Novel 18F Labeling Strategy for Polyester-Based NPs for in Vivo PET-CT Imaging

Article abstract of DOI:10.1021/acs.bioconjchem.5b00040

Drug-loaded nanocarriers and nanoparticulate systems used for drug release require a careful in vivo evaluation in terms of physicochemical and pharmacokinetic properties. Nuclear imaging techniques such as positron emission tomography (PET) are ideal and

Full text of DOI:10.1021/acs.bioconjchem.5b00040

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Article
Novel 18F labeling strategy for polyester-
based NPs for in vivo PET-CT imaging
Primiano Pio Di Mauro, Vanessa Gómez-Vallejo, Zurine Baz, Jordi Llop, and Salvador Borros
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00040 • Publication Date (Web): 24 Feb 2015
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Novel F labeling strategy for polyester-based NPs for
in vivo PET-CT imaging
1,2
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Primiano Pio Di Mauro , Vanessa Gómez-Vallejo , Zuriñe Baz Maldonado ,
3,*
1,2,**
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Jordi Llop Roig , Salvador Borrós
1 SagetisꢀBiotech;, Barcelona
2 Grup d'Enginyeria de Materials (GEMAT), Institut Quimic de Sarria, Universidad Ramon Llull,
3 Radiochemistry and Nuclear imaging, CIC biomaGUNE, San Sebastian, Spain.
4 Radiochemistry Department, CIC biomaGUNE, San Sebastian, Spain
*Coꢀcorresponding author jllop@cicbiomagune.es
** Coꢀcorresponding author salvador.borros@iqs.url.edu
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ABSTRACT
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Drugꢀloaded nanocarriers and nanoparticulate systems used for drug release require a
careful in vivo evaluation in terms of physicochemical and pharmacokinetic properties.
Nuclear imaging techniques such as Positron Emission Tomography (PET) are ideal and
nonꢀinvasive tools to investigate the biodistribution and biological fate of the
nanostructures, but the incorporation of a positron emitter is required. Here we describe a
novel approach for the 18Fꢀradiolabeling of polyesterꢀbased nanoparticles. Our approach
relies in the preparation of the radiolabeled active agent 4ꢀ[18F]fluorobenzylꢀ2ꢀ
bromoacetamide ([18F]FBBA), which is subsequently coupled to block coꢀpolymers under
mild conditions. The labeled block coꢀpolymers are ultimately incorporated as constituent
elements of the NPs by using a modified nano coꢀprecipitation method. This strategy has
been applied in the current work to the preparation of peptideꢀfunctionalized NPs with
potential applications in drug delivery. According to the measurements of particle size and
zeta potential, the radiolabeling process did not result in a statistically significant alteration
of the physicꢀchemical properties of the NPs. Moreover, radiochemical stability studies
showed no detachment of the radioactivity from NPs even at 12 hours after preparation.
The radiolabeled NPs enabled the in vivo quantification of the biodistribution data in rats
using a combination of imaging techniques, namely PET and Computerized Tomography
(CT). Low accumulation of the nanoparticles in the liver and their elimination mainly via
urine was found. The different biodistribution pattern obtained for the “free” radiolabelled
polymer suggests chemical and radiochemical integrity of the NPs under investigation. The
strategy here reported may be applied to any polymeric NPs containing polymers bearing a
nucleophile, and hence our novel strategy may find application for the in vivo and non
invasive investigation of a wide range of NPs.
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INTRODUCTION
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Nanomaterials (NMs) can be broadly classified as organic/polymeric, carbon based,
inorganic and hybrid. Inorganic NMs can have very different surfaceꢀtoꢀvolume ratios and
shapes1,2 and are ubiquitously utilized as food or paint additives,3,4 in the construction and
semiꢀconductor industries,5,6 in cosmetic applications7 and in many other industrial and
societal sectors. Carbon based NMs formed by carbon in sp2 conformation have recently
gained significant attention for their potential applications in a varied range of industries
such as spintronics,8,9 and sensor development.10,11 Organic/polymeric NMs are frequently
applied in nanomedicine.12 Finally, hybrid NMs combine materials of different nature, e.g.
as core/shell NMs or hierarchical structures, leading thus to a limitless number of scenarios
in terms of chemical composition, potential complexity of the NMs and their applications.
Among NMs, polymer based nanoparticles (PNPs) have been proposed as suitable drug
delivery vehicles. Since many drugs and therapeutic agents have poor pharmacokinetic
properties, their entrapment into PNPs can be anticipated as a valuable alternative to
enhance their therapeutic worth by improving their bioavailability, solubility in
physiological environment or circulation time.13,14 Additionally, carrier NPs can enable
delayed drug release and improve selective accumulation in specific tissues, by attachment
of specific targeting moieties on the NPs surface.
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Nanocarriers designed for the selective delivery of drugs to specific tissues or organs after
systemic administration require long circulation times in the bloodstream, in order to reach
the targeted organ or tissue before clearance or degradation occurs.15–18 This is not likely to
happen if the carrier is taken up by the mononuclear phagocyte system (MPS, also called
reticuloendothelial system or RES) immediately after intravenous administration, or if rapid
renal excretion occurs. Similar considerations are needed for nanosystems meant to be a
circulating reservoir for sustained release of therapeutic cargoes. Hence, investigation of
the pharmacokinetic properties of PNPs with biomedical applications is of paramount
importance.
Different approaches can be used to investigate the pharmacokinetic properties of PNPs.
Invasive visualization methods such as optical or electron microscopies are often employed.
Alternatively, in vivo imaging techniques such as Positron Emission Tomography (PET),
Single Photon Emission Computerized Tomography (SPECT), Magnetic Resonance
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Imaging (MRI) or Fluorescence Tomography can also be applied. Among them, PET offers
unparalleled in vivo sensitivity; in addition, due to the high penetration capacity of the highꢀ
energy (511 keV) gamma rays resulting from positron annihilation, it can be applied both to
small and large animal species as well as humans. Hence, PET represents an ideal tool to
investigate, in vivo and nonꢀinvasively, the biological fate of NPs, including stability in
biological environments (stealthiness), pharmacokinetics, and specific organ uptake.19
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The investigation of the pharmacokinetic properties of NPs using PET requires the
incorporation of a positron emitter in the NP (radiolabeling). To date, different PNPꢀ
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radiolabeling strategies have been developed, being attachment of a radiometal (e.g., Ga
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or Cu)20,21 by means of a bifunctional chelator (BFC) the most commonly used. Despite
very useful in a wide variety of scenarios, attachment of a radionuclide using BFCs has
several drawbacks. First, the formation of the chelatorꢀradiometal complex may be
hampered if the chelators (which are usually attached to the NPs during preparation) are
sterically hindered or not accessible. Second, the presence of non radioactivce metals with
the capacity to form complexes with the chelator all along the production process may
compromise the radiochemical yields and the specific radioactivity of the resulting
radiolabeled PNPs. Finally, release of the radiometal or transchelation due to the presence
of certain proteins during in vivo investigations may lead to completely wrong conclusions,
as the presence of the free radionuclide might be mistakenly interpreted as a signature of
the PNPs presence. This limitation can be circumvented by using appropriate chelators with
low dissociation constants; previous knowledge regarding the biodistribution pattern for the
free radionuclide may also help in interpretation of the results.
One alternative to the use of radiometals consists of radiolabeling the NPs with 18F, a
relatively long halfꢀlived positron emitter (T1/2 = 109.7 min), which can be produced as
[18F]Fꢀ in the GBq scale in all biomedical cyclotrons. Unfortunately, direct labeling with
[18F]Fꢀ requires harsh conditions (for example, high temperature and waterꢀfree organic
solvent), and long reaction times;22 alternatively, indirect labeling methods can be used. In
these, a radiolabeled prosthetic group is prepared in a first step and subsequently coupled to
the molecule of interest, usually under mild conditions. This strategy has been applied to
the preparation of radiolabelled NPs;23,24 of note, the presence of a reactive group on the
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surface of the NPs able to react with the labeled prosthetic group is required, and hence
this strategy cannot be directly translated to certain types of polymeric NPs. The
development of alternative synthetic routes is thus particularly convenient.
Very recently, we have reported the preparation of Paclitaxelꢀloaded PNPs using a
modified nanoprecipitation method.25 In this work, and following a similar synthetic
strategy, we present an unprecedented strategy for the preparation of analogous
radiolabeled PNPs. Our method relies on radiolabeling block coꢀpolymers using the
labeling agent 4ꢀ[18F]fluorobenzylꢀ2ꢀbromoacetamide ([18F]FBBA). These block coꢀ
polymers are ultimately incorporated in the PNPs as constituent elements. The strategy has
been applied to the preparation of radiolabeled, peptideꢀfunctionalized PNPs with potential
applications in drug delivery. Finally, quantitative biodistribution data was obtained in vivo
after intravenous administration in rodents using a combination of imaging techniques. The
strategy reported here relies in the condensation of the labeling agent [18F]FBBA with a
polymer containing a nucleophile, and subsequent utilization of the labeled polymer for the
preparation of radiolabeled NPs. Hence, our approach may find application in the
preparation of a wide range of radiolabelled polymeric NPs, and become thus an interesting
tool for the evaluation of the pharmacokinetic properties of newly developed PNPs.
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RESULTS AND DISCUSSION
Synthesis and characterization of [18F]FBBA and 18F-labeled polymers
The labeled active agent [18F]FBBA was synthesized following a well established 3ꢀstep
procedure (Figure 1).26 First, 4ꢀ[18F]fluorobenzonitrile ([18F]2) was prepared by reaction of
4ꢀCyanoꢀN,N,Nꢀtrimethylanilinium trifluoromethanesulfonate (1) with [18F]Fꢀ. After
purification using solid phase extraction and water removal using Na2SO4 and molecular
sieve, the cyano group in [18F]2 was efficiently reduced with LiAlH4 to yield [18F]3, which
was further reacted with bromoacetyl bromide to yield [18F]4. After purification using
HPLC, the presence of the desired labeled species was confirmed by HPLC and coꢀelution
with reference standard (Figure 2). Radiochemical yields similar to those previously
reported in the literature (12%, decay corrected; total synthesis time approximately 90 min
including purification) were achieved. It is worth mentioning that an alternative
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methodology for the preparation of [18F]FBBA with overall decay corrected radiochemical
yield of 75% (total synthesis time: 1 h) has been reported 27. In this work, the reduction step
was based on borohydride exchange resin in substitution of LiAlH4. This methodology was
not implemented in our lab; however, its utilization would lead to higher overall
radiochemical yields in the preparation of labeled polymers and NPs (vide infra).
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Figure 1 Scheme of the reaction for the preparation of [18F]FBBA ([18F]4); (i) azeotropically dried [18F]Fꢀ,
K2CO3, K2.2.2., MeCN, 130°C, 10 min; (ii) 0.1M LiAlH4, 2 min, 120 °C; (iii) bromoacetyl bromide in CH2Cl2,
2 min, RT.
Figure 2 HPLC chromatograms corresponding to: A) FBBA (reference standard solution, UV detector) and
B) [18F]FBBA (purified fraction, radiometric detector).
The synthesis of radiolabelled polymers using preꢀlabeled prosthetic groups has been
previously reported in the literature. For example, Herth and coꢀworkers28 [REF] reported
the preparation of labeled Nꢀ(2ꢀhydroxypropyl)ꢀmethacrylamide (HPMA)ꢀbased polymers
by aminolyzing functional reactive ester polymers, which were subsequently reacted with
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the labeling synthon 2ꢀ[18F]fluoroethylꢀ1ꢀtosylate ([18F]FETos). These polymers showed
excellent radiochemical stability and were used to perform biodistribution studies in
healthy and tumorꢀbearing animals 29,30
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Following a similar strategy to those previously reported, we approached the preparation of
18Fꢀlabelled PEGꢀthiolꢀacids by condensation of [18F]4 with the corresponding αꢀThioꢀωꢀ
carboxy poly (ethylene glycol) (MWs of 458.6 and 3300 Da). These experiments were
conducted to have a proof of principle of the feasibility of the condensation reaction, to be
translated later on to the radiolabeling of block coꢀpolymers P and 3P (vide infra). The
coupling reaction (see Figure 3 for PEGꢀthiolꢀacid with MW=458.6 Da) was optimized by
using different temperatures and reaction times, and the radiochemical conversion (RCC)
was determined by direct integration of the radiometric chromatographic profiles at
different reaction times. For the low MW PEGꢀthiolꢀacid, RCC values at 90°C reached a
plateau (>90% RCC) at t>10 min. A similar kinetic profile was also observed at T=70°C,
although lower conversion values (<85%) could be achieved and the plateau was slightly
delayed (t>20 min) (Figure S1). Very similar results were obtained for the higher MW
PEGꢀthiolꢀacid. A plateau was reached at t>10 min at 90ºC, with RCC values in the range
84ꢀ87%. These values dropped to 71ꢀ78% at T=70ºC (Figure S2).
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O
HN
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O
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HS
O
OH
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F
PBS / MeCN
T = 90ºC, 10 min
O
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Figure 3 Synthesis of [18F]ꢀFBBAꢀPEGꢀthiolꢀacid by the condensation of [18F]FBBA with PEGꢀthiolꢀacid
with Mw 458.6 Da
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Large scale synthesis under optimal conditions resulted in overall radiochemical yields
(referred to [18F]FBBA) of 51±7% (uncorrected) after purification (see Figure S3 for
example of chromatographic profiles). UPLCꢀESI/MS and MALDIꢀTOFꢀMS analysis of
the collected fractions confirmed the presence of the condensation products. Major peaks at
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+
m/z = 624.3 (M + H+) and 641.3 (M + NH4 ) were detected for low MW PEGꢀthiol acid
(Figure S4) and m/z = 1691.26 for high MW PEGꢀthiol acid, the latter corresponding
approximately to half of the molecular weight of the condensation product (Figure S5).
The first and second strategies used for the preparation of radiolabelled NPs (vide infra)
required the preparation of radiolabelled block coꢀpolymers P and 3P (namely, [18F]P and
[18F]3P). Incorporation of the labelled active agent [18F]FBBA into blockꢀco polymers P
and 3P was carried out following the chemical reaction schematized in Figures 4 and 5,
respectively. Radiochemical conversion values of 52% and 65% were achieved for polymer
P at 90°C and 120°C, respectively. However, at 120°C the reaction occurred faster,
reaching a plateau after 30 minutes. The plateau was reached only after 120 min when the
reaction was conducted at 90°C (Figure S6). For 3P block coꢀpolymer, radiochemical
conversion was approximately 60% after 1 hour when the reaction was carried out at 120°C
(Figure S7). After purification by HPLC, [18F]P and [18F]3P could be obtained in overall
decayꢀcorrected radiochemical yields of 17 ± 4% and 27± 7%, respectively, referred to the
starting amount of [18F]FBBA (total synthesis time starting from [18F]FBBA: 50 min for
[18F]P and 80 in for [18F]3P) .
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18F
P
PBS / MeCN
T = 90ºC, 30 min
O
O
O
O
O
O
H
O
O
O
HN
3
3
n
O
O
[18F]P
18F
Figure 4 Synthesis of [18F]FBBAꢀP block coꢀpolymer ([18F]P)
O
O
HO
HO
OH
OH
+
1,8-octanediol excess
MW reactor, 100W, 120oC, 1h
O
O
O
O
OH
HO
HS
NH
O
OH
+
n
O
MW reactor, 100W, 120oC, 1h
O
O
O
O
HN
Br
O
O
HS
NH
O
OH
+
n
O
3P
PBS / MeCN
18F
T = 90ºC, 30 min
O
O
O
O
O
O
S
NH
O
OH
n
HN
O
[18F]3P
18F
Figure 5 Synthesis of [18F]FBBAꢀ3P block coꢀpolymer ([18F]3P)
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Synthesis and characterization of [18F]-labeled NPs
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In a previous work we have developed a peptideꢀtargeted nano system able to modulate and
control the release of PTX across an in vitro BBB model based on human glioma cells
(results pending from publication). In continuation of this previous work, we selected here
AGBBB015F (15F) as the targeting peptide. 15F was introduced in 2007 by Demeule and
coꢀworkers31, and belongs to a family of peptides named Angiopeps, which were derived
from the Kunitz domain of aprotinin. Angiopeps are known to be able to pass through the
BBB via a mechanism which involves the LDLR (low density lipoprotein receptor).
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Here, by modifying the nano coꢀprecipitation method, 18Fꢀlabeled/peptideꢀfunctionalized
NPs were obtained following three different methods as specified in the experimental
section (see Figure 6 and Materials and Methods section for the preparation of 2P). These
three strategies resulted in values of radiolabeling efficiency ranging from 5 to 14 %,
related to the amount of starting radiolabeled polymer (strategies 1 and 2) or starting
amount of [18F]FBBA (strategy 3) (Table 1). Considering the radiochemical yields of all
individual steps, this results in overall decayꢀcorrected radiochemical yields of
approximately 0.1%, 0.45% and 1.2% (referred to [18F]Fꢀ) in overall preparation times of
200, 230 and 150 min for strategies 1ꢀ3, respectively. In other words, with a starting [18F]Fꢀ
activity of 37GBq (which is easily achieved in commercially available cyclotrons), the final
amount of radioactivity in the NPs is 10, 39 and 170 MBq, respectively, which is low but
still sufficient to approach in vivo experiments.
According to the measurements of particle size and zeta potential, the incorporation of the
radiolabel did not result in statistically significant alteration of the physicꢀchemical
properties of the resulting NPs. All the formulations exhibited average diameters in the
desired range (below 200 nm) with a narrow size distribution (polydispersity ranging from
0.05 to 0.282) (Table 1). Moreover, all formulations exhibited a net negative charge with
zetaꢀpotential values approximately of ꢀ30 mV.
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To confirm that the bioactivity of the coupled peptide is retained after incorporation into the
PNPs, a transendothelial transport experiments in an in vitro BBB model was performed
(see supplementary data, Figure S8)
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Figure 6 (A) Schematic illustration of the three strategies employed for the fabrication of 18Fꢀlabeled and
peptideꢀfunctionalizedꢀNPs via coꢀprecipitation. Labelled parts are (B) block coꢀpolymer P (strategy 1), (C)
block coꢀpolymer 3P (strategy 2) and (D) entrapped [18F]FBBA (strategy 3).
Radiochemical stability studies showed no detachment of the radioactivity for NPs
prepared following strategies 1 and 2 (radiochemical purity > 95% after 12 hours). Slow
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detachment of [18F]FBBA was observed at long times for NPs synthesized following
strategy 3 (% of radioactivity attached to the NPs: 78±12% at t= 1 h, 64±4% at t= 3 h,
42±8% at t= 12 h). As shown in a previous study,25 degradation of the NPs may occur
during the first days through uniform bulk degradation of the matrix. However, the
relatively fast detachment of [18F]FBBA could be attributed to a simple diffusion process
through the polymer core matrix, as a result of water penetration.
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Table 1 Radiochemical yields for the preparation of [18F]FBBA, [18F]polymers and [18F]NPs, for each of the
strategies of preparation of NPs (radionuclide anchored to polymers P and 3P, and encapsulation of
[18F]FBBA). Hydrodynamic diameters of the NPs, as determined by DLS, are also included.
18F-labelling
strategy
DC RCY1 for
[18F]FBBA2 (time)
DC RCY1 for
[18F]polymer3 (time)
DC RCY1 for
[18F]NPs4 (time)
Hydrodynamic
diameter (nm)5
[18F]P
[18F]3P
12% (90 min)
12% (90 min)
12% (90 min)
17% (50 min)
27% (80 min)
NA
5% (60 min)
14% (60 min)
10% (60 min)
173±13 nm
185±20 nm
168±27 nm
[18F]FBBA
1Decayꢀcorrected radiochemical yield; 2Referred to [18F]Fꢀ; 3Referred to [18F]FBBA; 4Referred to
[18F]polymer (strategies 1 and 2) or [18F]FBBA (strategy 3); 5as determined by DLS.
In vivo biodistribution in rats (PET-CT Imaging)
In vivo biodistribution studies using PET imaging were carried out using the labelled NPs
prepared according to strategy 2 (this is, using [18F]3P as labeled precursor). This decision
was made based on radiochemical yields during particle preparation (see Table 1) and
stability studies. The labelled precursors (namely, [18F]FBBA and [18F]3P) were also
investigated as controls. PET dynamic images were acquired after intravenous
administration of the labelled species and quantified by drawing volumes of interest (VOIs)
on the CT images for selected organs (i.e. kidneys, liver, stomach and brain).
Figure 7 displays the percentage of injected dose per gram of tissue (%ID/g) obtained after
analysis of the PET data. As can be seen, [18F]FBBA showed a high uptake in the brain a
short times after administration, followed by a progressive washꢀout at t>16 minutes. Such
high uptake may be due to the hydrophobic character of the molecule. [18F]FBBA also
showed significant uptake in the liver (approximately 10%ID/g at 9 min < t < 37 min) and
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in the stomach, while accumulation in the kidneys was lower than accumulation observed
for [18F]3P and [18F]NPs. This suggests a preferential hepatobiliary elimination pathway in
combination with renal excretion. Significant accumulation in the lungs (not shown in
Figure 7) was also observed, especially at short times after administration. This
accumulation decreased over time, although at long times the presence of radioactivity in
the lungs could still be detected (see Figure 8A for PETꢀCT images).
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Brain
Liver
FBBA
3P
FBBA
3P
NP
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Time (min)
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8
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Time (min)
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0
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Time (min)
150
200
Figure 7 Accumulation of [18F]FBBA (FBBA in the figure), [18F]3P (3P in the Figure) and 18Fꢀlabelled NPs
(NP in the Figure) in brain, liver, kidneys and stomach, expressed as percentage of injected dose (%ID) per
gram of organ (mean ± standard deviation, n=3) at different time points after intravenous administration.
[18F]3P showed low accumulation in the brain with fast washꢀout and very high
accumulation in the kidneys, although uptake in the liver and the stomach was also
significant. Again, this suggests elimination via the hepatobiliary system in combination
with renal excretion.
Interestingly, labeled NPs showed a low accumulation in the liver and elimination mainly
via urine. Accumulation in the liver increased fast to reach values around 4%ID/g; these
values rapidly decreased at longer times after administration until values close to 1%ID/g
were reached. Timeꢀactivity curves in the brain and in the kidneys followed a similar
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pattern to [18F]3P, while lower accumulation was observed in the stomach, with values
close to 1%ID/g all over the duration of the study.
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Actually, when NPs are administered, a variety of plasma proteins (e.g. opsonins) tend to
bind to their surfaces. The conjugates can then be recognized by the MPS and internalized
in macrophages, leading to a significant loss of NPs from circulation. The decoration of the
NPs with a PEG layer prevents opsonisation and sequestration by the MPS, resulting in
prolonged blood circulation.32,33 Hence, our results are in good agreement with the fact that
renal excretion of the NPs becomes the preferred route when the NPs are surfaceꢀ
functionalized with PEG; despite the negative superficial charge of the developed peptideꢀ
functionalizedꢀNPs, which favours the interaction with the MPS, opsonization and
consequently liver accumulation was clearly avoided thanks to stealthy behaviour awarded
to our nanoparticulate system. The low accumulation in the liver should also prevent
hepatotoxicity, which might be a consequence of high liver uptake.34
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Contrary to NPs, a marked initial renal uptake for [18F]3P block coꢀpolymer was observed.
These results confirm that low molecular weight block coꢀpolymers have a major tendency
to be eliminated via urine, contrary to larger molecules or NPs.34 Of note, the differences in
the uptake values obtained for [18F]3P and [18F]NPs suggest the chemical and
radiochemical stability of the NPs in vivo, at least during the duration of the study. As
shown in Figures 7 and 8, a low accumulation of NPs was observed in the brain despite the
presence of the peptide. The low uptake observed at short times after administration might
be due to the contribution of the presence of labeled NPs in the blood, and hence animal
sacrifice, organ removal and subsequent determination of the amount of radioactivity using
a gamma counter may provide more accurate information about the real concentration of
NPs in the brain. However, due to invasiveness of the technique, different animals have to
be utilized to obtain uptake values at different time points after administration.
It is worth mentioning that the residence time of the NPs in the body is longer that the timeꢀ
window in which images can be acquired (up to 8ꢀ10 hours), due to the relatively short
halfꢀlife of 18F. In order to obtain data at longer times after administration, different
labeling strategies using alternative radioisotopes would be required. A similar labeling
strategy can be envisaged by using radioiodinated prosthetic groups bearing
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bromoacetamide, such as Nꢀ(4ꢀiodobenzyl)ꢀ2ꢀbromoacetamide35. In this case, the positron
emitter 124I (T1/2 = 4.18 days) or the gamma emitter 123I (T1/2 = 13.22 hours) might be used,
enabling the acquisition of images at longer times after administration using PET or
SPECT, respectively. If SPECT is used, lower resolution images are obtained and absolute
quantification is more challenging. However, qualitative information about the
biodistribution pattern and biological fate of the NPs may be obtained.
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Interestingly, the synthetic route proposed here requires harsh conditions only for the
preparation of the labeling agent ([18F]FBBA) and subsequent formation of the labeled
polymers. However, preparation of the NPs takes place under mild conditions. Hence,
despite having the inconvenient that the label is incorporated in the first step of the process,
with the consequent decrease in radiochemical yields and potential increase in dose
exposure, NPs with functionalities sensitive to temperature or pH can be prepared. Hence,
this is a valid alternative when the NPs to be labeled do not have functionalities suitable for
the attachment of radiolabeled tags, or when the addition of chelators for subsequent
incorporation of radiometals is not feasible or convenient.
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Figure 8. PET images (Maximum intensity projections) for [18F]FBBA (A), [18F]3P (B) and [18F]NPs (C) at
different time points after administration. Frames within each timeꢀgap have been averaged to create the
images displayed. Computerised tomography (CT) images were adjusted along the Y axis for an appropriate
fitting with the tracer distribution image. The intensity of the PET images was adjusted by normalizing with
respect to injected dose.
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EXPERIMENTAL PROCEDURES
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The experimental part of this work has been carried out in the Molecular and Functional
Imaging Facility of CIC biomaGUNE. 1,8ꢀOctanediol (98%), 2,2‘ꢀdithiodipyridine (≥97%)
and polyethyleneglycol (PEG, Mw 1.5 KDa) were purchased from Sigma (Sigma–Aldrich,
USA). Glutaric acid (99%) was obtained from Alfa Aesar (USA). AGBBB015F
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(CGGKTFFYGGSRGKRNNFKTEEY))
peptide,
fluorescently
labeled
with
carboxyfluorescein, was synthesized by Innovagen AB (Sweden). PEG Thiol acids, αꢀThioꢀ
ωꢀcarboxy poly (ethylene glycol) (MW 458,6 and 3.300 Da) were supplied by Iris Biotech
GmbH (Germany). Ultrapure water (Type I water, ISO 3696) was obtained from a MilliꢀQ®
system (Merck Millipore). All other chemicals, unless otherwise specified, were of
analytical grade and were purchased from Sigma (Sigma–Aldrich, Germany).
Synthesis of the 18F-labeled prosthetic group [18F]FBBA
The prosthetic group 4ꢀ[18F]fluorobenzylꢀ2ꢀbromoacetamide ([18F]FBBA, [18F]4, Figure 1)
was synthesized following a previously reported methodology26 with minor modifications.
4ꢀ[18F]fluorobenzonitrile ([18F]2) was prepared by reaction of 4ꢀCyanoꢀN,N,Nꢀ
trimethylanilinium trifluoromethanesulfonate (1) with [18F]Fꢀ generated by proton
irradiation of [18O]H2O (>98%, Rotem Industries Ltd.); [18F]Fꢀ was first trapped in an anion
exchange resin and subsequently eluted with K2CO3/H2O and Kryptofix2.2.2/MeCN. After
azeotropic evaporation of the solvent, the dried potassium [18F]fluorideꢀKryptofix2.2.2
complex was dissolved in dry DMSO and the resulting solution was reacted with 1 (130ºC,
10 min). The reaction mixture containing [18F]2 was cooled, diluted with purified water (9
mL) and flushed through a Cꢀ18 cartridge (SepꢀPak®, Waters). The retained labeled
species was eluted with 2.5 mL of THF, dried over Na2SO4 and molecular sieve and reacted
with LiAlH4 in THF (120°C, 2 min) to yield [18F]3, which was further reacted with
bromoacetyl bromide (solution in CH2Cl2). After addition of 2 mL of MeCN/water 1/1, the
mixture was purified by high performance liquid chromatography (HPLC) using a
VP125/10 Nucleosil 100ꢀ7C18 semipreparative column (MachereyꢀNagel) as stationary
phase and MeCN/water 1/1 as the mobile phase to yield [18F]4 (retention time = 13 min) in
overall yield of 12% (decay corrected). The collected fraction was used without further
treatment for subsequent labeling steps. The presence of the desired product was confirmed
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by HPLC and coꢀelution with reference standard, using an Agilent 1200 Series HPLC
system with a multiple wavelength UV detector (λ = 254 nm) and a radiometric detector
(Raytest). A RPꢀC18 column (Eclipse XDB C18, 4.6x150 mm, 5 ꢁm particle size) was used
as stationary phase and water/MeCN (60/40) was used as the mobile phase at a flow rate of
1 mL/min (retention time = 9.5 min, injected volume = 20 µL).
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Synthesis of 18F-labeled PEG-thiol-acids
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The preparation of Fꢀlabeled PEGꢀthiolꢀacids was carried out by condensation of [18F]4
with the corresponding αꢀThioꢀωꢀcarboxy poly (ethylene glycol). These experiments were
conducted to have a proof of principle of the feasibility of the condensation reaction, to be
translated later on to the radiolabeling of block coꢀpolymers P and 3P (vide infra).
Experimentally, a 1 mg/mL solution of PEGꢀthiolꢀacid in 1:1 (v/v) phosphate buffer saline
and acetonitrile (total volume = 250µL), was mixed with a solution of [18F]FBBA in
water/MeCN (1/1, 250 µL). Two different PEGꢀthiolꢀacids with molecular weights of 458.6
(Figure 3) and 3300 Da were used, and experimental conditions were optimized (T = 70ꢀ
90°C, t=10ꢀ30 min) in both cases. The radiochemical conversion (RCC) was determined by
radioꢀHPLC, using the same experimental conditions as above.
Large scale syntheses were carried out using the optimal reaction conditions (T= 90°C, t =
10 min); the radiolabeled species were purified by HPLC under analytical conditions
described above using a 500 µL injection loop. The fractions containing the pure labeled
polymers were collected, allowed to complete decay and the identity of the labeled species
was confirmed by UPLC/ESIꢀMS (low MW PEGꢀthiolꢀacid) or MALDIꢀTOFꢀMS (high
MW PEGꢀthiolꢀacid). UPLCꢀMS analyses were performed using an AQUITY UPLC
separation module coupled to a LCT TOF Premier XE mass spectrometer (Waters,
Manchester, UK). An Acquity BEH C18 column (1.7 µm, 5 mm, 2.1 mm) was used as
stationary phase. The elution buffers were A (water and 0.1% formic acid) and B (Methanol
and 0.1% formic acid). The column was eluted with a gradient: t=0 min, 95% A, 5% B;
t=0.5 min, 95% A, 5% B; t=7.5 min, 1% A, 99% B; t=10min, 1% A, 99% B. Total run was
10 min, injection volume was 5 µL and the flow rate 300 µL/min. The detection was
carried out in negative ion mode, monitoring the most abundant isotope peaks from the
mass spectra. For MALDIꢀTOFꢀMS analyses, the sample preparation was carried out by
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deposition of 0.5 µL of sample directly onto a preꢀspotted Anchorchip (PACꢀII 96, Bruker
Daltonics) plate that contains αꢀcyanoꢀhydroxycinnamic acid matrix (HCCA). MALDIꢀ
TOF analysis was performed using an UltrafleXtreme III timeꢀofꢀflight mass spectrometer
equipped with a Nd:YAG laser (Smartbeam II, 355 nm, 1 kHz) and controlled by Flex
Control 3.3 software (Bruker Daltonics, Bremen, Germany). The acquisitions were carried
out in positiveꢀion reflectron mode at a laser frequency of 500 Hz. The spectrum was
acquired at 30% laser fluency and was recorded in the m/z range from 1400 to 2000. The
deflector cutoff was set at m/z 1000 and the spectrum resulted from accumulation of 1000
laser shots.
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Synthesis of 18F-labeled P block co-polymer ([18F]P)
The first strategy for the synthesis of radiolabeled NPs required the preparation of
radiolabeled block coꢀpolymer P, which was prepared by the esterification of 1,8ꢀ
octanediol with glutaric acid and subsequent reaction with PEG 1500. Glutaric acid (6 g, 45
mmol) and 1,8ꢀOctanediol (5.53 g, 37 mmol) were reacted in a microwave reactor
(Discovery CEM) at 100 W for 1 hour. The reaction was performed under vacuum (100
mbar) with continuous stirring by airꢀcooling to maintain the temperature constant at 120°C
to form the preꢀpolymer. Subsequently, PEG 1500 was added (1:1 weight ratio with the
preꢀpolymer) and the polymerization reaction was conducted in the microwave reactor with
the conditions described above (100 W, 1 hour) to yield P as a white solid.
For the radiosynthesis of [18F]FBBAꢀlabeled P block coꢀpolymer ([18F]P), a 1 mg/mL
solution of P in 1:1 (v/v) phosphate buffer saline and acetonitrile (total volume = 250 µL),
was mixed with an equal volume of a solution of [18F]FBBA in water/acetonitrile
(Figure 4). The reaction was incubated at a preꢀdetermined temperature and the
radiochemical conversion was monitored at different times from chromatographic profiles
(chromatographic conditions described above). Different incubation times (10ꢀ180 min)
and temperatures (90°C and 120°C) were tested in order to achieve optimal reaction
conditions.
Large scale syntheses for in vivo experiments were carried out using the optimal reaction
time (30 min) and temperature (120°C); the radiolabeled species was purified by HPLC
under conditions described above, using a 500 µL injection loop.
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Synthesis of 18F-labeled 3P block co-polymer ([18F]3P)
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The second strategy for the preparation of radiolabeled NPs (vide infra) required the
preparation of a novel block coꢀpolymer (3P) labeled with 18F (Figure 5). First, the
microwaveꢀassisted production of a preꢀpolymer (P*) was approached by reacting glutaric
acid and 1,8ꢀoctanediol at a molar ratio of 1:1.2 at 100 W for 1 hour. The reaction was
performed under vacuum with continuous stirring and airꢀcooling to maintain the
temperature at 120°C. The resulting preꢀpolymer P*, which contained an excess of 1,8ꢀ
octanediol, was reacted with the PEGꢀthiolꢀacid (3.3 KDa) to obtain 3P block coꢀpolymer.
This second reaction was conducted in a microwave reactor under experimental conditions
described above (100 W, 1 hour), using a 1:1 (w/w) ratio between P* and PEGꢀthiolꢀacid.
Finally, radiolabeling of 3P was achieved by condensation with [18F]FBBA, under the same
experimental conditions used for the preparation of 18Fꢀlabeled PEGꢀthiolꢀacids.
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Synthesis of block co-polymer 2P
In order to achieve targeted NPs, a block coꢀpolymer attached via a linker to peptide
AGBBB015F was used during NP preparation. For the preparation of the linker, 2,2’ꢀ
dithiodipyridine (MW:220.31, 66.4 mg, 2.14 mmol) in THF (5 mL) and AcOH (5 mL)
were added to an oven dried oneꢀnecked flask under nitrogen atmosphere. αꢀThioꢀωꢀ
carboxy poly (ethylene glycol) (MW 3317 Da, 500 mg, 1.07 mmol) dissolved in THF was
added dropwise and the solution was allowed to stir for three days. The product was then
precipitated by addition of hexane (100 mL) under low magnetic stirring. The solution was
refrigerated overnight at 8ꢀ10°C, and subsequently vortexed and centrifuged (two cycles,
2700 g, 15’ and 1300 g, 20’). After each cycle the supernatant was removed, the same
amount of hexane was added and a short vortex agitation to redisperse the pellet was
performed. Residual solvent was removed under vacuum to yield the product as a yellow
solid.
The coupling between Cysteine residues present at AGBBB015F and linker was carried out
in 5 ml PBS (10 mM, pH 7.4). Linker and peptide (1:1.05 molar ratio) were accurately
mixed and the reaction was stirred at 0 °C for 4 hours to reach quantitative conversion. The
solution was finally lyophilized to yield PeptideꢀTargeted Linker as a white powder.
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Finally, to obtain the block coꢀpolymer 2P, a preliminary step for the production of the preꢀ
polymer P* was needed. Glutaric acid and 1,8ꢀoctanediol (molar ratio of 1:1.2) were
reacted under microwave heating at 100 W for 1 hour. The preꢀpolymer P*, containing an
excess of 1,8ꢀoctanediol, was reacted with the linker bearing AGBBB015F in the
microwave reactor under identical conditions to those described above and using a ratio 1:1
(w/w) between preformed peptideꢀtargeted linker and preꢀpolymer P*. The resulting
targeted block coꢀpolymer was obtained as a white solid.
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Synthesis and characterization of [18F]-labeled/peptide-functionalized NPs
The synthesis of 18Fꢀlabeled/peptide functionalized NPs was approached following a
previously reported nano coꢀprecipitation method,25 with some modifications. Three
different strategies were followed, depending on the labeled species used. In all of them,
peptideꢀfunctionalized block coꢀpolymer 2P (Figure 6A) was used as one of the starting
materials. The first strategy (Figure 6B) consisted of dissolving block coꢀpolymers [18F]P
and 2P at 9:1 (w/w) ratio in 1 ml of acetone to form the diffusing phase. This phase was
then added to 20 ml of ultrapure water by means of a syringe controlled by a syringe pump
(KD Scientific) under magnetic stirring at room temperature; the needle was positioned
directly in the medium and a flow rate of 50 µL/min was used. The resulting suspension
was allowed to stir uncovered, in order to allow the evaporation of acetone. The suspension
was purified by centrifugation (Hettich Centrifuge, EBA 21, 4000 g, 45 min at RT) with
ultracentrifugal filters (Amicon Ultraꢀ15, Ultracel membrane with 100.000 MWCO,
Millipore, USA). The second strategy (Figure 6C) consisted of dissolving block coꢀ
polymers P, 2P and [18F]3P in the organic phase (acetone) at a ratio 8:1:1 (w/w), and
following exactly the same experimental procedure. Finally, and taking advantage of the
hydrophobic character of the labeled prosthetic group, a third strategy (Figure Error!
Reference source not found.6D) consisting of encapsulating [18F]FBBA into the peptideꢀ
functionalized NPs was approached. With that aim, the labeled species ([18F]FBBA) was
dissolved in the organic phase (acetone, 1 mL) together with the starting polymers (P and
2P), and the solution was added dropwise to the aqueous phase under continuous stirring,
following the same experimental procedure.
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In all cases, the physicochemical properties (size and zeta potential) of the resulting
18Fꢀlabeled/peptideꢀfunctionalized NPs were determined after complete decay as described
in our previous work.25 To assess the radiolabeling efficiency (R.E., %), the amount of
radioactivity in the pellet and the supernatant after centrifugation were determined, and
R.E. was calculated as the ratio between the amount of radioactivity in the pellet and total
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amount of radioactivity (pellet + supernatant)
.
To assess the radiochemical stability, NPs were prepared and purified as described above
and subsequently resuspended in 200 µL of physiologic NaCl 0.9% solution (Braun
Medical S.A.). The suspension was then divided into 3 different aliquots containing 50 µL
of the NPs each. The aliquots were kept at 37°C for 1, 3 and 12 hours, respectively. The
samples were then filtered (Amicon Ultraꢀ15, 100.000 MWCO) and radioactivity was
measured in a 2470 WIZARD2 Automate Gamma Counter (PerkinElmer). The dissociation
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of F (expressed in percentage) at each time point was calculated as the ratio between the
amount of radioactivity in the filtrate (decayꢀcorrected) and the starting amount of
radioactivity.
Animals
Healthy, 9ꢀ10 weeks aged SpragueꢀDawley rats (Harlan, Udine, Italy) were used to
examine the biodistribution pattern of radiolabeled NPs after intravenous injection. The
animals were maintained and handled in accordance with the Guidelines for
Accommodation and Care of Animals (European Convention for the Protection of
Vertebrate Animals Used for Experimental and Other Scientific Purposes) and internal
guidelines, and experimental procedures were approved by the local authorities. Rats were
acclimated to the housing facility at 22ꢀ24°C and 40ꢀ60% of humidity under light/dark
conditions for at least 5 days prior to experiments.
In vivo biodistribution in rats (PET-CT Imaging)
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Animals were anesthetized with 4.5% isoflurane in pure oxygen as carrier gas. Fꢀlabeled
NPs were suspended in a certain volume of saline solution (NaCl 0.9%) to obtain the
required concentration of radioactivity (37ꢀ74 MBq/mL) and intravenously administered
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via one of the lateral tail veins. Formulations of 18Fꢀ3P and 18FꢀFBBA were also
administered and used as controls.
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PETꢀCT imaging was performed on an eXplore VistaꢀCT camera (GE Healthcare).
Acquisition of the scans was started immediately after dose administration. Three beds
were defined to acquire whole body images (frames: 4 x 4 min, 4 x 7 min, 4 x 20 min, 4 x
30 min, total acquisition time = 244 min). After acquisition, a CT scan was performed for
later attenuation correction during image reconstruction. Random and scatter corrections
were also applied to the reconstructed images (2DOSEM iterative algorithm, 4 iterations).
PETꢀCT images were coꢀregistered and analyzed using PMOD image processing tool.
Volumes of interest (VOIs) were drawn in the kidneys, liver, stomach and brain, using the
CT images for anatomical reference. Time–activity curves (decay corrected) were obtained
for each organ as cps/cm3. Curves were transformed into real activity (Bq/cm3) curves by
using a calibration factor. Injected dose and organ mass normalizations were finally applied
to data to achieve percentage of injected dose per gram of tissue (%ID/g).
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CONCLUSIONS
This work describes a novel approach for the radiolabeling of PNPs and subsequent
utilization for the in vivo determination of the biodistribution pattern using a combination
of imaging techniques, i.e. Positron Emission Tomography (PET) and Computerized
Tomography (CT). These techniques represent an ideal tool for investigations of
pharmacological profiles of new nanosystems. The labeling approach is based on
radiolabeling one of the precursor polymers, which is finally integrated in the NP using a
nanoprecipitation method. Radiolabeled NPs showed values of size and zeta potential
equivalent to those of the unlabeled analogues, within the experimental error. The labeled
NPs enabled the quantification of the biodistribution data in rats up to 4 hours after
intravenous administration. PET images show low accumulation in the liver and
elimination mainly via urine. The different biodistribution pattern obtained for the “free”
radiolabeled polymer ([18F]3P) suggest chemical and radiochemical integrity of the NPs
under investigation. The strategy reported here might be applied to the preparation of other
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radiolabeled polymeric NPs, in order to determine their pharmacokinetic properties and
evaluate their potential therapeutic capabilities.
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ACKNOWLEDGMENTS
The authors would like to thank GEMAT group of IQS for preliminary synthesis work,
SagetisꢀBiotech for promoting the investigation, Mikel González and Mikel Errasti for
radioisotope production and radiochemical synthesis, Maria Puigivila for technical
assistance during in vivo experiments and image analysis and Dr. Javier Calvo for
assistance in mass spectrometry. The in vivo work was partially funded by Ministerio de
Economía y Competitividad (Project reference MAT2013ꢀ48169ꢀR).
SUPPORTING INFORMATION
Radiochemical conversions of [18F]FBBA into [18F]FBBAꢀPEGꢀthiolꢀacid (MW = 458.6
and 3300 Da) as a function of time and temperature, HPLC chromatograms, MS spectrum,
MALDIꢀTOFꢀMS spectrum of [18F]FBBAꢀPEGꢀthiolꢀacids, radiochemical conversion of
[18F]FBBA into [18F]P and [18F]3P as a function of time and temperature (Figure S1ꢀS7).
This material is available free of charge via the Internet at http://pubs.acs.org
ABBREVIATIONS
[18F]FBBA, 4ꢀ[18F]fluorobenzylꢀ2ꢀbromoacetamide; UV, ultraviolet; PEGꢀthiolꢀacid, αꢀ
Thioꢀωꢀcarboxy poly (ethylene glycol); RCC, radiochemical conversion; HPLC, highꢀ
performance liquid chromatography; UPLC, ultraꢀperformance liquid chromatography;
ESI, electrospray ionization; MS, mass spectroscopy; TOF, time of flight; THF,
tetrahydrofuran; AcOH, acetic acid; PBS, phosphate buffer solution; MW, molecular
weight; MWCO, molecular weight cutꢀoff; PTX, paclitaxel; BBB bloodꢀbrain barrier;
MPS, mononuclear phagocyte system; RES, reticuloendothelial system.
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