated dimerization of the cinnamoyl groups yielded stable nanoparticles. Incorporation of fluorine-18 was achieved by nucleophilic displacement of the mesylates by the radioactive fluoride ion with 31% incorporation of radioactivity. The resulting positron-emitting nanoparticles are to be used as in vivo molecular imaging agents for use in tumor imaging. Copyright

Full text of DOI:10.1021/ja802010d

Published on Web 05/02/2008
Synthesis of Fluorine-18 Functionalized Nanoparticles for use as in vivo
Molecular Imaging Agents
John B. Matson and Robert H. Grubbs*
NanoSystems Biology Cancer Center, DiVision of Chemistry and Chemical Engineering, MC 127-72, California
Institute of Technology, Pasadena, California 91125
Received March 18, 2008; E-mail: rhg@caltech.edu
Scheme 1. Monomer Synthesesa
Nanostructures are the subject of intense study toward applica-
tions in cancer diagnosis, imaging, and treatment.1 Research in this
area is driven by the observation that particles in the nanometer
size range are known to localize more heavily in tumor tissue than
in healthy tissue. This phenomenon, known as the enhanced
permeability and retention effect (EPR effect), is due to the leaky
vasculature exhibited in the tumor tissue.2 Exploitation of the EPR
effect is a common strategy for targeting tumor cells, using
nanostructures including liposomes,3 quantum dots,4 dendrimers,5
a Reaction conditions: (i) H2NCH2CH2OH, NEt3, toluene, DS-trap; (ii)
trans-cinnamic acid, EDC, DMAP, CH2Cl2, rt; (iii) H2N(CH2CH2O)13OH,
polymeric micelles,6 and other molecular conjugates.7
Imaging of nanostructures designed to detect tumors by exploiting
the EPR effect has been accomplished using several in vivo imaging
techniques, including magnetic resonance (MR),8 near-IR fluores-
cence (NIR),9 and positron emission tomography (PET).10 Of these
techniques, PET is the most sensitive and accurate method of
measuring the temporal pattern in the biodistribution of nanopar-
ticles as PET is a specific, highly sensitive, and versatile three-
dimensional molecular imaging technique that is used broadly in
tumor imaging. The most widespread radionuclide used in PET
imaging is fluorine-18, which is the positron-emitting isotope in
the commonly used PET tracer 18-fluorodeoxyglucose. Due to its
relatively long half-life (t1/2 ) 109 min), many more fluorine-18-
containing radiotracers are synthetically accessible than are ra-
diotracers with other small positron-emitting nuclides, such as
carbon-11 (t1/2 ) 20 min), nitrogen-13 (t1/2 ) 10 min), and oxygen-
15 (t1/2 ) 2 min). This has led to a dramatic increase in recent
years in the production of fluorine-18, which can only be synthe-
sized in a cyclotron.
To our knowledge, no reports of tumor-targeting nanoparticles
functionalized with fluorine-18 have been published. While nano-
particles incorporating positron-emitting metals such as copper-64
(t1/2 ) 12.7 h)10a–c have been synthesized, rapid and efficient
incorporation of fluorine-18 into nanoparticles remains elusive.
Incorporation of this common PET nuclide into nanoparticles would
pave the way for precise and accurate in vivo PET imaging using
nanostructured materials. With this goal in mind, we sought to
synthesize fluorine-18-containing nanoparticles by mimicking well-
known small molecule chemistry. Many fluorine-18-containing
molecular imaging agents rely on nucleophilic displacement of a
sulfonate ester by the nucleophilic fluoride-18 anion.11 These
reactions are typically run in acetonitrile, using kryptofix-222 to
bind potassium. In our nanoparticle design, we chose to incorporate
sulfonate esters to take advantage of this well-developed chemistry.
Our long-term goals include probing the limits of the EPR effect
and determining the optimal nanoparticle size for tumor targeting.
To this end, we selected amphiphilic block copolymers as scaffolds
from which to build nanoparticles due to their predictable self-
assembly behavior in forming micelles, the well-defined nature of
such nanostructures, and the ability to control micelle size by
modifying block lengths and ratios.12 Ring opening metathesis
C6H6, DS-trap; (iv) MsCl, NEt3, -30 °C.
polymerization (ROMP) provides a method for producing very low
polydispersity amphiphilic block copolymers without protecting
group chemistry.13 This is a distinct advantage over other living
polymerization techniques, such as anionic polymerization and
controlled free radical polymerization. Syntheses of nanoparticles
using these techniques typically require multiple polymerization
steps, followed by one or more deprotection or functionalization
reactions. Many of the steps performed after polymerization require
lengthy purification procedures such as dialysis. Additionally, living
ROMP using substituted norbornenes produces polymers whose
degrees of polymerization can be easily and precisely controlled
by adjusting the monomer to catalyst ratio.14 When polymerizing
substituted norbornenes, ROMP is free of chain transfer and
termination events. Reactions are therefore typically run to complete
conversion, allowing for extremely precise control over polymer
molecular weight. As a result, factors affecting nanoparticle size
and shape, such as the length and relative ratio of the hydrophilic
and hydrophobic blocks, can be easily modified to produce a wide
variety of nanoparticle architectures in a short amount of time. We
show here that cross-linked micelles can be easily synthesized and
efficiently functionalized with fluorine-18 using amphiphilic block
copolymers made by ROMP.
The synthesis of nanoparticles containing functional groups for
cross-linking, biocompatibility, and facile radiofluorination required
the development of block copolymers that exhibited all of these
elements. Substituted exo-norbornenes were chosen as the mono-
mers due to their ability to undergo living ROMP, as well as their
ability to be easily functionalized. Specifically, norbornene-imides
were used because the condensation of exo-anhydride 1 with
functionalized amines is a versatile reaction capable of forming a
variety of monomers (Scheme 1).
As the cross-linking element, we chose the cinnamoyl group
because it has been used previously as a photo-cross-linker in
nanoparticle syntheses.15 Cross-linking of the micelles is necessary
to ensure that the nanoparticles stay intact upon dilution in the
bloodstream, as the polymer concentration can drop below the
critical micelle concentration (cmc) in vivo. Under irradiation with
ultraviolet light, the trans-olefin of the cinnamoyl group undergoes
9
10.1021/ja802010d CCC: $40.75
2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 6731–6733 6731

C O M M U N I C A T I O N S
Scheme 2. Polymer Synthesis
Scheme 3. Fluorinated Nanoparticle Synthesisa
Table 1. GPC Characterization of Block Copolymers
entry
m
n
Mn (theo)
Mn (GPC)
PDI
1
2
3
4
50
100
200
400
150
300
600
140400
280500
560100
1124000
133200
280000
544000
1222000
1.01
1.03
1.18
1.73
1200
Table 2. Characterization of Nanoparticles
a Black lines represent polymer backbone; blue lines and red lines
represent pendent PEG and cinnamoyl groups, respectively. Purple balls
represent mesylate groups, and green balls represent fluorine atoms.
Conditions: (i) dialysis against H2O; (ii) hν, 3 min; (iii) (1) K18F, kryptofix-
222, K2CO3, BHT, MeCN, 120 °C, 60 min; (2) K19F, kryptofix-222, MeCN,
80 °C, 30 min.
entry
polymer Mn
diameter (AFM)
diameter (DLS)
1
2
3
4
133200
280000
544000
1222000
12.7 ( 2.6 nm
16.4 ( 4.5 nm
21.1 ( 3.9 nm
39.7 ( 4.0 nm
47.4 ( 7.5 nm
58.1 ( 1.8 nm
79.7 ( 9.7 nm
142.5 ( 6.8 nm
due to catalyst death. A block ratio of 3:1 hydrophilic/hydrophobic
was chosen after extensive study of the solubility of nanoparticles
with various block lengths.
a [2 + 2] dimerization to afford a tetrasubstituted cyclobutane
ring.16 Hydrophobic monomer 3 was synthesized by reaction of
exo-norbornene anhydride 1 with aminoethanol to produce nor-
bornene-imide 2, followed by coupling with trans-cinnamic acid
using EDC.
Micelle formation was accomplished by dissolving a given
polymer in THF, a good solvent for both blocks, followed by slowly
adding water to the solution. The resulting micelle solution was
then dialyzed against water to remove the THF (Scheme 3). The
micelles were analyzed by dynamic light scattering (DLS) and
atomic force microscopy (AFM) before cross-linking to ensure that
no structural or significant size changes occurred during irradiation
(Figure S1).
The cross-linking reaction was accomplished by irradiation of
the micelles with UV light in degassed water at room temperature.
The extent of the reaction was kept between 15 and 25% as longer
reaction times caused the nanoparticles to become insoluble.
Typically, only 3 min of irradiation was necessary to achieve this
conversion. Absorption at the peak absorbance of 278 nm was
measured to evaluate the extent of cross-linking. While a small
amount of intrachain cross-linking is inevitable, this contribution
is expected to be small due to the compact nature of the micelle
core.
Characterization of the micelles and nanoparticles (Table 2) was
accomplished in the solid state by AFM, as shown in Figure 1,
and in solution by DLS. The expected trend of increasing
nanoparticle diameter with increasing polymer molecular weight
is observed, with nanoparticle diameters ranging from 12.7 to 39.7
nm by AFM and 47.4 to 142.5 nm by DLS. The apparent diameter
of the nanoparticles is 2-3 times larger when measured using DLS
than when measured using AFM. This effect is likely due to the
swelling of the polymer chains in solution as well as the hydration
sphere surrounding the particles in aqueous environments. The
hydrodynamic diameter is a good indicator of the particle size in
vivo. DLS measurements show that the particles fall into the desired
range to effectively probe the limits of the EPR effect.1d
Lyophilized nanoparticle samples were used for the radiofluori-
nation experiments. Fluoride-18 was transported in its hydrated form
into the reaction vessel, and solutions of kryptofix-222, K2CO3,
and BHT were added. The water was removed by three consecutive
We chose to incorporate PEG into the hydrophilic monomer
because PEG is known to be nonimmunogenic and nontoxic, and
both linear and grafted PEG chains are known to provide a stealth
coating for nanoparticles in the bloodsteam.17 Mesylate leaving
groups were added to the end of the PEG chain for later
displacement by radioactive fluoride. The mesylate group was
preferred after examination of a variety of leaving groups due to
its high stability and solubility. The PEGylated norbornene imide
4 was synthesized by reaction of a previously reported monoami-
nated poly(ethylene glycol) (PEG) chain with exo-norbornene
anhydride 1, followed by reaction with mesyl chloride to produce
hydrophilic monomer 5. Many lengths of PEG chains were
examined, but PEG600 was found to provide the desired solubility
while retaining high reactivity during ROMP.
To demonstrate the capability of this synthetic method in forming
a broad range of nearly monodisperse nanoparticles, four different
polymers of varying molecular weights were made (Scheme 2).
Sequential copolymerization of the two monomers was carried out
under argon on the benchtop in THF. The ruthenium olefin
metathesis catalyst (H2IMes)(pyr)2(Cl)2RudCHPh (6) was chosen
as the initiator due to its ability to produce extremely low
polydispersity polymers18 and its good benchtop stability. Polym-
erization of the first monomer was complete after 1-2 min, at which
point the second monomer was added to the reaction mixture. All
block copolymerizations were complete in 30 min. Quenching with
ethyl vinyl ether and precipitation into ether/hexanes (1:1) afforded
the desired products in excellent yields. The materials showed PDIs
that were as low as polymers produced by other similar initiators.14
Characterization by gel permeation chromatography (GPC) showed
monomodal, very low polydispersity peaks for most of the polymers
(Table 1, entries 1-3). Broadening of the PDI was observed with
higher MW polymers (Table 1, entry 4). This broadening is likely
9
6732 J. AM. CHEM. SOC. VOL. 130, NO. 21, 2008

C O M M U N I C A T I O N S
Acknowledgment. We acknowledge the National Cancer
Institute (grant No. 5U54 CA119347) and the National Science
Foundation (grant No. CHE-0410425) for support of this research.
We would also like to thank Peigen Cao and Jim Heath for help
with the AFM images and size distribution measurements, Mark
Davis for assistance with DLS measurements, and Nagichettiar
Satyamurthy for help and advice with the radiolabeling experiments.
Supporting Information Available: Experimental procedures,
characterization data, AFM images, and radioTLC. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Brigger, I.; Dubernet, C.; Couvreur, P. AdV. Drug DeliVery ReV. 2002,
54, 631–651. (b) Sinha, R.; Kim, G. J.; Nie, S. M.; Shin, D. M. Mol. Cancer
Ther. 2006, 5, 1909–1917. (c) Ferrari, M. Nat. ReV. Cancer 2005, 5, 161–
171. (d) Heath, J. R.; Davis, M. E. Annu. ReV. Med. 2008, 59, 251–265.
(2) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release
2000, 65, 271–284.
(3) (a) Lee, S. M.; Chen, H.; Dettmer, C. M.; O’Halloran, T. V.; Nguyen,
S. T. J. Am. Chem. Soc. 2007, 129, 15096–15097. (b) Torchilin, V. P. Nat.
ReV. Drug DiscoVery 2005, 4, 145–160.
(4) (a) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.;
Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science
2005, 307, 538–544. (b) Smith, A. M.; Ruan, G.; Rhyner, M. N.; Nie, S. M.
Ann. Biomed. Eng. 2006, 34, 3–14. (c) Bagalkot, V.; Zhang, L.; Levy-
Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C.
Nano Lett. 2007, 7, 3065–3070.
(5) (a) Ihre, H. R.; De Jesus, O. L. P.; Szoka, F. C.; Frechet, J. M. J.
Bioconjugate Chem. 2002, 13, 443–452. (b) Lee, C. C.; MacKay, J. A.;
Frechet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23, 1517–1526. (c)
Jain, N. K.; Asthana, A. Exp. Opin. Drug DeliVery 2007, 4, 495–512.
(6) (a) Torchilin, V. P. J. Controlled Release 2001, 73, 137–172. (b) Sun, X. K.;
Rossin, R.; Turner, J. L.; Becker, M. L.; Joralemon, M. J.; Welch, M. J.;
Wooley, K. L. Biomacromolecules 2005, 6, 2541–2554. (c) Licciardi, M.;
Giammona, G.; Du, J. Z.; Armes, S. P.; Tang, Y. Q.; Lewis, A. L. Polymer
2006, 47, 2946–2955.
Figure 1. AFM images of nanoparticles. The nanoparticle diameters are
shown to increase with increasing molecular weight of the constituent block
copolymers.
additions and evaporations of acetonitrile. The nanoparticles were
added as a solution in acetonitrile, and the reaction mixture was
heated in a sealed vessel for 1 h at 120 °C. Because fluoride-18 is
the limiting reagent, the remaining mesylates were displaced with
additional fluoride-19 to avoid undesired reactions in vivo. The
radiofluorinated particles were isolated by diluting the reaction
mixture with water and passing this solution through neutral alumina
and strongly acidic cation exchange resin. These conditions
effectively removed all of the kryptofix and most of the unreacted
fluoride. Measurement of the specific activity of the nanoparticles
showed that 50% of the fluoride was incorporated into the product.
Radiochemical purity was measured by radioTLC (Figure S2),
which showed that the nanoparticles accounted for 61% of the total
specific activity, meaning that 31% of the fluoride was incorporated
into the nanoparticles. The bulk of the remaining activity came from
unreacted fluoride. 1H NMR has shown that the mesylates are
completely displaced during this process.
In conclusion, fluorine-18 functionalized nanoparticles have been
synthesized for use as in vivo molecular imaging agents. Block
copolymer micelles were formed by the ROMP of two new,
functional norbornene-based monomers. Cross-linking of the mi-
celles yielded discrete nanoparticles that exhibited hydrodynamic
diameters from 47 to 142 nm. Fluorine-18 was incorporated into
the nanoparticles using standard nucleophilic fluorination chemistry
in 61% radiochemical purity. Ongoing in vivo studies in mice will
establish the optimal size range of nanoparticles for exploitation
of the EPR effect.
(7) Heidel, J.; Mishra, S.; Davis, M. E. AdV. Biochem. Eng. Biotechnol. 2005,
99, 7–39.
(8) (a) Morawski, A. M.; Lanza, G. A.; Wickline, S. A. Curr. Opin. Biotechnol.
2005, 16, 89–92. (b) Seo, W. S.; Lee, J. H.; Sun, X. M.; Suzuki, Y.; Mann,
D.; Liu, Z.; Terashima, M.; Yang, P. C.; McConnell, M. V.; Nishimura,
D. G.; Dai, H. J. Nat. Mater. 2006, 5, 971–976. (c) Kobayashi, H.;
Brechbiel, M. W. AdV. Drug DeliVery ReV. 2005, 57, 2271–2286.
(9) Yang, Z.; Zheng, S. Y.; Harrison, W. J.; Harder, J.; Wen, X. X.; Gelovani,
J. G.; Qiao, A.; Li, C. Biomacromolecules 2007, 8, 3422–3428.
(10) (a) Bartlett, D. W.; Su, H.; Hildebrandt, I. J.; Weber, W. A.; Davis, M. E.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15549–15554. (b) Fukukawa, K.;
Rossin, R.; Hagooly, A.; Pressly, E. D.; Hunt, J. N.; Messmore, B. W.;
Wooley, K. L.; Welch, M. J.; Hawker, C. J. Biomacromolecules 2008, 9,
1329–1339. (c) Schipper, M. L.; Cheng, Z.; Lee, S.-W.; Bentolila, L. A.;
Iyer, G.; Rao, J.; Chen, X.; Wu, A. M.; Weiss, S.; Gambhir, S. S. J. Nucl.
Med. 2007, 48, 1511–1518.
(11) Lasne, M. C.; Perrio, C.; Rouden, J.; Barre, L.; Roeda, D.; Dolle, F.;
Crouzel, C. Top. Curr. Chem. 2002, 222, 201–258.
(12) Stubenrauch, K.; Moitzi, C.; Fritz, G.; Glatter, O.; Trimmel, G.; Stelzer,
F. Macromolecules 2006, 39, 5865–5874.
(13) (a) Bielawski, C. W.; Grubbs, R. H. Prog. Polym. Sci. 2007, 32, 1–29. (b)
Maynard, H. D.; Okada, S. Y.; Grubbs, R. H. Macromolecules 2000, 33,
6239–6248. (c) Rawat, M.; Gama, C. I.; Matson, J. B.; Hsieh-Wilson, L. C.
J. Am. Chem. Soc. 2008, 130, 2959–2961.
(14) Choi, T. L.; Grubbs, R. H. Angew. Chem., Int. Ed. 2003, 42, 1743–1746.
(15) Henselwood, F.; Liu, G. J. Macromolecules 1997, 30, 488–493.
(16) Khan, M.; Brunklaus, G.; Enkelmann, V.; Spiess, H. W. J. Am. Chem.
Soc. 2008, 130, 1741–1748.
(17) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V. P.;
Langer, R. Science 1994, 263, 1600–1603.
(18) (a) Camm, K. D.; Castro, N. M.; Liu, Y. W.; Czechura, P.; Snelgrove,
J. L.; Fogg, D. E. J. Am. Chem. Soc. 2007, 129, 4168–4169. (b) Slugovc,
C.; Demel, S.; Stelzer, F. Chem. Commun. 2002, 2572–2573.
JA802010D
9
J. AM. CHEM. SOC. VOL. 130, NO. 21, 2008 6733