Synthesis, biodistribution, and microsingle photon emission computed tomography (SPECT) imaging study of technetium-99m labeled PEGylated dendrimer poly(amidoamine) (PAMAM)-folic acid conjugates

Article abstract of DOI:10.1021/jm901910j

Three conjugates based on dendrimer PAMAM generation five were synthesized and radiolabeled successfully. To investigate their tumor targeting, the in vitro and in vivo stability, cell uptake, in vivo biodistribution, and micro-SPECT imaging were evaluate

Full text of DOI:10.1021/jm901910j

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3262 J. Med. Chem. 2010, 53, 3262–3272
DOI: 10.1021/jm901910j
Synthesis, Biodistribution, and Microsingle Photon Emission Computed Tomography (SPECT)
Imaging Study of Technetium-99m Labeled PEGylated Dendrimer Poly(amidoamine)
(PAMAM)-Folic Acid Conjugates
Yuanqing Zhang,†,‡ Yanhong Sun,† Xiaoping Xu,†,‡ Xuezhu Zhang,†,‡ Hua Zhu,†,‡ Liliang Huang,†,‡ Yujin Qi,† and
Yu-Mei Shen*,†,§
†Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 2019 Baojia Road, Shanghai 201800, China, ‡Graduate School of the
Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China, and §Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong
University, 800 Dongchuan Road, Shanghai 200240, China
Received December 23, 2009
Three conjugates based on dendrimer PAMAM generation five were synthesized and radiolabeled
successfully. To investigate their tumor targeting, the in vitro and in vivo stability, cell uptake, in vivo
biodistribution, and micro-SPECT imaging were evaluated, respectively. The conjugate of 99mTc
labeled PEGylated dendrimer PAMAM folic acid conjugate (99mTc-G5-Ac-pegFA-DTPA) shows
much higher uptake in KB cancer cells and accumulated more in the tumor area than that of the other
two conjugates. The uptake in KB cells depends on the incubation time. The results of in vivo
biodistribution agree with the data obtained from micro-SPECT imaging. These studies show that
PEGylation of PAMAM dendrimer folic acid conjugate improves the tumor targeting. Folate-
conjugated dendrimer maybe developed to be potential radiopharmaceuticals and targeted drug
delivery systems.
Introduction
of dendrimers as targeting carriers in cancer therapy and
imaging. It is well established that the conjugation of special
targeting moieties to dendrimers can lead to preferential
distribution of the cargo in the targeted tissue or cells. Exam-
ples of these special targeting moieties include folic acid,8-10
antibody,11,12 peptide,13 and epidermal growth factor.14,15
Folate receptors (FR), which are 38 kDa glycosylphospho-
tidylinositolanchored proteins, exist in three major forms,
namely FR-R, FR-β, and FR-γ. The FR-R form is over-
expressed in many types of tumors including ovarian, endo-
metrial, breast, renal cell carcinomas, and so forth. The fact
that high affinity FR binding is retained when folate is
covalently linked via its γ-carboxyl group to a foreign mole-
cule, combined with the prevalence of FR overexpression
among tumors, so folate-based targeting systems present an
effectivemeans of selectively delivering therapeutic or imaging
agents to tumors.16-20 Folate-targeted technology has been
successfully applied to drug delivery, therapeutic agents, MRI
contrast agents, and gadolinium liposomes and radioimaging
of cancer cells.21-30
Nanoparticles are able to accumulate within tumor tissue
due to the widely reported enhanced permeation and retention
(EPRa) effect which relies on the passive accumulation of
colloidal macromolecules of 40 kDa and above in tumors.1,2
The EPR effect arises due to aberrant tumor endothelium,
which is a result of its “leakiness”, allows the penetration into
tumor tissue. Dendrimers are artificial macromolecules with
tree-like structures.3-6 They are hyperbranched and mono-
disperse three-dimensional molecules with defined molecular
weights and host-guest entrapment properties. Dendrimers
are synthesized from branched monomer units in a stepwise
manner, thus it is possible to precisely control their molecule
properties, such as size, shape, dimension, density, polarity,
flexibility, and solubility, by choosing different building/
branching units and surface functional groups.7 Furthermore,
the large numbers of surface functional groups on dendrimer’s
outer shell can be modified or conjugated with a variety of
interesting guest molecules. These specific properties make
dendrimers suitable for drug delivery systems. In the recent
years, increasing interest has been attracted to the application
A lot of literature has already reported that dendrimer folic
acid conjugates were fluorescein labeled and bioevaluated.8-10
However, the fluorescein labeled dendrimer conjugates is very
difficult to be detected in a live animal study. At the same time,
radiolabeled dendrimer conjugates is very convenient in small
animal single photon emission computed tomography
(SPECT) or positron emission tomography (PET) imaging
studies. The conjugate of dendrimer monoclonal antibodies
*To whom correspondence should be addressed. Phone: þ86-21-
39194692. Fax: þ86-21-39194691. E-mail: shenyumei@sinap.ac.cn.
a Abbreviations: PAMAM, poly(amidoamine); EPR, enhanced per-
meability and retention; FA, folic acid; FR, folate receptors; SPECT,
single photon emission computed tomography; PET, positron emission
tomography; PEG, poly(ethylene glycol); NHS, N-hydroxysuccinimide;
DCC, N,N-dicyclohexyl carbodiimide; EDC, 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide HCl triethylamine; DMSO, dimethyl sulf-
oxide; DMF, dimethylformamide; 1B4M-DTPA, 2-(p-isothiocyanato-
benzyl)-6-methyl-diethylenetriaminepentaacetic acid; ACN, acetonitrile;
TFA, trifluoroacetic acid.
conjugates have been radiolabeled with 111In or 153Gd or 125
I
radionuclides.31-34 Radiolabeled dendrimer-biotin and den-
drimer-avidin conjugates were also investigated in vivo.35,36
To the best of our knowledge, the live animal micro-SPECT
r
pubs.acs.org/jmc
Published on Web 03/30/2010
2010 American Chemical Society

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Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3263
Scheme 2. Preparation of NHS Ester from Folic Acida
Scheme 1. Synthesis of Diaminopolyoxyethylene1540a
a Reagents and conditions: (1) CH3SO2Cl, Et3N, CH2Cl2, rt, 12 h; (2)
NH4OH, rt, 48 h.
imaging study of the dendrimer complex has not been reported
so far. Technetium-99m is so far the most commonly used
radionuclide in nuclear imaging. More than 80% of all usually
used radiopharmaceuticals contain this short-lived metastable
radionuclide. This is due to the highly interesting physical
properties of 99mTc among which are its short half-life (6 h) and
γphoton emission of 140 keV, which is very important for both
effective imaging and patient safety perspectives. Technetium-
99m can be derived as a column elute from a 99Mo/99mTc
generator, which makes it readily available.37 Furthermore,
99mTc possesses latent chemical properties, facilitating thereby
the labeling of several types of kits for versatile diagnostic
applications. Recently, we reported radiolabeled dendrimer
folic acid conjugate has certain accumulation in KB tumor
tissue.38 To bind efficiently with the folate receptor positive
tumors, the structure of the dendrimer folic acid conjugate was
modified by PEGylation. Both of the biodistribution and
micro-SPECT imaging study revealed that the PEGylated
conjugate has highest concentration on the tumor while the
radiolabeled dendrimer without folic acid shows the lowest
accumulation in the three conjugates, which will be described
in this paper. Herein we report our detailed results on radio-
labeled dendrimer conjugates for potential SPECT imaging
agent.
a Reagents and conditions: DCC, NHS, DMSO, rt, 12 h.
the synthesis was shown in Scheme 3. H2N-PEG-FA was
synthesized as previously described for the synthesis of NHS
folate ester. The conjugation of folic acid to the unprotected
primary amine of PEG was a complicated process. The
byproduct of H2N-PEG-FA was accompanied with FA-
PEG-FA due to no selectivity of the primary amine group in
H2N-PEG-NH2. Additionally unreacted folic acid and H2N-
PEG-NH2 interfered with the purity of H2N-PEG-FA.
Because of the ionic difference of these molecules, we decided
to adopt Sephadex G-25 column for separation. The purity
(>95%)wasconfirmedbyanalyticalHPLC(datanotshown).
ESI mass spectroscopic techniques further confirmed the
conjugation of folic acid to the unprotected primary amine
of PEG. Compared to that of H2N-PEG-NH2, the molecular
weight of H2N-PEG-FA increased about 400-500, which
indicated only one folic acid molecular conjugated to the
H2N-PEG-NH2. Next, H2N-PEG-FA reacted with succinic
anhydride, and to control the process more easy, the mount of
succinic anhydride was 5-fold excess of H2N-PEG-FA. The
side product was separated by washing with acetone. The FA-
PEG-COOH was characterized through UV, IR spectroscopy,
and 1H NMR. In UV spectra, the peak at 282 nm indicates the
presence of molecular folic acid . Important peaks obtained for
the folic acid were at 1657.8 cm-1 (aromatic CdC bending and
stretching), 1477.2 cm-1 (CH-NH-CdO amides bending),
836.4 cm-1 (aromatic C-H bending and benzene 1,4-disub-
stitution). The 1H NMR spectra confirmed the conjugation of
the aromatic protons of folic acid (6.65, 7.55, and 8.66 ppm)
and the multiplets of PEG (O-CH2-) at 3.6 ppm.
Results
Chemistry. PEG bis amine (NH2-PEG 1540-NH2) was
synthesized in two successive steps (Scheme 1) in which PEG-
1540 was converted to CH3SO2-PEG 1540-SO2CH3 using
CH3SO2Cl, then CH3SO2-PEG 1540-SO2CH3 was con-
verted to NH2-PEG 1540-NH2 using ammonia hydroxide.
PEG bis amine synthesis was confirmed by IR spectroscopy
and 1H NMR. The IR spectrum of PEG 1540 revealed peaks
at 3440.1 cm-1 (O-H stretch), 1286.5 cm-1 (O-H deflec-
tion), 2857.0 cm-1 (C-H stretch), 1251.7 cm-1 (C-O
deflection), 1110.6 cm-1 (strong peak of C-O stretch of
ether), and so forth. The second step in the synthesis of PEG
bis amine was conversion of CH3SO2-PEG-SO2CH3 to H2N-
PEG-NH2, where one of the important peaks obtained was
at 3145.8 cm-1 (N-H stretch antisymmetric of substituted
1
primary amine). H NMR (CDCl3) δ (ppm): 3.91 (t, 4H,
NH2CH2CH2), 3.14 (t, 4H, NH2CH2), which were not found
in the 1H NMR of PEG 1540.
The active ester NHS-PEG1540-FA was obtained from FA-
PEG-COOH in a similar manner to synthesis of FA-NHS in an
earlier step. The NHS ester of PEG1540-FA was characterized
through IR spectroscopy. Important peaks obtained for the
NHS conjugated PEG-FA: 2930.8 cm-1 (carboxylic acid CdO
and O-H stretching unconjugated), 1699.6 cm-1 (ketones
CdO unconjugated stretch), which confirmed the synthesis
of NHS ester of folic acid. 1H NMR (CDCl3, 500 MHz): folic
acid 8.64 (1H), 7.62 (2H), 6.68 (2H), 4.31 (2H); PEG 3.50
(140H); NHS 2.54 (4H); all of them confirmed the successfully
syntheses of NHS ester of PEG-FA.
NHS ester of folic acid (Scheme 2) was synthesized using
DCC and NHS following by a reported method.18 The NHS
ester of folic acid was characterized through IR spectroscopy.
Important peaks obtained for the folic acid were at 1660.8
cm-1 (aromatic CdC bending and stretching), 1487.9 cm-1
(CH-NH-CdO amides bending), 828.4 cm-1 (aromatic C-H
bending and benzene 1,4-disubstitution), and for the NHS
conjugated folic acid 3689.9 cm-1 (amide N-H and CdO
stretching), 3001.5 cm-1 (carboxylic acid CdO and O-H
stretching unconjugated), 1711.6 cm-1 (ketones CdO uncon-
jugatedstretch), whichconfirmed the synthesis of NHS ester of
folic acid. This NHS activated folic acid was conjugated to
PEG bis amine (NH2-PEG 1540-NH2). The general strategy of
To increase the solubility and decrease the nonspecific
cellular uptake, the primary amine on the surface of PAMAM
dendrimer were partially converted to acetamide moieties in
the presence of acetic anhydride and triethylamine (Figure 1).

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3264 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhang et al.
Scheme 3. Synthesis of NHS-PEG1540-Folic Acid Conjugate from NHS-Folatea
a Reagents and conditions: (a) DMSO, 0 °C-rt, 12 h; (b) succinic anhydride, pyridine, DMSO, rt, 12 h; (c) DCC, NHS, DMSO, 0 °C-rt, 12 h.
1
1
The degree of acetylation was measured by H NMR. H
NMR spectrum of the acetylated dendrimer showed the
proton signal at d 2.35 ppm, which corresponded to the
methylene protons of -CH2C(O)- in dendrimer PAMAM
G5. The specific signal at d 1.93 ppm corresponded to the
methyl protons of induced acetyl groups. The integration ratio
of these two kinds of proton signals in the acetylated dendrimer
suggested that an average of 77 acetyl groups are present on
the surface of each G5 PAMAM dendrimer (Ac77-G5). There
is a little difference with the reported method due to reaction
conditions, especially temperature.26 Conjugation of FA to the
partially acetylated dendrimers was carried out via condensa-
tion between the γ-carboxyl group of FA and the primary
amine of the dendrimer. The active ester of FA, formed by
reaction with EDC in DMSO-DMF (1:3, volume ratio), was
added dropwise to a solution of DI water containing G5-Ac
(77) and vigorously stirred for 3 days to confirm the reaction of
FA and the G5-Ac (77) completes. The number of FA
molecules attached to one dendrimer G5-Ac (77) was deter-
1
mined also by H NMR.10,11,26 The aromatic folate proton
peaks could be observed at 6.83, 7.74, and 8.73 ppm. From the
integral ratio of the folate proton at 8.73 ppm to the methylene
protons of -CH2C(O)- in dendrimer PAMAM G5, approxi-
mately 4.5 folic acid molecular were found attached to each
molecule of PAMAM dendrimer. UV spectroscopy, utilizing
the free folic acid concentration calibration curve, determined
the number of folic acid molecules to be 4.9.42 By the other
method, attachment of folic acid was accomplished through
use of a PEG spacer. In the 1H NMR spectrum of the
conjugates, the aromatic folate proton peaks could be ob-
served at 6.87, 7.73, and 8.78 ppm, along with PEG protons at
3.64 ppm. The multiple proton peaks from PAMAM were

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Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3265
Figure 1. Synthetic procedure for multifunctional PAMAM dendrimer conjugates. Reagents and conditions: (a) Ac2O, Et3N, 18 h, rt;
(b) 1B4M-DTPA, pH = 9-10, 40 °C water; (c) FA, EDC, DMF/DMSO = 3:1; (d) FA-PEG-NHS, DMSO; (e) 1B4M-DTPA,
-
pH = 9-10, 40 °C water; (f) 1B4M-DTPA, pH = 9-10, 40 °C water; (g) Sn2þ
,
99mTcO4
.
found between 2.4 and 3.7 ppm. From the integral ratio of the
folate proton at 8.63 ppm to the multiplets of PAMAM,
approximately 5.1 folate-PEG moieties were found attached
to each molecule of PAMAM dendrimer. The number of FA
molecules (5.2) was also determined by UV spectroscopy. For
detecting the conjugates in live animals, we employed 99mTc as
radioactive nuclide, which is commercially available and easy
to coordinate with bifunctional chelating agent DTPA. The
partially acetylated dendrimer was reacted with 1B4M-DTPA,
whereas isothiocyanates are active enough to react very easily
with terminated primary amine of PAMAM, and the degree of
functionalization can be controlled by stoichiometric control
of reagents ratio and determined by a similar manner above
(1H NMR). In summary, each Ac-G5-DTPA contained 77
acetyl and eight DTPA molecules, Ac-G5-FA-DTPA con-
tained 77 acetyl, 4.8 folic acid molecules, and seven DTPA
molecules, and Ac-G5-pegFA-DTPA contained 77 acetyl, 5.2
folic acid molecules, and eight DTPA molecules. So three
compounds (Ac-G5-pegFA-DTPA, Ac-G5-FA-DTPA, and
Ac-G5-DTPA) contained the similar number (7-8) of DTPA
molecules. Ac-G5-pegFA-DTPA and Ac-G5-FA-DTPA con-
tained the similar number (4.8-5.2) of folic acid molecules.
Radiochemistry. All of the conjugates gave excellent radio-
chemical yield (above 95%), which can be used directly
without further purification (Figure 2). A high-performance
liquid chromatograph, equipped with a radioactivity γ detec-
tor, was used to monitor the conversion. The HPLC chro-
matograms in Figure 2 show that 99mTc-G5-Ac-pegFA-
DTPA (compound 10) had a retention time of 26.54 min,
the 99mTc-G5-Ac-FA-DTPA (compound 9) had a retention
time of 12.36 min, and the 99mTc-G5-Ac-DTPA (compound
8) had a retention time of 11.78 min. The mean recovery of
five determinations of radioactivity was 88.3% ( 1.0. It was
because the nuclides decay and there were inevitable losses.
In Vitro/in Vivo Stability Studies and Partition Coeffi-
cients. The in vitro stability in PBS and new-born calf serum
was studied (Table 1). The radioactive conjugates for all of the
radiolabeled compounds keeps excellent in vitro stability in
PBS and newborn calf serum at 37 °C within 6 h, and at least
86% and 84% conjugate still keeps the original structure,
respectively. In vivo, at least 80% of conjugate keeps good
stability within 6 h in the blood of normal mice for all of the
three conjugates.
Partition coefficients of the radiolabeled conjugates were
determined by the ratio between n-octanol and water. The
partition ratio of the three compounds: for compound 8 the
Log P is -2.167, for compound 9 the Log P is -2.030, and
for compound 10 the Log P is -2.061. The property of
excellent water solubility further improves the radiolabeled
yield. Octonol/water partition has a direct relationship with
radiochemical yield and in vivo distribution. Similar lipid-
water partition coefficient for studying whether folic acid
played a target role is a more objective tool.
Blood Clearance and Internalization. We next evaluated
the pharmacokinetic blood clearance of the three labeled
compounds. Thus, the normal healthy mice received an
intravenous dose of 0.74 MBq of the radiolabeled com-
pounds, and blood samples were collected at various time
intervals thereafter. As shown in Figure 3, 99mTc-G5-Ac-
pegFA-DTPA, 99mTc-G5-Ac-FA-DTPA, and 99mTc-G5-
Ac-DTPA were rapidly removed from circulation in the
mouse. The plasma half-life of the radiolabeled conjugates
were estimated to be 13.15 min for 99mTc-G5-Ac-pegFA-
DTPA, 13.75 min for 99mTc-G5-Ac-FA-DTPA, and 12.73
min for 99mTc-G5-Ac-FA-DTPA, and less than 10% of the
injected 99mTc dose remained in circulation after 6 h
(assuming that blood represents 5.5% of the total body
mass). These data indicate that dendrimers conjugates are
rapidly removed from circulation following intravenous
administration and that valuable tissue biodistribution data
can be obtained after only a few hours postinjection without
the concern for nonspecific tissue uptake due to bloodborne
radioactivity.
Cell uptake of 99mTc labeled compounds was quantitatively
measured, as shown in Figure 4. In the KB cell that express high
levels of FR, in vitro cell binding of 99mTc-G5-Ac-pegFA-
DTPA was approximately 15% cell binding of total added
radioactivity after incubation for 6 h at 37 °C. At the same time,
the cell binding of 99mTc-G5-Ac-FA-DTPA and 99mTc-G5-Ac-
DTPA were almost 11% and 10%, respectively. The data