Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-Functionalized Radiolabeled Gold Nanoparticles in Tumor Mice

Article abstract of DOI:10.1021/acs.bioconjchem.6b00102

(Graph Presented) To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of ⁶⁷Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7-14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice indicated that the presence of BBN influences to some degree the biological profile of the nanoconstructs. For IV administration, the receptor-mediated pathway appears to be outweighed by the EPR effect. By contrast, in IP administration, it is reasoned that the GRPr-mediated mechanism plays a role in pancreas uptake.

Full text of DOI:10.1021/acs.bioconjchem.6b00102

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Article
Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate
of Peptide-functionalized Radiolabeled Gold Nanoparticles in Tumor Mice
Francisco Silva, Ajit Prakash Zambre, Maria Paula Cabral Campello,
Lurdes Gano, Isabel Rego Santos, Ana Maria Ferraria, Maria João Ferreira,
Amolak Singh, Anandhi Upendran, António Paulo, and Raghuraman Kannan
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00102 • Publication Date (Web): 22 Mar 2016
Downloaded from http://pubs.acs.org on March 25, 2016
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Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-
functionalized Radiolabeled Gold Nanoparticles in Tumor Mice
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†
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‡§
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Francisco Silva , Ajit Zambre , Maria Paula Cabral Campello* , Lurdes Gano , Isabel Santos ,
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¥
‡
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⊥
Ana Maria Ferraria , Maria João Ferreira , Amolak Singh , Anandhi Upendran , António Paulo*
and Raghuraman Kannan*
‡¶€
†
Centro de Ciências e Tecnologias e Nucleares, Instituto Superior Técnico, Universidade de
Lisboa, Centro de QuímicaꢀFísica Molecular, Instituto Superior Técnico, Universidade de
#
,
¥
Lisboa, Portugal Centro de Química Estrutural, Instituto Superior Técnico, Universidade de
Lisboa, Portugal
‡
¶
€
⊥
Departments of Radiology, MUꢀiCATS, BioEngineering, and International Center for
Nano/Micro Systems and Nanotechnology, University of Missouri, Missouriꢀ65211, USA
Keywords: Peptide, GRP receptors, Gallium, gold nanoparticles, tumor.
Corresponding Authors: Raghuraman Kannan, Department of Radiology, One Hospital Dr.
Columbia, MO 65211. USA Eꢀmail: kannanr@health.missouri.edu
António Paulo and Maria Paula Cabral Campello,
Centro de Ciências e Tecnologias e Nucleares, Instituto Superior Técnico, Universidade de
Lisboa, Portugal Eꢀmail: apaulo@ctn.tecnico.ulisboa.pt
§
Contributed equally to this manuscript
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Abstract
To get a better insight on the transport mechanism of peptideꢀconjugated nanoparticles to tumor,
we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold
nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare
AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types.
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The DTPA derivatives were unable to provide a stable coordination of Ga and, therefore, the
functionalization with the BBN analogs was pursued only for the DOTAꢀcontaining AuNPs. The
DOTAꢀcoated AuNPs were functionalized with BBN[7ꢀ14] using a unidentate cysteine group or
a bidentate thioctic group to attach the peptide. AuNPs functionalized with thiocticꢀBBN
displayed highest in vitro cellular internalization (≈ 25%, 15 min) in GRP expressing cancer cells.
However, these results fail to translate to in vivo tumor uptakes. Biodistribution studies following
intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice
indicated that the presence of BBN influences to some degree the biological profile of the
nanoconstructs. For IV administration, the receptorꢀmediated pathway appears to be outweighed
by EPR effect. By contrast, in IP administration, it is reasoned that GRPrꢀmediated mechanism
plays a role in pancreas uptake.
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INTRODUCTION
Targeting nanoparticles selectively to tumor site remains a significant challenge. To overcome
this challenge, several research studies focused on understanding the transport mechanism of
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nanoparticles to tumor. Two types of mechanism, passive and active targeting are reported in
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the literature to explain delivery of nanoparticles to tumor. Passive targeting is based on the
leakiness characteristics of the tumor; whereas, active targeting is based on the receptors that are
overexpressed on the tumor. Nanoparticles choose either one or both of these mechanisms to
reach the tumor site. Previous studies demonstrate that numerous parameters influence the choice
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of mechanism for in vivo delivery of nanoparticles to tumor.
Several endogenous parameters
including size, charge, and surfaceꢀbound ligand play significant roles in deciding the mechanism
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of delivery of nanoparticles.
In addition, exogenous factors such as reticuloendothelial
system (RES) sequestration and bioꢀcorona formation are also responsible in the transport
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mechanism of NPs to tumor.
With regard to passive targeting, Xia and coworkers have
demonstrated that 30 nm gold cages performed better in vivo when compared with 55 nm
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nanocages. In sharp contrast, Chan and coworkers have shown that smaller size gold
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nanoparticles (20 nm) exhibit a lower degree of tumor accumulation than did 100 nm particles.
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These studies further demonstrate that multiple factors in addition to size play role in deciding
final in vivo destination of nanoparticles in passive targeting. A similar observation is noted in the
case of active targeting. Several novel approaches were developed for directing the nanoparticles
toward receptors that are over expressed on the surface of the tumor. A variety of biomolecules
including antibodies, ScFv fragments, affibodies, peptides, aptamers, and carbohydrates have
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been attached to nanoparticles and their selective uptake in tumor studied.
Even though
these approaches are relatively successful, it is not yet clear that surface bound biomolecules have
a convincing role in targeting the tumor. Pioneering work by Park and coworkers demonstrate
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that antibody targeted liposomes have not shown any increase in tumor uptake when compared
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with the nonꢀtargeted liposomes. Nevertheless, Davis and coworkers have demonstrated that
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targeted nanoparticles showed enhanced efficacy than the nonꢀtargeted analogue. As noted
above, the ground rules for developing a successful targeted nanoparticles are still emerging.
To obtain further insight on the role of surface bound biomolecules in targeted delivery, herein
we have systematically studied the in vivo tumor targeting characteristics of radiolabeled gold
nanoparticles (AuNPs) covalently conjugated with bombesin peptides. Bombesin (BBN) peptide
recognizes Gastrin Releasing Peptide (GRP) receptors that are over expressed in a variety of
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human tumors including prostate, breast and lung cancer.
In this study, the in vivo tumor
uptake of these AuNPs is studied in prostate tumor bearing mouse models using Gaꢀ67 as
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radiolabel. Ga is a γꢀemitting radionuclide that presents physical properties suitable for in vivo
imaging by Single Photon Emission Computed Tomography (SPECT); moreover, the congener
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Ga is an emerging positron emitter with increasing clinical relevance, namely for tumor
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imaging by Positron Emission Tomography (PET). For radiolabeling, galliumꢀchelating ligands
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DTPA or DOTA were surface attached aiming at a fast and stable coordination of Ga. Before
proceeding with the functionalization of the nanoparticles surface with the targeting peptides, it
has been investigated if each type of ligand was able to achieve the desired stable coordination of
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Ga. To confirm that active targeting plays a crucial role, receptorꢀblocking studies were
performed. We studied whether the route of administration, intravenous and intraperitoneal, plays
any role in active targeting mechanism. Our previous studies were focused more on in vitro
demonstration of nanoconjugates in recognizing receptors on the cell surface and peptide
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conjugated gold nanoparticles internalize primarily via receptor mediated endocytosis.
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addition, we have also shown that radioactive bombesin gold nanoconjugates ( AuNPs) localize
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in pancreas and have poor uptake in tumor in mice models. In the present study, we report the
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following results: (i) design, synthesis, and characterization of Gaꢀlabeled targeted AuNPs; (ii)
understanding the in vitro stability, GRP receptor recognition and internalization efficacy of
targeted AuNPs in human cancer cells; (iii) measuring the quantitative biodistribution of
nanoparticles in tumor bearing mice models; (iv) comparing uptake of targeted nanoparticles in
tumor mice by blocking receptors; and (v) evaluating the route of administration of nanoparticles
for optimal tumor uptake.
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RESULTS AND DISCUSSION
It is important to design a robust and tunable nanoconstruct to effectively study the mechanism of
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tumor uptake in mice models. We have set out to obtain a library of BBN targeted Gaꢀlabeled
nanoparticles of general formula [AuNP(L)(R)], where L represents a polyamino carboxylic
chelator and R can be a BBN derivative. These nanoconstructs would contain three important
components: (i) Component 1 (AuNPs): the nanoparticle core is comprised of rigid small core (3 ꢀ
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nm) sized gold nanoparticles (AuNPs). The variation in core size was minimized throughout
study. Several investigations have demonstrated that AuNPs are nonꢀtoxic, nonꢀimmunogenic,
and are ideally suited for our study; (ii) Component 2 (L): We investigated both DTPA and
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DOTA as ligands (L) for irreversibly chelating radiolabeledꢀ Ga and surface anchored to AuNPs.
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L contains both ‘SH’ group and amino carboxylates for attaching with AuNP and to chelate Ga
respectively. The radiolabeling method was chosen for performing quantitative in vivo
biodistribution studies, since the measurement of radioactivity is a much more reliable method
than the elemental analysis of gold (e.g. by fAAS, ICP or NAA). However, it is important to
ensure irreversible attachment of the radionuclide to the surface of nanoparticle; and (iii)
Component 3 (BBN): the targeting molecule chosen for the study is a receptorꢀavid peptide, a
bombesin derivative. Our rationale for choosing BBN is as follows: GRP receptors (called
bombesin type 2 (BB ) receptor) have high affinity for its natural ligand, bombesin (BBN).
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Several clinical studies have shown that BBN analogues have superior targeting characteristics.
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Scheme 1. Synthesis of TDOTA: i) MeOH, 24 h, RT; ii) Toluene, 24 h, reflux; iii) CH CN,
Na CO , 8 h, 70 ºC; iv) TFA/CH Cl , 2 h, RT; v) THF, Ph CSH, NaH, 30 min, RT; vi)
CH Cl , NHS, EDC, DIPEA, 24 h, RT; vii) DMF/CH Cl , DIPEA, 24 h, RT; viii)
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THF/MeOH/H O, LiOH, 24 h, RT; ix) TFA:H O:thioanisole:1,2-dithiolethane, 2 h, RT.
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The ligand framework – DTPA or DOTA – was attached on the surface of the nanoparticle by a
stable thiol bond. Such proximity between ligand and nanoparticle provide rigidity and enhanced
stability to the final construct. Several research studies have shown that both DTPA and DOTA
are excellent chelating ligands for stabilizing Gaꢀ67 in both molecular or nanosized
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frameworks.
As such, both DTPA and DOTA ligands lack chemical functionality to directly
attach with gold atoms. Hence, we devised synthetic strategies to incorporate thiol groups in the
ligand structure being aware that the modification of these polyamine chelators (L) can influence
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the in vivo stability of GaꢀL that directly depends on the coordination saturation and trans-
chelation with transferrin. We have focused on the already described thiolated DTPA derivative
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ꢀ[bis[2ꢀ[carboxymethylꢀ[2ꢀoxoꢀ2ꢀ(2ꢀsulfanylethylamino)ethyl]amino]ethyl]amino]acetic
(DTDTPA) and on a new thiolated DOTA derivative. This new DOTA derivative, trimethyl
,2’,2’’ꢀ(10ꢀ2(3ꢀ(tritylthio)propamido)ethyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀ
acid
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trityl)triacetate (TDOTA), relied on the introduction of a single 2ꢀethylamine group in the cyclen
framework, for further coupling to a thiolated pendant arm (Scheme 1). The synthesis of
TDOTA started with the Nꢀalkylation of cyclen by reaction with tertꢀbutyl Nꢀ(2ꢀbromoethyl)
carbamate (1). The resulting monoalkylated cyclen derivative (2), containing a BOC protected
pendant arm, was treated with methyl 2ꢀbromoacetate to afford 3. Then, removal of the BOC
protecting group from 3 with TFA gave compound 4 displaying a terminal amino function for
coupling of 3ꢀmercaptopropionic acid. Treatment of 4 with a NHS activated ester of 3ꢀ
mercaptopropionic acid (6), having the terminal thiol protected with a trityl group, yielded the
amide derivative 7. Basic hydrolysis of the methyl ester functions of 7 followed by acid
deprotection of the trityl protecting groups led to the desired final compound.
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Scheme 2. Synthesis of the non-targeted nanoconstructs stabilized with DTDTPA or
TDOTA, and the respective peptide conjugated and/or radiolabeled particles.
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Synthesis of targeted and non-targeted nanoconstructs
For synthesizing the nanoconstructs, we developed a stepꢀwise ligand/peptide incorporation
methodology. In the first step, the ligand framework was attached to the surface of AuNPs using
the thiol groups of the modified DOTA or DTDTPA. Previously developed procedure was used
to attach the DTPA derivative (DTDTPA) onto the surface of AuNPs, which led to the AuNP-
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DTDTPA (NP1) nanoconstructs (Scheme 2).
Details of the synthetic procedure are presented
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in the experimental section (see Supplementary Information). Similar to NP1, the strategy for the
synthesis of the AuNPs stabilized with TDOTA was also based on the modified procedure of
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Brust et al. As shown in Scheme 2, this involved the reduction of HAuCl •3H O with NaBH in
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the presence of TDOTA using a molar ratio of 1:2.5 (Au:TDOTA) that resulted in the formation
of a dark brown solution of DOTA stabilized gold nanoconstructs designated as AuNP-TDOTA
(NP2). AuNPꢀL (L = DTDTPA and TDOTA) were purified by centrifugation methods, washed
and used for subsequent steps.
As a next step, we focused on synthesizing radiogallium conjugates of NP1 and NP2 by reacting
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the nanoconstructs with GaCl . We optimized the procedure by radiolabeling nanoconjugates at
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different pH and reaction times. In a typical reaction, NP1 or NP2 (11.4 µmol) was dissolved in
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acetate buffer at pH 7 and mixed with GaCl (70ꢀ120 MBq) in 0.1 M HCl, the reaction mixture
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was left undisturbed at 50 C. Each reaction mixture was purified by ultraꢀfiltration and the
respective radiolabeled conjugate analyzed using RadioꢀTLC. RadioꢀTLC control provided a
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quantifiable data on the radiochemical yield and purity. The final Gaꢀlabeled nanoconstructs
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NP1- Ga and NP2- Ga were obtained with radiochemical yields of 86 and 88%, respectively,
and with a radiochemical purity > 95%.
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It is important to study the stability of the radiolabelled NP- Ga conjugates in the presence of
biologically relevant media and apoꢀtransferrin. Gaꢀ67 attached to weakly stable chelate is
susceptible for transꢀchelation with apoꢀtransferrin, resulting in leaching of gallium from the
chelate. Thus, this test acts as an independent measure to evaluate the stability of the
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nanoconjugate. For the stability studies, NP- Ga nanoconjugates were suspended in 0.1 M PBS,
NaCl 0.9%, cell culture medium and apoꢀtransferrin (3 mg/mL, in 10 mM NaHCO ) and
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incubated at 37 ºC. At different intervals of time, a small amount of solution was removed and
analyzed using radioꢀTLC (up to 24 hours). The details are presented in a graph (ESI-Figure 6).
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The Gaꢀlabelled nanoconstruct functionalized with DTDTPA (NP1- Ga) showed poor stability
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in apoꢀtransferrin when compared to the one functionalized with TDOTA (NP2- Ga). In
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addition, NP2- Ga has shown a moderate to high stability in all biological media, although,
there is some decrease in the percentage of radiolabeled nanoconstruct for the shortest incubation
times but for longer incubation times such percentage remained fairly constant. Due to the
inability of NP1 to provide a stable coordination of Gaꢀ67, we did not use this nanoplatform for
further functionalization with the BBN peptides. The observed instability would lead to unreliable
data regarding the biological profile of the corresponding BBNꢀconjugated AuNPs when
performing cellular (in vitro) and animal (in vivo) studies, due to a high probability that the
radioactivity distribution would not reflect that of the nanoparticles.
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After confirming that AuNPꢀTDOTA (NP2) nanoconstruct possess suitable coordinating
properties to attach Gaꢀ67, NP2 was functionalized with BBN peptides. We used two different
BBN analogs in our study (SSꢀBBN, SꢀKKKꢀBBN; Scheme 2). In both the analogs the amino
acid sequence BBN[7ꢀ14] remain unaltered; however, the functional group that allows for the
binding to the gold surface is altered. In SSꢀBBN, the thioctic acid group ligate with
nanoparticles, while for SꢀKKKꢀBBN, it was through cysteine ꢀSH. We hypothesized that
cysteine would allow increased loading of peptide on AuNPs. The possibility of increasing the
peptide loading was expected to enhance the targeting ability of the BBNꢀcontaining AuNPs.
We used a 1:2 ratio (w/w) of AuNP:BBN for functionalizing NP2 (Scheme 2), both with SSꢀ
BBN and SꢀKKKꢀBBN, to obtain NP3 and NP4, respectively. After reaction, the amount of
unreacted BBN analogs was estimated using HPLC. The concentration of SSꢀBBN attached in
NP3 was 0.86 µmol (0.98 mg), while the amount of SꢀKKKꢀBBN attached in NP4 was 0.17
µmol (0.24 mg) per mg of nanoparticle. As mentioned above, the conjugation of SꢀKKKꢀBBN to
the AuNPs involves uniquely the formation of one AuꢀS bond and, therefore, the coupling of the
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same number of peptide molecules involves less number of gold atoms if compared with SSꢀ
BBN. This is why one could expect that it should be possible to attach a high number of peptide
molecules to the AuNPs in the case of SꢀKKKꢀBBN. However, the involvement of two sulfur
atoms per molecule in the conjugation of SSꢀBBN to the AuNPs can lead to a more stable
binding, which eventually justifies the higher payload that has been achieved in the case of this
BBN derivative. The characterization details of these BBNꢀcontaining nanoconjugates and
respective precursor AuNPs (UVꢀvisible, TEM analysis, charge and hydrodynamic size
distribution, nanoparticle tracking analysis, XPS, proton and HSQC NMR) are presented in
supporting information (ESI-Figure 1 – ESI-Figure 5; Table 1; and ESI-Table 1).
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Table 1. Physico-chemical characterization of nanoconjugates synthesized in the present
study.
Nanoparticle* UV-Vis (nm)
TEM (nm)
Hydrodynamic size
nm) (PDI)
Zeta-potential
(mV)
(
NP1
NP2
NP3
NP4
520
520
520
520
2.28 ± 1.32
4.29 ± 1.60
4.79 ± 1.50
4.04 ± 1.52
100.6 (0.111)
20.6 (0.342)
22.5 (0.420)
47.35 (0.370)
ꢀ80.7 ± 15.6
ꢀ62.6 ± 18.6
ꢀ60.5 ± 16.4
ꢀ30.1 ± 16.8
*All measurements were performed by preparing the nanoparticle solution in DI water ( pH 6)
≈
Nanoconstructs NP3 and NP4 were successfully radiolabeled with Gaꢀ67 following the same
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procedure described previously for NP2 (Table 2). The resulting nanoparticles, NP3- Ga and
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NP4- Ga, were obtained with radiochemical yields of 69% and 71%, respectively, and with a
radiochemical purity > 95%.
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7
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The stability of NP3- Ga and NP4- Ga was studied in the presence of relevant biological media
and apoꢀtransferrin (ESI-Figure 6). Overall, NP3 and NP4 maintain a moderate to high stability
as seen previously for their precursor NP2. However, among these radiolabelled DOTA
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conjugated NP2-NP4, NP4 showed least stability. Nevertheless, more than 60% of NP4ꢀ Ga did
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not release Ga even after 24 h of incubation with cell medium. It is possible that the positively
charged amino acids (lysine) present in peptide backbone of NP4 repel with positive charged
gallium ions in the close proximity resulting in their leaching. Gallium ions farther from lysine
residues are stably coordinated and stay intact with no release during the study period.
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Table 2. Reaction conditions for the Ga-labelling of nanoconjugates and respective
radiochemical yields.
Reaction time
Radiochemical
yield (%)
Nanoparticle
Temperature (ºC)
(
min)
67
NP1- Ga
50
50
50
50
15
86
89
69
71
67
NP2- Ga
30
30
30
67
NP3- Ga
67
NP4- Ga
Receptor affinity and cellular uptake of NP3 and NP4
We used competitive binding assay to estimate the specificity of BBN conjugated nanoconstructs
to GRP receptors overexpressed on the surface of cancer cells. In this assay, the ability of the
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nanoparticles to displace radioiodinated specific binding ligand was estimated. We used IꢀTyr4ꢀ
BBN as radioligand to assess the affinity of NP(2-4) in human prostate cancer cells (PC3).
(Figure 1). As expected, NP3 and NP4 display a significant affinity towards GRP with IC50
values of 0.045 ± 0.003 and 0.160 ± 0.027 µg/mL, respectively. In a similar fashion, NP2 showed
no affinity for GRP receptors. Among all the nanoconjugates, NP3 has a lower IC and it can be
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attributed to the increased number of BBN peptide present in this construct.
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NP2
NP3
NP4
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IC50 = 0.16 ꢁg/mL
5
0
IC50 = 0.045 ꢁg/mL
0
ꢀ3
ꢀ2
ꢀ1
0
1
2
log [AuNP](ꢁg/mL)
Figure 1. Representative binding affinity (mean ± SD, n=4) of nanoconjugates NP2, NP3 and
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NP4 in PCꢀ3 cells by competitive assays with IꢀTyr ꢀBBN.
4
One of the crucial experiments to measure the specificity of radiolabeled nanoconjugates toward
GRP receptors is to understand the internalization pattern. We performed receptor uptake studies
in GRPrꢀpositive human prostate cancer PC3 cells, by exposing the cells to a solution of the
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radiolabeled NPs (NP(2-4)- Ga) in cell culture medium and incubation at 37 ºC for different
intervals of time (Figure 2). The internalization observed for NP2 and NP4 showed a similar
profile with a slow uptake reflected from the increase in radioactivity that reaches relatively low
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plateau values (< 2%). By contrast, NP3- Ga has shown a very high and rapid internalization
into the cells with almost 25% internalization after 15 min of incubation; thereafter, there is a
slow decrease in the uptake of radioactivity suggesting the release of the Ga ions from the cells.
As stated before, the peptide load of NP3 is roughly fiveꢀfold higher than NP4, which is certainly
related with the trend observed for the cellular internalization as well for the binding affinities of
the nanoconstructs towards GRPr in PC3 cells. Altogether, these findings indicated that the
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internalization of NP3- Ga should involve a receptorꢀmediated process. To further confirm that
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NP3- Ga is retained within PC3 cells, we investigated the efflux of radioactivity from the cell.
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Typically, NP3- Ga was incubated in PC3 cells for 15 minutes, and the unbound nanoconjugates
were washed. The radioactivity retained by the cells was measured. Then, the medium was
replaced with culture medium without any radioactive compound and the radioactivity release
was monitored at different intervals of time by washing and measuring the radioactivity
associated with the cells again. A fast washout of the radioactivity has been observed during the
first 2 h. However, thereafter, the efflux rate significantly decreases and the activity present in the
cells remains essentially constant after 4 h of incubation, reaching roughly 40% of the initial
radioactivity (ESI- Figure 8). Therefore, one can consider that the PC3 cells retained a
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reasonable amount of NP3- Ga, which was an encouraging result to further study the targeting
ability of these AuNPs in GRP receptor positive tumors.
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0
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NP2ꢀ Ga
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NP3ꢀ Ga
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NP4ꢀ Ga
20
10
0
6
7
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Figure 2. Cellular uptake (mean ± SD, n=4) of NP2- Ga, NP3- Ga and NP4- Ga in PC3 cells,
after incubation at 37 ºC at different intervals of time. Internalization is expressed as the
percentage of the applied radioactivity internalized by the cells.
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Figure 3. (a) Cellular uptake (mean ± SD, n=4) of NP3- Ga at 37 ºC in PC3 cells in the
presence and absence of i) BBN (1 µM/well) and ii) cold NP3 (8 µg/mL and 20 µg/mL per well);
(
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b) Comparative cellular uptake (mean ± SD, n=4) for NP3- Ga at 37 ºC in PC3, MCF7 and
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B16F1 cell lines; (c) Comparison of cellular uptake of NP3- Ga at 37 and 4ºC in PC3 cells; (d)
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Cellular uptake of NP3- Ga in the presence of amiloride, phenylarsine and cadaverine at 37 ºC
in PC3 cells. Internalization is expressed as the percentage of the applied radioactivity
internalized by the cells.
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Mechanism of Cellular Internalization of NP4- Ga Conjugate
As the focus of this study is to understand whether peptide conjugated nanoparticles undergo
active targeting mechanism, we have performed a very systematic study to evaluate the
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mechanism of uptake of NP3- Ga in GRPr expressing PC3 cells. Several studies described
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above demonstrate that NP3- Ga exhibit high stability, cellular internalization, and retention.
Therefore, our mechanistic studies were performed utilizing only this conjugate. We performed
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four experiments using NP3- Ga: (i) evaluate the effect of internalization of NP3- Ga after
blocking the receptors with free peptide or cold NP3; (ii) compare the internalization of NP3-
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Ga in GRP receptor positive (PC3) and GRP receptor negative cells lines (human breast cancer
MCF7 and mouse melanoma B16F1 cell lines); (iii) evaluate the energy dependence of the NP3-
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Ga in internalization; and (iv) evaluate the effect of internalization of NP3- Ga in the presence
of cellular pathway blockers. First, GRP receptors on PC3 cells were blocked using free BBN
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and subsequently incubated with NP3- Ga. Thereafter, the amount of nanoparticles internalized
at different time intervals was quantified (Figure 3a(i)). These studies showed that there is no
significant difference in the amount of internalized radioactivity between the blocked and the
nonꢀblocked cells. The presence of BBN on the surface of the AuNPs clearly influences its
internalization into the cells, but apparently the process involved is not mediated merely by the
specific interaction with GRPr, as indicated by the inability of cold free BBN peptide to inhibit
the uptake. It was hypothesized that the moderately large hydrodynamic size of NP3 (22.5 nm
(
PDI = 0.420)) could contribute for an easier saturation of the GRPr receptors in the cell
membrane. As invoked by other authors, due to their size, a single nanoparticle can block the
access to several receptor molecules, even without being involved in direct and specific
46
interactions with such molecules. On the flip side, a single NP3 having several copies of the
bioactive peptide can specifically interact with more than one GRP receptor, leading to a strong
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binding as found often for multimerized constructs, due to the soꢀcalled concept of avidity. For
all these reasons, monomeric BBN can be a less effective competitor in the GRP binding of NP3-
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Ga if compared with the cold NP3 itself. Therefore, blockade experiments using increasing
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concentrations of cold NP3 were performed (Figure 3a (ii)). No significant decrease of the
amount of internalized radioactivity was observed, even using a 5ꢀfold greater concentration of
NP3, compared to the concentration of the radiolabeled AuNPs. It should also be taken into
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ꢀ9
account that due to the low amount of Ga present in the radiolabeling mixture (< 4.7x10
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7
mmol) it is common to have nonꢀradiolabeled AuNPs in the final NP3- Ga solution. These nonꢀ
radiolabeled AuNPs will compete for the binding with GRP receptor. So far, the separation of the
radiolabeled AuNPs from the nonꢀradiolabeled ones was not possible with the available
methodologies. In summary, blocking the GRP receptors on the surface does not affect
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internalization of NP3- Ga in PC3 cells. Second, internalization ability of NP3- Ga was
investigated in both GRP receptor positive (PC3) and negative cell lines (human breast cancer
MCF7 and mouse melanoma B16F1). It is observed that the initial rate of internalization is
significant for the GRP positive PC3 cells (with > 20% of internalization observed), whereas in
the GRP receptor negative cell lines less than 5% of internalization was observed at 15 min
incubation. Moreover, there is a slow decrease in radioactivity from PC3 cells upon increasing
incubation time, while a reverse trend (i.e.) a steady increase of internalization with the time of
incubation was observed for the MCF7 and B16F1 cell lines. These results are indicative that the
presence of GRPr influences the cellular uptake of the nanoparticles, taking into account the
much faster internalization observed for the PC3 cells (Figure 3b). Altogether, these results
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prompt us to presume that the cellular internalization of NP3- Ga in PC3 cells is fairly mediated
by the GRP receptors. Third, in order to gain more insight into the involvement of different
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energy dependent pathways in the internalization of NP3- Ga in PC3 cells, cellular uptake was
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monitored at two different temperature conditions. The uptake of NP3- Ga in PC3 cells at 4 ºC
is less that at 37 ºC; these results confirm that its cellular uptake involves energy dependant
mechanisms, like phagocytic and endocytic pathways (Figure 3c). Similar results are observed in
our previous study on BBN conjugated gold nanocages; these cages showed decreased cellular
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uptake at 4 ºC in GRP expressing cancer cells.
Fourth, to investigate if the cellular internalization pathway is depended on receptorꢀmediated
endocytosis, cellular uptake was monitored in the presence of inhibitors of different cellular
transport processes such as amiloride, phenylarsine oxide or cadaverine (endocytosis or
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phagocytosis inhibitors). These inhibitors decreased the cellular uptake of NP3- Ga up to 20%
+
+
in PC3 cells (Figure 3d). Amiloride is a Na /H channel inhibitor that is known to block
macropinocytosis and phagocytosis pathways that correspond to nonꢀreceptor mediated
pathways. Phenylarsine oxide or cadaverine, are both clathrinꢀmediated endocytosis (CME)
4
8
inhibitors. In general, receptor based internalization of molecules involves clathrin machinery.
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These results indicate that the uptake of NP3- Ga occurs most probably via active phagocytic
and endocytic pathways, which in the latter case might involve the internalization of GRPr.
Recently, we have shown that the uptake in PC3 cells of BBNꢀcontaining gold nanocages is
mediated by CME, and confirmed the formation of characteristic clathrin coated pits with
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lysosomal release of the nanocages. For NP3- Ga, the cellular uptake mechanistic study was
not so detailed, and other internalization possibilities include other endocytic transport processes,
namely caveolae (lipid transport) or alternative pathways need to be investigated. As proposed by
other authors, the involvement of these alternative pathways may result from the aggregation of
3
3, 49, 50
the individual nanoparticles, since clathrin mediation is size dependent.
In fact, it is
considered that nanoparticles can utilize CME to internalize the cells when their dynamicalꢀ
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aggregates are not superior to a 300 nm size limit. As the hydrodynamic size of NP3 was 22.5
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nm (PDI = 0.420), it is conceivable that the internalization of NP3- Ga will occur through CME,
with involvement of GRP, only when these AuNPs present themselves to the cell surface as
individual “monomeric” nanoparticles.
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Figure 4. Biodistribution of (a) NP3- Ga and (b) NP4- Ga in tumor and organs in BALB/c
nude mice bearing human prostate PC3 xenografts, injected intravenously via tail vein injection.
Data are expressed as percentages of injected activity per gram of tissue (%ID/g), except for the
GI tract, which is expressed as percentages of injected activity per organ. Error bars represent the
mean ± standard deviation (n=3).
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Figure 5. Comparison of tumor uptake of the BBNꢀcontaining radiolabelled nanoconstructs at 24
hrs post injection time point.
In vivo Studies
It is important to understand the biodistribution of nanoconjugates in tumor bearing mice before
initiating mechanistic studies. Previous studies demonstrate that the nanoparticles preferentially
accumulate in RES organs –liver and spleen. For example, Gaꢀ67 labeled dextranꢀcoated iron
4
0
oxide showed 75% of ID accumulated in liver and spleen after 15 minutes post injection. Our
previous investigation showed that gum Arabic coated radioactive gold nanoparticle accumulates
51
in liver and spleen. Of interest to the present study, we have demonstrated that gold
1
nanoparticleꢀbombesin conjugates accumulate more than 50% ID/g in liver and spleen. If the
size of the nanoparticles is large, accumulation in lungs also predominate; however, smaller sized
52
particles excrete via urine. As noted in previous sections, size alone does not dictate the fate and
distribution of nanoparticles in vivo. Other factors such as charge, surface coating, zeta potential,
and protein corona around nanoparticles play crucial roles in their accumulation and excretion. In
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addition to the above, radiolabeled conjugates should establish in vivo structural integrity for
effective uptake. Poor stability leads to release of radiolabel from conjugate resulting in
diminished uptake in target organs, thus would draw wrong conclusions. Liu and coworkers have
shown that Cuꢀ64 alloyed gold nanoparticles showed uptake in spleen, instead of liver, due to
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release of the radiolabel and formation of smaller size particles. Smaller sized radiolabeled
particles were filtered by spleen and they used biodistribution data as a tool to establish the in
vivo stability. In the present study, the structural integrity of the nanoconjugate was evaluated by
measuring the uptake of radioactivity in RES and other nonꢀtarget organs. Particularly, we
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investigated the biodistribution and tumor uptake of NP3- Ga and NP4- Ga (Figure 4 and 5;
ESI-Table 2).
One of the important factors that represent the in vivo integrity of the nanoparticle is their uptake
in RES organs. In general, most nanoconjugates show very high uptake in these organs and,
therefore, it may serve as an independent parameter to evaluate their in vivo stability. For both
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Ga labeled AuNPs reported herein (NP3- Ga and NP4- Ga), the general trend of the RES
uptake remains similar for different time points, and the additional data are presented in the
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supporting information. At 4 hours postꢀinjection time point, NP3- Ga showed the lowest uptake
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in liver and spleen (27.1 and 10.6% ID/g) when compared with NP4- Ga (61.9 and 12.7 %ID/g).
A recent study with Gaꢀ68 labeled NOTA ligand conjugated to hydrophilic bombesin showed
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less than 5% ID/g uptake in liver and spleen. In another study, GaꢀDOTAꢀGABAꢀBBN
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showed less than 1% ID/g uptake in RES organs. These studies confirm that small molecule
BBN conjugates labeled with Gaꢀ67 or Gaꢀ68 show lower RES organ uptake, when compared
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with our Ga labeled BBNꢀcontaining AuNPs.
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To further evaluate the GRP receptorꢀtargeting efficacy of the radiolabeled nanoconstructs, the
uptake of NPs in pancreas, tumor and intestine were analyzed at different post injection time
points. It is known in the literature that the GRP receptor density in mice follow the order:
pancreas > tumor > intestine, and consequently it is common to observe high uptake in pancreas
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for BBN derivatives.
At 24h post injection (p.i.) the pancreas uptakes for NP3- Ga and NP4-
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Ga were 4.5 and 1.5% ID/g, respectively. Previous studies show that Gaꢀ68ꢀNOTAꢀBBN
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conjugate has an uptake of ~5 % ID/g in pancreas within 1 hour of injection and GaꢀDOTAꢀ
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GABAꢀBBN derivative showed 1.2% of uptake.
A very high uptake of 27% ID/g was
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observed in pancreas with Gaꢀlabeled NOTAꢀ8ꢀAocꢀBBN(7ꢀ14)NH2. In general, BBN
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conjugates showed 5 to 20 % ID/g of uptake in GRP receptor over expressing tissues.
When
compared with these values, the uptakes observed for our nanoconjugates in pancreas are
relatively close to the low range values that have been found in the reported studies. Additional
evidence for receptor targeting was obtained by comparing the tumorꢀuptake data of these
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conjugates. At 24h p.i, NP3- Ga and NP4- Ga showed 3.7 and 3.3 %ID/g of radioactivity
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uptake in tumor, respectively (Figure 5). GaꢀNOTAꢀBBN and GaꢀDOTAꢀGABAꢀBBN
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conjugates reported in literature showed more than 8% and 1.2% ID/g in tumor, respectively.
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Overall, nanoconstructs NP3- Ga and NP4- Ga display uptake in tumor, in line with values
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found from other BBN compounds reported. Although NP3- Ga showed a remarkably high
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cellular internalization compared to NP4- Ga that did not translate to the biodistribution profile.
At this point we had questioned whether it was EPR or receptor mediated uptake mechanisms that
were predominant for these nanoparticles. In order to answer this, further studies were performed
and described as follow.
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NP3ꢀ67Ga
NP4ꢀ67Ga
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Pancreas
Tumor
Pancreas
Tumor
Figure 6. Uptake of NP3ꢀ67Ga and NP4ꢀ67Ga in tumor and pancreas with and without blocking
by bombesin in BALB/c nude mice bearing human prostate PC3 xenografts at 4 h post injection.
Error bars represent the mean ± standard deviation (n=3). Statistical analysis of the data was done
with GraphPad Prism and the level of significance was set at 0.05. For Pancreas p=0.5434; for
Tumor p=0.5677. Thus values are not significantly different.
Mechanism of Tumor Uptake
To unravel the mechanism of passive versus active tumor targeting by peptideꢀconjugated
nanoparticles, we studied the uptake of nanoparticles in pancreas and tumor in mice after
blocking all nonꢀEPR pathways. In our experiment, we blocked receptors in tumors by injecting
free BBN in mice 30 minutes prior to administration of nanoconjugates. After 4 hours of
administration, mice were sacrificed and the radioactivity measured in pancreas and tumor
(
Figure 6). Our results indicate that blocking of GRP receptors by BBN showed no effect in
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uptake of nanoconjugates in both these organs. Between both nanoconstructs, NP3- Ga showed
a decrease of ~1% ID/g after blocking with BBN. The results suggest following important
conclusions: (i) BBN peptide conjugated nanoparticles are not utilizing receptor mediated
pathway as the primary route for targeting tumor; or (ii) EPR pathway is dominant in uptake of
NPs in tumor. In addition to the above, the removal of nanoparticles by RES organs also plays a
3
decisive role because the amount of nanoparticles accessible to tumor is largely limited. The
protein binding characteristics of nanoconjugates may also possibly decide the in vivo tumor
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uptake. Several reports have already suggested that the biomolecular corona around nanoparticles
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play important role in deciding the in vivo fate of the nanoparticle.
Protein corona becomes
very relevant in nanoconjugates that are not functionalized with PEG on the surface, as PEG
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molecules prevent corona effects. In fact, preformed albumin corona has been utilized as
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protective coating for delivery of nanoparticles. Dawson and coworkers have recently shown
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that protein corona can significantly alter the targeting characteristics of nanoparticles.
In
summary, even though targeting by bombesin nanoconjugates was achieved in vitro, the in vivo
targeting efficacy was dampened possibly due to the exposure of nanoconstructs to complex
biochemical milieu.
BBN Nanoconjugates recognize GRP receptor in vivo
To evaluate whether the route of administration of nanoparticles in mice have any effect in
minimizing protein corona formation which in turn would increase tumor uptake, we chose to
administer the conjugate through intraperitoneal (IP) route to mice. IP administrations of
nanoconjugates provide immediate access due to proximity of injection site to GRP receptors
present in pancreas. IP administration route limits the exposure of nanoconjugates to in vivo
milieu and decrease the protein corona formation. Previous studies have shown that exposure
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time of nanoparticles to serum protein is also an important factor in protein corona formation.
Furthermore, IP administration would decrease the RES uptake and increase the accessibility of
concentration of nanoparticles to target organs. Therefore, we performed following additional
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studies: (i) Biodistribution of NP3- Ga after IP administration. We have selected NP3- Ga due
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to its augmented in vitro cell uptake, stability and tumor uptake; (ii) Biodistribution of NP2- Ga
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after IP administration; (iii) IP injection of NP3- Ga after blocking the receptors with bombesin
(30 minutes prior to injection of nanoconjugate).
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Figure 7. Biodistribution results (mean, n=3; expressed as %I.D./g of organ) for (a) NP2- Ga
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and (b) NP3- Ga after i.p. administration in BALB/c nude mice bearing human prostate PC3
xenografts.
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The biodistribution profile of NP3- Ga after IP injection and IV administrations are different. In
particular, the radioactivity uptakes in the liver, spleen and lung are significantly lower, with
values < 5% %ID/g for all the postꢀinjection times. Moreover, there is a significant pancreas
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uptake of 9.7 ± 1.6 %ID/g at 4 h p.i.. The biodistribution profile of NP2- Ga can be considered
similar but displayed a lower pancreatic uptake (5.6 ± 0.6 %I.D./g at 4 h p.i.). The tumor uptake
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for NP3- Ga after IP administration is only slightly higher when compared with NP2- Ga (0.95
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±
0.03 vs 0.65 ± 0.11 %I.D./g at 24 h p.i.) (Figure 7; ESI-Table 3). Also the tumor uptake is
lower than that exhibited by the same nanoconjugates following IV administration (3.7 ± 0.5 %
I.D./g at 24 h p.i.). Altogether, the study showed that there is a slightly increase in uptake of NP3-
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Ga in pancreas. To understand whether this slight increase is receptor mediated, we performed
the receptor blocking experiment. In this experiment, BBN peptide was injected IV, 30 minutes
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prior to injection of NP3- Ga through IP. As shown in Figure 8, there is a decrease (≈ 34%) of
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the pancreas uptake of NP3- Ga, when blocked with BBN. The result is in accordance with
previously reported receptor saturating experiments utilizing antibody labeled nanoparticles. For
example, Cai and coworkers have demonstrated that antibody conjugated radiolabeled silica
nanoparticles showed a ~30ꢀ40% decrease in tumor uptake upon saturating the receptors with free
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antibody. These results suggest that uptake of NP3- Ga in pancreas is possibly being mediated
by GRP receptors, but other factors play crucial role in tumor uptake. Nanoconjugates need to
travel a longer distance to reach tumor and would meanwhile encounter with large number of
serum proteins. This is most likely the reason why the IP administration leads to a lower tumor
uptake in comparison to IV administration.
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Nonꢀblocked
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Tumor
Figure 8. Comparison of the pancreas and tumor uptake (mean ± SD, n=2; expressed as %I.D./g of organ)
for NP3- Ga after i.p. administration in BALB/c nude mice bearing human prostate PC3 xenografts,
treated (blocked) or nonꢀtreated with BBN, at 4 h p.i.
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CONCLUSIONS
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Our initial work was focused on studying the Gaꢀcoordination capability of two distinct AuNPs
platforms, one stabilized with a DTPA derivative (NP1) and another with a DOTA derivative
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(
NP2). Nanoconstruct NP1 lack adequate capacity to maintain optimal Gaꢀ67 coordination in the
presence of biological media and apoꢀtransferrin. On the other hand, NP2 showed moderate to
high stability, indicating that DOTAꢀcontaining AuNPs were optimal for Gaꢀ67 delivery, and
hence, were chosen for BBNꢀconjugation (NP3 and NP4).
Previous studies in the literature have resulted in conflicting conclusions on the tumor targeting
mechanism of nanoparticles, and the factors that govern targeting property of nanoparticles are
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poorly understood. Nanconstruct NP3- Ga displays a remarkably higher cellular internalization
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compared with NP4- Ga. However, this did not translate to the biological profile, as it was
observed that both nanoconjugates have a similar tumor uptake. We decided to investigate the
mechanism of these targeted nanoparticles and performed blocking experiments, wherein the
receptors were blocked by free peptide prior to administration of the BBNꢀconjugated AuNPs. No
difference in tumor uptake was observed. These results suggest that active targeting mechanism is
not playing a key role. Therefore, we believe that other factors, such as EPR and protein corona
influence the transport mechanism of targeted AuNPs to reach tumor. Aiming to obtain further
understanding on whether the BBNꢀconjugated AuNPs recognize receptors in vivo, we performed
experiments in which the nanoparticles were injected close to organs, other than tumor, that
express receptors for bombesin peptide. It is very well known that pancreas in mice overexpress
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GRP receptors, and therefore we administered NP3- Ga through IP route. IP injection provided
AuNPs in a close proximity to the receptors present in pancreas.
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Our results suggest that NP3- Ga interaction seems to occur with the GRP receptors in pancreas.
These nanoconstructs showed up to 3% ID/g more in pancreas compared with nonꢀtargeted ones
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(
NP2- Ga). To further probe on this hypothesis, we blocked the receptors with free bombesin.
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Here we observed a decrease of ∼2.5 to 3% in NP3- Ga uptake in tumor. Taken together, it
appears that active targeting plays a role – BBN nanoconjugates target GRP receptors in
pancreas, and BBN nanoconjugates showed more uptakes in tumor than nonꢀtargeted
nanoparticles. On the other hand, receptor blocking experiments reveal that uptake of the BBNꢀ
conjugated AuNPs in tumor is mediated by passive mechanism. The contribution of both the
mechanism operates in targeting of the AuNPs to tumor. However, the results pointed out that the
BBNꢀconjugated AuNPs are not utilizing the receptorꢀmediated pathway as the primary route for
targeting tumor, playing most probably the EPR pathway a predominant role. In the case of IP
administration, the nanoconjugates travel a longer distance to reach tumor than to reach intra
peritoneal or retroperitoneal organs. Hence, they have a better chance of getting involved in
interactions with circulating serum proteins during their way to the tumor sites, which may cause
the corona effect. In brief, it is our reasoning that the receptor mediated pathway in vivo is
outweighed by the passive EPR effect, or even hindered due to the possible formation of a protein
corona enveloping the nanoparticles. In summary, both active and passive targeting play a role in
governing the final in vivo fate of peptide conjugated nanoparticles. However, it is our reasoning
that the significance of these mechanisms is highly dependent on the nanoparticle structure and
its physicoꢀchemical properties. Our results encourage further evaluation of these nanoconstructs,
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considering their proven suitability to retain a stable coordination of Ga under a biological
milieu. We anticipate that such suitability will also apply for other medically relevant trivalent
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radiometals, such as In, Y or Lu just to cite a few, conferring a high potential to this newly
synthesized AuNPs in the design of multimodal tools for cancer theranostics. To fully explore
this possibility, still is crucial to provide the nanoparticle structure with the means to overcome its
inability to optimally reach the desired target tumor with adequate payload.
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EXPERIMENTAL PROCEDURES
All details of materials and methods including experimental procedures are provided in the
supplementary data.
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Supporting Information
Supporting Information is available on the ACS Publication website.
ACKNOWLEDGEMENTS
RK thanks Coulter Foundation for providing financial support. Fundação para a Ciência e
Tecnologia (FCT) is acknowledged for financial support (EXCL/QEQꢀMED/0233/2012).
2
C TN/IST authors gratefully acknowledge the FCT support through the UID/Multi/04349/2013
project and COST Action TD1004. C. Fernandes is acknowledged for the ESIꢀMS analyses
which were run on a QITMS instrument acquired with the support of the Programa Nacional de
Reequipamento Científico (Contract REDE/1503/REM/2005 ꢀ ITN) of FCT and is part of RNEM
ꢀ
Rede Nacional de Espectrometria de Massa. A. M. Ferraria would like to thank Prof. A. M.
Botelho do Rego for the discussion of XPS results and, also FCT for financial support of PEstꢀ
OE/CTM/LA0024/2013. F. Silva thanks FCT for the doctoral research grant
(SFRH/BD/47308/2008).
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