99mTc-Labeled Multifunctional Low-Generation ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

99mTc-Labeled Multifunctional Low Generation Dendrimer-Entrapped Gold Nanoparticles for Targeted SPECT/CT Dual-Mode Imaging of Tumors Xin Li, Zuogang Xiong, Xiaoying Xu, Yu Luo, Chen Peng, Mingwu Shen, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04827 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

ACS Applied Materials & Interfaces 99m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tc-Labeled Multifunctional Low Generation Dendrimer-Entrapped Gold

Nanoparticles for Targeted SPECT/CT Dual-Mode Imaging of Tumors Xin Li

a, 1

, Zuogang Xiong

b, 1

, Xiaoying Xu a, Yu Luo a, Chen Peng

b,

*, Mingwu Shen

a,

*,

Xiangyang Shi a, * a

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China b

Department of Radiology, Shanghai Tenth People's Hospital, School of Medicine, Tongji

University, Shanghai 200072, P. R. China

*

Corresponding

author.

E-mail

addresses:

[email protected]

[email protected] (M. Shen), [email protected] (X. Shi). 1

Authors who had eaqual contribution to this work.

1

ACS Paragon Plus Environment

(C.

Peng),

ACS Applied Materials & Interfaces

Abstract: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Development of cost-effective and highly efficient nanoprobes for targeted tumor single photon emission computed tomography (SPECT)/computed tomography (CT) dual-mode imaging still remains to be a challenging task. Here, multifunctional dendrimer-entrapped gold nanoparticles (Au DENPs) modified with folic acid (FA) and labeled with 99mTc are synthesized for targeted dual-mode SPECT/CT imaging of tumors. Generation 2 (G2) poly(amidoamine) (PAMAM) dendrimers (G2.NH2) conjugated with cyclic diethylenetriamine pentaacetic anhydride (cDTPAA) via an amide linkage and FA via a spacer of polyethylene glycol (PEG) were used for templated synthesis of Au core NPs, followed by labelling of 99mTc via chelation. The thus created multifunctional Au DENPs were well characterized. It is shown that the particles with an average Au core diameter of 1.6 nm can be dispersed in water, display stability under different conditions, and are cytocompatible in the studied concentration range. Further results demonstrate that the multifunctional nanoprobe is able to be utilized for targeted SPECT/CT dual-mode imaging of cancer cells having FA receptor (FAR)-overexpression in vitro and the estabilished subcutaneous tumor model in vivo within a time frame up to 4 h. The formed multifunctional Au DENPs synthesized using dendrimers of low generation may be employed as an effective and economic nanoprobe for SPECT/CT imaging of different types of FAR-expressing tumors. Keywords: Dendrimers; Gold nanoparticles; Folic acid targeting; SPECT/CT imaging; Tumors

2


Page 2 of 31

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Many common imaging technologies, such as magnetic resonance, computed tomography (CT), positron emission tomography, and single-photon emission computed tomography (SPECT), play important roles in tumor diagnosis. Each imaging modality possesses its own inherent advantages and disadvantges. For instance, CT imaging possesses high spatial and density resolution and can give information of the anatomic structure, but it lacks sufficient sensitivity and molecular details.1-4 SPECT imaging has the merits of high sensitivity and is capable to obtain information for functional imaging, but it is unable to provide anatomical information and lacks resolution.5-9 Therefore, it is necessary to integrate two different imaging modes together to surmount the limitations and combine advantages of each imaging mode.10-12 SPECT/CT has been identified as a powerful new molecular imaging technology due largely to the fact that it can obtain the metabolic information from SPECT and the anatomic diagnostic information from CT at the same time, thus significantly improving the accuracy of diagnosis.13 For efficient dual mode SPECT/CT imaging, it is necessary to develop the desired contrast agents that can integrate imaging elements of both SPECT and CT. Nanotechnology has been showing promising potentials in a wide variety of biomedical fields, such as disease diagnosis and treatment,14-17 biological sensing,18-20 drug

21-23

or gene delivery.24-26

Particularly, for the application of single-mode and dual-mode imaging, nanoparticles (NPs)-based contrast agents have been developed due to their unique properties including the facile surface functionalization, controllable in vivo behaviors, and extended circulation time in the blood.27 For SPECT/CT dual mode imaging applications, one has to label a radionuclide (e.g.,

99m

Tc) onto or

within the NP system. In most cases, the mode of CT imaging is just used to locate the skeleton or the whole bone structure of the animals, and no CT imaging contrast agents have been integrated onto or within the NPs; while the mode of SPECT is used to locate the targeted organ or disease site. For instance, the recently reported NP systems labeled with

99m

Tc have been employed for dual

modality SPECT/CT imaging of sentinel node,28 lung carcinoma of small animals,29 or the in vivo biodistribution of cubosomes and hexosomes after administration.30 Currently, there are few 3



Page 4 of 31

examples to integrate both SPECT and CT imaging components within or onto a single nanoparticle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

system for the applications of dual modality SPECT/CT imaging.31-32 To develop nanoscale contrast agents, one has to select an appropriate carrier system that is able to load the imaging agents. Poly(amidoamine) (PAMAM) dendrimers are known to be a family of branched macromolecules with good monodispersity that can be considered to be an ideal carrier system to load different imaging agents for biomedical applications.33 For instance, dendrimers or functionalized dendrimers can be used as templates to synthesize gold (Au) NPs or as stabilizers to stabilize Au NPs for blood pool, major organ, or tumor CT imaging.34-37 This is because Au NPs possess an X-ray attenuation property superior to a commerical iodine-based CT contrast agent (e.g., Omnipaque), which is believed to be ascribed to the fact that Au possesses a higher atomic number than iodine.1 Furthermore, through the simultaneous dendrimer periphery modification of chelator/gadolinium complexes and dendrimer interior Au NP entrapment, dendrimer-based multifunctional nanoplatforms have been explored for dual mode animal organ and tumor CT/MR imaging.38-40

Lastly, dendrimer periphery

diethylenetriaminepentaacetic acid (DTPA) for

can also 99m

be

modified

with a chelator of

Tc labelling, thus generating a nanoprobe for

SPECT imaging of tumors.41 These studies highlight the significance to utilize dendrimers as a unique nanoscaffold to create multifunctional nanoprobes for different imaging applications. For translational medicine application, it is desirable to using cost-effective low generation dendrimers, instead of highly expensive high generation (e.g., generation 5, G5) dendrimers. In our recent study, we have demonstrated that low-generation G2 PAMAM dendrimers having surface partially modified with polyethylene glycol (PEG) can be used to entrap Au NPs for tumor CT imaging.42 The major advantage to use PEGylated G2 dendrimers is that the PEGylation modification of dendrimer termini significantly improves the entrapment capability of Au NPs.42-43 The previous successes relevant to the use of dendrimer chemistry to fabricate multifunctional nanoprobes stimulate us to speculate that G2 dendrimers could also be utilized as a platform to generate a nanoprobe for SPECT/CT imaging applications. In this current study, multifunctional dendrimer-entrapped Au NPs (Au DENPs) modified with 4


Page 5 of 31


folic acid (FA) and labeled with 99mTc were generated for SPECT/CT dual mode imaging of tumors. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Amine-terminated G2 PAMAM dendrimers (G2.NH2) pre-functionalized with 99mTc chelator (DTPA) via an amide linkage and targeting ligand FA via a PEG spacer were used to entrap Au core NPs. Followed by

99m

Tc labelling via chelation, FA-targeted

99m

Tc-chelated Au DENPs were formed

(Figure 1a). The formed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} NPs were well characterized in terms of their structure, composition, colloidal stability, radio stability, and cytocompatibility. The developed nanoprobe was then used for dual-mode targeted SPECT/CT imaging of cancer cells in vitro and a tumor model in vivo. To the best of our knowledge, our work is the first to descibe the development of low-generation dendrimer-based nanoprobes for SPECT/CT dual mode imaging applications.

Experimental Section Materials. All chemicals and materials were purchased from commercial resources and were used as received. In all experiments, water used was purified according to our previous work.37 FA-PEG-COOH was created according to previous protocols reported in the literature.44 Synthesis of the {(Au0)6-G2-DTPA-PEG-FA} DENPs. G2.NH2 (20.0 mg) dissolved in 3 mL water was mixed with 3 molar equiv. of cDTPAA (6.6 mg) dissolved in water (2 mL) under stirring for 12 h to get a raw product of G2-DTPA. Then, 15 molar equiv. of FA-PEG-COOH (184.3 mg) pre-activated by EDC (38.5 mg) in water (5 mL) was reacted with the above G2-DTPA solution under stirring. Three days later, we dialyzed the mixture against water (9 times, 2 L) for 3 days via a membrane having an MWCO of 3 000. The dialysis liquid was freeze dried to get the G2-DTPA-PEG-FA product. The synthesized G2-DTPA-PEG-FA conjugates were utilized for templated synthesis of Au NPs through NaBH4 reduction. We selected the molar ratio of dendrimer/gold salt at 1:6. The procedure to synthesize the {(Au0)6-G2-DTPA-PEG-FA} NPs is the same as that described in our previous work.42-43 The {(Au0)6-G2-DTPA-PEG-FA} DENPs and {(Au0)6-G2-DTPA-mPEG} DENPs without 5



Page 6 of 31

FA modification were obtained. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Formation

of

the

{(Au0)6-G2-DTPA(99mTc)-PEG-FA}

DENPs.

99m

Tc-labeled

{(Au0)6-G2-DTPA-PEG-FA} DENPs were formed according to the previously reported method.45 Typically, {(Au0)6-G2-DTPA-PEG-FA} DENP (0.2 mg) and SnCl2 (100 µg) were added to a 5 mL vial containing 200 µL PBS (pH = 7.4), and sterile

99m

Tc-pertechnetate (740 MBq, 1 mL) was

rapidly added to the above mixture. After incubation for 30 min at room temperature, the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs were purified using PD-10 desalting columns. The non-targeted {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs were also prepared under the same experimental

conditions

for

comparison.

In

addition,

G2-PEG-FA

dendrimers

and

{(Au0)6-G2-PEG-FA} DENPs without DTPA modification were also prepared and labeled with 99m

Tc, respectively under the same experimental conditions. Characterization Techniques. 1H NMR, UV-Vis spectrometry, TEM imaging, dynamic light

scattering (DLS), ζ-potential measurements, X-ray attenuation property evaluation, and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were performed according to standard procedures reported in the literature.42-43 The radioactivity was measured with a CRC-15R radioisotope dose calibrator (Capintec, Inc., Ramsey, NJ). Radiochemical

Purity

The

Analysis.

radiochemical

purity

of

the

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} was measured by instant thin-layer chromatography (ITLC) using silica gel-coated fiber glass sheets (Macherey-Nagel, GmbH & Co. KG, Düren, Germany). Saline was utilized as the mobile phase, and the sheets were analyzed with a thin-layer chromatogram scanner (Bioscan Inc., Tucson, AZ). In Vitro Stability Assay. The colloidal stability of the {(Au0)6-G2-DTPA-PEG-FA} DENPs was evaluated by UV-Vis spectroscopy under varying pH and temperature conditions according to our previous protocol described in the literature.1 In order to further prove the stability of the {(Au0)6-G2-DTPA-PEG-FA} DENPs dispersed in PBS, their hydrodynamic size was measured by DLS within 7 days. The radio stability of the products after

99m

Tc labeling was tested by ITLC.

6


Page 7 of 31


{(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs (200 µL, 1 mg•mL-1) were dispersed in 2 mL of PBS. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ITLC was performed to estimate the radiochemical purity after the particles were incubated for 1 h, 4 h, and 8 h, respectively at 37 oC. Cell Culture. HeLa cells were routinely cultivated in MEM according to our previous work.14 HeLa cells cultured in FA-free medium overexpress high folic acid receptor (FAR), which were identified as HeLa-HFAR, while HeLa cells cultured in free FA-containing MEM ([FA] = 2 mM) have low-level expression of FAR (shortened as HeLa-LFAR). Without specific declaration, HeLa cells always represent HeLa-HFAR cells. In Vitro Cytotoxicity and Cellular Uptake Assays. MTT assay and morphology observation of HeLa cells were used to evaluate the cytotoxicity of the {(Au0)6-G2-DTPA-PEG-FA} or {(Au0)6-G2-DTPA-mPEG} DENPs at different concentrations according to protocols described in the literature.42, 46 Specific cellular uptake of the {(Au0)6-G2-DTPA-PEG-FA} DENPs within HeLa cells was evaluated by quantitative ICP-OES analysis and qualitative TEM observation according to the literature.46-47 In Vitro CT and SPECT Imaging of HeLa Cells. HeLa-HFAR or HeLa-LFAR cells were cultured according to the above conditions and incubated with fresh medium containing PBS, {(Au0)6-G2-DTPA-PEG-FA} or {(Au0)6-G2-DTPA-mPEG} DENPs at different concentrations (1000 and 4000 nM) for 3 h. After appropriate treatments as described in the literature,42 the cells were imaged by CT according to our previous work.42 For SPECT imaging, the cells cultured under the same conditions described above were incubated with the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} and {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs with different doses (100 and 500 µCi) for 3 h. Then the cells were treated under the above conditions and were scanned according to our previous work.48 In Vivo Micro-CT and SPECT/CT Imaging of a Xenografted Tumor Model. Animal experiments were carried out following the protocols authorized by the institutional animal care committee and also the policy of the National Ministry of Health. For in vivo micro-CT imaging, male 5-week-old nude mice (22-25 g, Shanghai Slac Laboratory Animal Center, Shanghai, China) 7



Page 8 of 31

were subcutaneously injected with 1 × 106 HeLa cells/mouse in the right foreleg. The tumor had a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

volume of 0.4-0.7 cm3 at about 3 weeks postinjection. {(Au0)6-G2-DTPA(99mTc)-PEG-FA} or {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs dispersed in PBS ([99mTc] = 740 MBq·mL-1, [Au] = 0.08 M, 100 µL) were injected intravenously via tail vein. We anesthetized the mice using 2% isoflurane through a mask while on the imaging bed. The tumor-bearing mice were CT scanned at 30, 90, 150 and 240 min postinjection using a NanoSPECT/CT In Vivo Animal Imager (Bioscan Ltd.,Washington, D. C.) with a tube voltage of 80 kV, tube current of 450 µA, and slice thickness of 45 µm. For

micro-SPECT/CT

imaging,

after

the

intravenous

injection

of

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} or {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs with the same dose as described above, SPECT/CT scans were performed using the same imaging system. All image data was reconstructed and analyzed by In Vivo Scope software supplied by the manufacturer. In Vivo Biodistribution. We used ICP-OES to analyze the tissue/organ biodistribution of the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} or {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs in the mice bearing tumors. At 24 h postinjection of the DENPs ([99mTc] = 740 MBq.mL-1, [Au] = 0.08 M, in 100 µL PBS), the mice were sacrificed and the tumor and major organs (heart, liver, spleen, lung and kidney) were extracted and weighed. Then, the Au uptake in different organ pieces was quantitatively analyzed by ICP-OES. Results were displayed as the percentage injected dose per gram of wet tissue (mean ± standard deviation, n = 4). Some more experimental details are provided in Supporting Information.

Results and Discussion Synthesis and Characterization of the {(Au0)6-G2-DTPA-PEG-FA} DENPs. To validate our hypothesis that low-generation dendrimers modified with PEG enable the convenient integration of both Au NPs and

99m

Tc within single NP system for SPECT/CT imaging applications, we

sequentially modified DTPA and PEGylated FA (FA-PEG-COOH) onto the G2 dendrimer surface 8


Page 9 of 31


via EDC coupling chemistry, followed by entrapment synthesis of Au NPs using the dendrimers as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

templated and 99mTc labelling (Figure 1a). Firstly, FA-PEG-COOH was synthesized by reacting NH2-PEG-COOH with FA and characterized by 1H NMR (Figure S1b, Supporting Information). Through NMR integration, we were able to estimate the number of FA conjugated to each PEG to be 0.7. The modification of DTPA and FA-PEG-COOH onto the G2 dendrimer surface was also characterized by 1H NMR. By integrating the related NMR peaks (Figures S1a and S1c, Supporting Information), the numbers of DTPA and FA-PEG-COOH attached onto each G2 dendrimer were measured to be 2.7 and 6.5, respectively. We also synthesized G2-DTPA-mPEG without FA for comparison. As shown in the 1H NMR spectrum (Figure S1d, Supporting Information), the number of mPEG-COOH linked onto each G2 dendrimer can be estimated to be 6.5, ensuring meaningful comparison. Then, the synthesized G2-DTPA-PEG-FA dendrimers were used for templated synthesis of Au NPs. It is noted that we selected the Au salt/dendrimer molar ratio of 6:1 to synthesize the Au DENPs, which was slightly lower than that used in our previous work (8:1).42-43 This ratio is very important for the formation of stabe Au DENPs using PEGylated G2 dendrimers as templates, since an Au salt/dendrimer ratio higher than 8:1 often leads to a certain degree of precipitation of the Au DENPs. The formation of Au core NPs led to a strong build up appearing in the range of 500-800 nm for the {(Au0)6-G2-DTPA-PEG-FA} DENPs in the UV-Vis spectrum (Figure 1b), which is ascribed to the light scattering effect induced by the NPs. In contrast, the G2-DTPA-PEG-FA dendrimers without Au NPs entrapped do not display such an absorption feature. The surface plasmon resonance band around 510-520 nm is not obvious, which is likely due to the quite small size of the Au NPs (< 2 nm) as shown in the TEM images (Figures 1c and 1d). This phenomenon has been reported in the literature.49-50 The absorption peak at 282 nm belonging to the typical FA absorption for both the G2-DTPA-PEG-FA dendrimers and {(Au0)6-G2-DTPA-PEG-FA} DENPs further confirmed the success of the FA conjugation to the dendrimer surface. The morphology, size and surface potential of the {(Au0)6-G2-DTPA-PEG-FA} and {(Au0)6-G2-DTPA-mPEG} DENPs were further characterized. High-resolution TEM (Figure 1c and 9



Page 10 of 31

Figure S2a, Supporting Information) show that both the {(Au0)6-G2-DTPA-PEG-FA} and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

{(Au0)6-G2-DTPA-mPEG} DENPs display a spherical or semi-spherical shape with a quite narrow size

distribution.

The

mean

diameters

of

{(Au0)6-G2-DTPA-PEG-FA}

the

and

{(Au0)6-G2-DTPA-mPEG} DENPs were estimated to be 1.3 nm and 1.6 nm, respectively (Figure 1d, Figure S2b, Supporting Information). High-resolution TEM image also reveals that both the {(Au0)6-G2-DTPA-PEG-FA} and {(Au0)6-G2-DTPA-mPEG} DENPs have high crystallinity as lattices can be clearly visualized (inset of Figure 1c and Figure S2a, Supporting Information). The hydrodynamic sizes of both DENPs were also analyzed by DLS and data reveal that the {(Au0)6-G2-DTPA-PEG-FA} and {(Au0)6-G2-DTPA-mPEG} DENPs have a hydrodynamic size of 311.5 ± 5.4 and 291.2 ± 7.5 nm, respectively (Table S1, Supporting Information). It is notable that the hydrodynamic sizes measured for both Au DENPs are much bigger than those measured by TEM. This is because TEM just measures the size of the single Au core particles, while DLS measures the clustered Au DENPs in aqueous solution, which may comprise of many single Au DENPs, in accordance with the literature data.42-43 Furthermore, zeta-potential measurements reveal that both the {(Au0)6-G2-DTPA-PEG-FA} and {(Au0)6-G2-DTPA-mPEG} DENPs have a surface potential of 10.6 ± 0.7 mV and 15.5 ± 1.2 mV, respectively, indicating the positive charge of G2 dendrimers is able to be effectively shielded via PEGylation modification, similar to our previous data.42-43 Stability of the {(Au0)6-G2-DTPA-PEG-FA} DENPs. Since the absorption feature change is directly associated with the change of the stability of the Au NPs, UV-Vis spectrometry was used to evaluate the colloidal stability of the {(Au0)6-G2-DTPA-PEG-FA} DENPs under varying temperature and pH conditions (Figure S3, Supporting Information). Our results reveal that the absorption feature of the {(Au0)6-G2-DTPA-PEG-FA} DENPs does not show any prominent changes, indicating that the particles possess good stability under the studied pH and temperature conditions.

Furthermore,

DLS

measurements

of

the

hydrodynamic

size

of

the

{(Au0)6-G2-DTPA-PEG-FA} DENPs were used to check their colloidal stability. Similarly, the particle hydrodynamic size does not have any appreciable changes after they were stored in PBS for a period of 7 days, further confirming their good colloidal stability (Figure S4, Supporting 10


Page 11 of 31


Information). The non-targeted {(Au0)6-G2-DTPA-mPEG} DENPs displayed similar stability (data 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

not shown), which ensures the meaningful comparison with the FA-targeted Au DENPs. Radiostability of the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs. Via a simple chelation step, 99m

Tc was labeled onto the surface of the {(Au0)6-G2-DTPA-PEG-FA} or {(Au0)6-G2-DTPA-mPEG}

DENPs. Instant thin-layer chromatography (ITLC) data revealed that the efficiency to label

99m

Tc

onto the {(Au0)6-G2-DTPA-PEG-FA} or {(Au0)6-G2-DTPA-mPEG} DENPs was 93% and 91%, respectively. In contrast, the efficiency to label 99mTc onto the G2-PEG-FA and {(Au0)6-G2-PEG-FA} DENPs without DTPA modification was less than 1%. Accordingly, for efficiecnt labeling of 99mTc, the modification of DTPA chelator onto dendrimers is essential. To ensure the effective SPECT imaging study, we investigated the radiostability of the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs via ITLC by assessing their radiochemical purity at different time periods (Figure S5, Supporting Information). The radiochemical purity of the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs is higher than 97.4% within 8 h after exposure to phosphate bufferer saline (PBS) solution (Table S2, Supporting Information), suggesting that the developed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs are radiostable, ensuring their further in vivo radionuclide imaging applications. Similarly, the non-targeted {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs also display good radio stability as confirmed by monitoring their radiochemical purity for the same time period (data not shown). Cytotoxicity Assay and Cell Morphology Observation. The cytocompatibility of the DENPs prior to

99m

Tc labelling was assessed by MTT assay of HeLa cell viability (Figure 2a). Clearly, the

viability of cells treated with the {(Au0)6-G2-DTPA-mPEG} or {(Au0)6-G2-DTPA-PEG-FA} DENPs at the concentration up to 4000 nM still remains 89.5% or above and does not have significant difference when compared with that of HeLa cells treated with PBS (p > 0.05). This suggests that both DENPs display good cytocompatibility in the concentration range studied. The cytocompatibility of the {(Au0)6-G2-DTPA-mPEG} or {(Au0)6-G2-DTPA-PEG-FA} DENPs was next validated by visualization of the morphology of HeLa cells (Figures S6b-f and S6h-l, Supporting Information). Clearly, HeLa cells treated with either non-targeted or targeted Au DENPs do not exhibit any prominent morphological changes when compared to the PBS control 11



Page 12 of 31

(Figures S6a and S6g, Supporting Information). Combined quantitative MTT assay data and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

qualitative

cell

morphology

observation

results

demonstrate

that

the

developed

{(Au0)6-G2-DTPA-mPEG} and {(Au0)6-G2-DTPA-PEG-FA} DENPs are non-cytotoxic in the investigated concentration range. X-ray Attenuation Property. For CT imaging applications, we explored the X-ray attenuation property of the developed {(Au0)6-G2-DTPA-PEG-FA} DENPs (Figure S7, Supporting Information). Omnipaque was also investigated for comparison. Clearly, the brightness of CT images increases with

the

Au

or

I

concentration

(Figure

S7a,

Supporting

Information),

and

the

{(Au0)6-G2-DTPA-PEG-FA} DENPs have significantly larger CT values than Omnipaque under the same Au or I concentrations (Figure S7b, Supporting Information). In both cases, the X-ray attenuation intensity increases with the Au or I concentration, and the considerably higher increasing trend of the {(Au0)6-G2-DTPA-PEG-FA} DENPs than that of Omnipaque suggests that the formed {(Au0)6-G2-DTPA-PEG-FA} DENPs possess an X-ray attenuation property better than Omnipaque. In Vitro Cellular Uptake. NPs modified with FA have been known to be rendered with targeting specificity to FAR-overexpressing cancer cells.35,

44

We next assessed the targeting

specificity of the {(Au0)6-G2-DTPA-PEG-FA} DENPs to FAR-expressing HeLa cells via the analysis of Au uptake in both HeLa-HFAR and HeLa-LFAR cells by ICP-OES (Figure 2b). Clearly, the Au uptake in HeLa-HFAR cells treated with the {(Au0)6-G2-DTPA-PEG-FA} DENPs is 2.0 times higher than that in HeLa-LFAR cells treated with the same Au DENPs and 3.5 times higher than that in HeLa-HFAR cells treated with the non-targeted Au DENPs (p < 0.01) at the particle concentartion of 1000 nM. At a higher concentration of 4000 nM, the targeting specificity of the {(Au0)6-G2-DTPA-PEG-FA} DENPs to HeLa-HFAR cells can be reserved, and the Au uptake is 1.8 times higher than that of the HeLa-LFAR cells treated with the targeted Au DENPs and 2.5 times higher than that of the HeLa-HFAR cells treated with the non-targeted Au DENPs (p < 0.01). Our results suggests that the modification of FA renders the Au DENPs with targeting specificity to HeLa-HFAR cells.

12


Page 13 of 31


The targeting specificity of the {(Au0)6-G2-DTPA-PEG-FA} DENPs was further validated by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TEM observation of cells (Figures 2c and 2d). It can be seen that both {(Au0)6-G2-DTPA-mPEG} and {(Au0)6-G2-DTPA-PEG-FA} DENPs can be uptaken by HeLa-HFAR cells after 3 h incubation; however, there are significantly more {(Au0)6-G2-DTPA-PEG-FA} DENPs uptaken in the cytoplasm of cells than the non-targeted {(Au0)6-G2-DTPA-mPEG} DENPs. This further proves that the developd {(Au0)6-G2-DTPA-PEG-FA} DENPs can be specifically taken up by HeLa-HFAR cells likely via receptor-mediated endocytosis. In Vitro Targeted CT and SPECT Imaging of Cancer Cells. The applicability to use the {(Au0)6-G2-DTPA-PEG-FA} DENPs for targeted cancer cell CT imaging in vitro was next tested (Figures 3a and 3b). Although the difference of the brightness of CT images of HeLa cells is not obvious (Figure 3a), similar to our previous study,34 quantitative CT value measurements show that HeLa-HFAR cells treated with the {(Au0)6-G2-DTPA-PEG-FA} DENPs have a CT value 1.8 times higher than that HeLa-LFAR cells treated with the same particles, and 2.2 times higher than HeLa-HFAR cells treated with the {(Au0)6-G2-DTPA-mPEG} at the same particle concentrations (1000 nM, p < 0.001, Figure 3b). At the particle concentration of 4000 nM, the CT contrast enhancement of HeLa-HFAR cells using targeted Au DENPs is 1.6 and 1.8 times higher than that of HeLa-LFAR cells treated with the targeted Au DENPs and that of HeLa-HFAR cells treated with the non-targeted Au DENPs, respectively. This suggests that the developed {(Au0)6-G2-DTPA-PEG-FA} DENPs enable targeted CT imaging of FAR-expressing HeLa cells in vitro. After

99m

Tc labelling, we used the formed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs for

targeted SPECT imaging of cancer cells (Figures 3c and 3d). It is clear that the SPECT images of HeLa-HFAR cells treated with the targeted Au DENPs are much brighter than those of HeLa-LFAR cells treated with the targeted Au DENPs and those of HeLa-HFAR cells treated with the non-targeted Au DENPs at the same radionuclide doses (Figure 3c). Quantative SPECT intensity measurements show that at a

99m

Tc dose of 100 µCi, the SPECT signal contrast enhancement of

HeLa-HFAR cells treated with the targeted Au DENPs is 1.7 times higher than that of HeLa-LFAR cells treated with same NPs, and 2.2 times higher than that of HeLa-HFAR cells treated with the 13



non-targeted 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Au

DENPs.

This

further

demonstrates

Page 14 of 31

that

the

formed

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs enable targeted SPECT imaging of FAR-expressing cancer cells in vitro. In Vivo Targeted CT and SPECT Imaging of a Tumor Model. With the success of the in vitro CT and SPECT imaging of cancer cells, we next used the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs for CT (Figure 4) and SPECT (Figure 5) imaging of a subcutaneous xenografted tumor model. As shown in Figure 4, the tumor CT reaches the peak value at 90 min for tumors treated with either the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} or the {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs (Figure 4c). However, the tumor CT value treated with the targeted Au DENPs is 1.3 times higher than that treated with the non-targeted Au DENPs (p < 0.001). Due to the FA-mediated specific targeting, the tumor CT value treated with the targeted Au DENPs is much higher than that treated with the non-targeted Au DENPs at the same time points (p < 0.01). The in vivo micro-CT imaging data suggest that the developed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs can be applied as a nanoprobe for targeted CT imaging of tumors. The potential to use the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs for targeted SPECT imaging of tumors was next validated. A dual mode of Micro-SPECT/CT was used, where CT was used to localize the animal skeleton and bones (Figure 5). Clearly, at each time point, the SPECT image of tumors treated with the targeted Au DENPs is much brighter than that treated with the non-targeted Au DENPs (Figures 5a and 5b). Quantitative SPECT signal intensity (Figure 5c) and SPECT intensity ratio (tumor/muscle, Figure 5d) measurements show that the tumor SPECT signal intensity and the SPECT signal ratio (tumor/muscle) of mice injected with the targeted Au DENPs is much higher than that of the non-targeted group at the same time points. For instance, at 90 min postinjection, the tumor SPECT signal intensity and SPECT signal ratio of tumor/muscle of mice injected with the targeted Au DENPs was 1.2 and 1.6 times higher than those injected with the non-targeted Au DENPs (p < 0.001). It should be noted that in Figure 5, the signal of both tumor and normal tissue in (a) was much higher than (b). In both cases, only one specific plane of the images was shown. In Fig. 5(c), we show the SPECT signal intensity of tumors that include all signals from 14


Page 15 of 31


different planes of the whole tumor area. Therefore, the quantitative SPECT signal intensity does not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

necessarily correlate well with the images shown in Figures 5a and 5b. Overall, our data indicate that the developed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs can be used as a nanoprobe for effective dual mode SPECT/CT imaging of tumors with a great FA-mediated targeting specificity. Due to the fact that FAR is overexpressed on the surface of different types of cancer cells such as ovary,51 human lung adencarcinoma,52 and human glioma,53 etc., it is expected that the developed {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs may also be used for targeted SPECT/CT imaging of these types of cancer cells. In Vivo Biodistribution. To investiagte the fate of the

99m

Tc-labeled Au DENPs in vivo,

ICP-OES analysis was performed (Figure 6). It is clear that at 24 h postinjection, all the organs especially the spleen and liver have higher Au content in the mice treated with either the targeted {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs or the non-targeted {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs than in mice treated with PBS. This suggests that both particles can be cleared by the reticuloendothelial system (RES) located in the liver and spleen, and metabolized through kidney. Importantly, due to the FA-mediated targeting, the Au uptake in the tumor region treated with the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs is 2.0 time higher than that treated with the non-targeted {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs, suggesting that more targeted Au DENPs are able to retain in the tumor region than non-targeted Au DENPs for an prolonged time period up to 24 h. It should be noted that due to the RES clearance of the particles, the uptake of either the targeted Au DENPs or the non-targeted Au DENPs in the spleen, lung, liver and kidney is still more significant than that in the tumors. However, due to the much higher tumor uptake of the FA-targeted

Au

DENPs

than

that

of

the

non-targeted

Au

DENPs,

the

developed

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs may be used as a promising radiopharmaceutical agent for tumor diagnosis.

Conclusion 15



We demonstrated a convenient approach to preparing multifunctional 99mTc-labeled Au DENPs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for targeted tumor dual-mode SPECT/CT imaging applications. G2 dendrimers covalently modified with DTPA and PEGylated FA onto their surface can be used to entrap Au NPs and be labeled with 99m

Tc via chelation. The developed multifunctional {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs can

be dispersed in water, display colloidal stability and radio stability, and are cytocompatible in the investigated concentration range. They can be used as a nanoprobe for targeted dual mode SPECT/CT imaging of FAR-expressing cancer cells and a tumor model. The developed cost-effective low generation dendrimer-based nanodevices may be used for accurate diagnosis of different types of cancer.

Acknowledgments This research is financially supported by the National Natural Science Foundation of China (21273032 and 81401458), the Science and Technology Commission of Shanghai Municipality (15520711400 for M. Shen), the Sino-German Center for Research Promotion (GZ899), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. X. Li thanks the Innovation Funds of Donghua University Master Dissertation of Excellence (15D310518).

Supporting Information Additional experimental details and data of DLS, zeta potential, radiochemical purity, 1H NMR, TEM, colloidal stability assessment, cell morphology, and CT phantom. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1) Guo, R.; Wang, H.; Peng, C.; Shen, M.; Pan, M.; Cao, X.; Zhang, G.; Shi, X. X-Ray Attenuation Property of Dendrimer-Entrapped Gold Nanoparticles. J. Phys. Chem. C 2010, 114, 50-56. (2) Hyafil, F.; Cornily, J. C.; Feig, J. E.; Gordon, R.; Vucic, E.; Amirbekian, V.; Fisher, E. A.; 16


Page 16 of 31

Page 17 of 31


Fuster, V.; Feldman, L. J.; Fayad, Z. A. Noninvasive Detection of Macrophages Using a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nanoparticulate Contrast Agent for Computed Tomography. Nat. Med. 2007, 13, 636-641. (3) Popovtzer, R.; Agrawal, A.; Kotov, N. A.; Popovtzer, A.; Balter, J.; Carey, T. E.; Kopelman, R. Targeted Gold Nanoparticles Enable Molecular CT Imaging of Cancer. Nano Lett. 2008, 8, 4593-4596. (4) Xiao, M.; Nyagilo, J.; Arora, V.; Kulkarni, P.; Xu, D.; Sun, X.; Dave, D. P. Gold Nanotags for Combined Multi-Colored Raman Spectroscopy and X-Ray Computed Tomography. Nanotechnology 2010, 21, 338-346. (5) Bailey, D. L.; Willowson, K. P. Quantitative SPECT/CT: SPECT Joins PET as a Quantitative Imaging Modality. Eur. J. Nucl. Med. Mol. Imaging 2014, 41, S17-S25. (6) Bhushan, K. R.; Misra, P.; Liu, F.; Mathur, S.; Lenkinski, R. E.; Frangioni, J. V. Detection of Breast Cancer Microcalcifications Using a Dual-Modality SPECT/NIR Fluorescent Probe. J. Am. Chem. Soc. 2008, 130, 17648-17649. (7) Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Coordinating Radiometals of Copper, Gallium, Indium, Yttrium, and Zirconium for PET and SPECT Imaging of Disease. Chem. Rev. 2010, 110, 2858-2902. (8) Jiang, D.; Sun, Y.; Li, J.; Li, Q.; Lv, M.; Zhu, B.; Tian, T.; Cheng, D.; Xia, J.; Zhang, L.; Wang, L.; Huang, Q.; Shi, J.; Fan, C. Multiple-Armed Tetrahedral DNA Nanostructures for Tumor-Targeting, Dual-Modality in Vivo Imaging. ACS Appl. Mater. Interfaces 2016, 8, 4378-4384. (9) Yang, Y.; Zhang, L.; Cai, J.; Li, X.; Cheng, D.; Su, H.; Zhang, J.; Liu, S.; Shi, H.; Zhang, Y.; Zhang, C. Tumor Angiogenesis Targeted Radiosensitization Therapy Using Gold Nanoprobes Guided by MRI/SPECT Imaging. ACS Appl. Mater. Interfaces 2016, 8, 1718-1732. (10) Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theragnosis. Chem. Soc. Rev. 2012, 41, 2656-2672. (11) Weissleder, R. Molecular Imaging in Cancer. Science 2006, 312, 1168-1171. (12) Weissleder, R.; Pittet, M. J. Imaging in the Era of Molecular Oncology. Nature 2008, 452, 580-589. 17



(13) Avram, A. M. Radioiodine Scintigraphy with SPECT/CT: An Important Diagnostic Tool for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Thyroid Cancer Staging and Risk Stratification. J. Nucl. Med. 2012, 53, 754-764. (14) Li, J.; Hu, Y.; Yang, J.; Wei, P.; Sun, W.; Shen, M.; Zhang, G.; Shi, X. Hyaluronic Acid-Modified Fe3O4@Au Core/Shell Nanostars for Multimodal Imaging and Photothermal Therapy of Tumors. Biomaterials 2015, 38, 10-21. (15) Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-Targeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2012, 134, 5722-5725. (16) Wang, X.; Chen, H.; Zheng, Y.; Ma, M.; Chen, Y.; Zhang, K.; Zeng, D.; Shi, J. Au-Nanoparticle Coated Mesoporous Silica Nanocapsule-Based Multifunctional Platform for Ultrasound Mediated Imaging, Cytoclasis and Tumor Ablation. Biomaterials 2013, 34, 2057-2068. (17) Yuan, H.; Fales, A. M.; Vo-Dinh, T. TAT Peptide-Functionalized Gold Nanostars: Enhanced Intracellular Delivery and Efficient NIR Photothermal Therapy Using Ultralow Irradiance. J. Am. Chem. Soc. 2012, 134, 11358-11361. (18) Kim, J. H.; Heller, D. A.; Jin, H.; Barone, P. W.; Song, C.; Zhang, J.; Trudel, L. J.; Wogan, G. N.; Tannenbaum, S. R.; Strano, M. S. The Rational Design of Nitric Oxide Selectivity in Single-Walled Carbon Nanotube Near-Infrared Fluorescence Sensors for Biological Detection. Nat. Chem. 2009, 1, 473-481. (19) Ray, P. C. Size and Shape Dependent Second Order Nonlinear Optical Properties of Nanomaterials and Their Application in Biological and Chemical Sensing. Chem. Rev. 2010, 110, 5332-5365. (20) Wu, C.; Bull, B.; Christensen, K.; McNeill, J. Ratiometric Single-Nanoparticle Oxygen Sensors for Biological Imaging. Angew. Chem., Int. Ed. 2009, 48, 2741-2745. (21) Fu, F.; Wen, S.; Zhu, J.; Shi, X. Encapsulation of Doxorubicin within Lactobionic Acid-Modified Multifunctional Poly(amidoamine) Dendrimers for Targeted Therapy of Liver Cancer Cells. J. Controlled Release 2015, 213, E31-E32. (22) Kumar, C. S. S. R.; Mohammad, F. Magnetic Nanomaterials for Hyperthermia-Based Therapy 18


Page 18 of 31

Page 19 of 31


and Controlled Drug Delivery. Adv. Drug Delivery Rev. 2011, 63, 789-808. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(23) Liang, Y.; Deng, X.; Zhang, L.; Peng, X.; Gao, W.; Cao, J.; Gu, Z.; He, B. Terminal Modification of Polymeric Micelles with Pi-Conjugated Moieties for Efficient Anticancer Drug Delivery. Biomaterials 2015, 71, 1-10. (24) Endres, T. K.; Beck-Broichsitter, M.; Samsonova, O.; Renette, T.; Kissel, T. H. Self-Assembled Biodegradable Amphiphilic PEG-PCL-lPEI Triblock Copolymers at the Borderline Between Micelles and Nanoparticles Designed for Drug and Gene Delivery. Biomaterials 2011, 32, 7721-7731. (25) Shan, Y.; Luo, T.; Peng, C.; Sheng, R.; Cao, A.; Cao, X.; Shen, M.; Guo, R.; Tomas, H.; Shi, X. Gene Delivery Using Dendrimer-Entrapped Gold Nanoparticles as Nonviral Vectors. Biomaterials 2012, 33, 3025-3035. (26) Xing, S.; Jiang, D. W.; Li, F.; Li, J.; Li, Q.; Huang, Q.; Guo, L. J.; Xia, J. Y.; Shi, J. Y.; Fan, C. H.; Zhang, L.; Wang, L. H. Constructing Higher-Order DNA Nanoarchitectures with Highly Purified DNA Nanocages. ACS Appl. Mater. Interfaces 2015, 7, 13174-13179. (27) Luo, Y.; Yang, J.; Yan, Y.; Li, J.; Shen, M.; Zhang, G.; Mignani, S.; Shi, X. RGDFunctionalized Ultrasmall Iron Oxide Nanoparticles for Targeted T-1-Weighted MR Imaging of Gliomas. Nanoscale 2015, 7, 14538-14546. (28)Brouwer, O. R.; Buckle, T.; Vermeeren, L.; Klop, W. M. C.; Balm, A. J. M.; van der Poel, H. G.; van Rhijn, B. W.; Horenblas, S.; Nieweg, O. E.; van Leeuwen, F. W. B.; Olmos, R. A. V. Comparing the Hybrid Fluorescent-Radioactive Tracer Indocyanine Green-Tc-99m-Nanocolloid with Tc-99m-Nanocolloid for Sentinel Node Identification: A Validation Study Using Lymphoscintigraphy and SPECT/CT. J. Nucl. Med. 2012, 53, 1034-1040. (29) Liu, Z.; Huang, J.; Dong, C.; Cui, L.; Jin, X.; Jia, B.; Zhu, Z.; Li, F.; Wang, F. Tc-99m-Labeled RGD-BBN Peptide for Small-Animal SPECT/CT of Lung Carcinoma. Mol. Pharmaceutics 2012, 9, 1409-1417. (30) Nilsson, C.; Barrios-Lopez, B.; Kallinen, A.; Laurinmaki, P.; Butcher, S. J.; Raki, M.; Weisell, J.; Bergstrom, K.; Larsen, S. W.; Ostergaard, J.; Larsen, C.; Urtti, A.; Airaksinen, A. J.; Yaghmur, A. 19



SPECT/CT Imaging of Radiolabeled Cubosomes and Hexosomes for Potential Theranostic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Applications. Biomaterials 2013, 34, 8491-8503. (31) Criscione, J. M.; Dobrucki, L. W.; Zhuang, Z. W.; Papademetris, X.; Simons, M.; Sinusas, A. J.; Fahmy, T. M. Development and Application of a Multimodal Contrast Agent for SPECT/CT Hybrid Imaging. Bioconjugate Chem. 2011, 22, 1784-1792. (32) Wu, Y.; Sun, Y.; Zhu, X.; Liu, Q.; Cao, T.; Peng, J.; Yang, Y.; Feng, W.; Li, F. Lanthanide-Based Nanocrystals as Dual-Modal Probes for SPECT and X-Ray CT Imaging. Biomaterials 2014, 35, 4699-4705. (33) Qiao, Z.; Shi, X. Dendrimer-Based Molecular Imaging Contrast Agents. Prog. Polym. Sci. 2015, 44, 1-27. (34) Peng, C.; Li, K.; Cao, X.; Xiao, T.; Hou, W.; Zheng, L.; Guo, R.; Shen, M.; Zhang, G.; Shi, X. Facile Formation of Dendrimer-Stabilized Gold Nanoparticles Modified with Diatrizoic Acid for Enhanced Computed Tomography Imaging Applications. Nanoscale 2012, 4, 6768-6778. (35) Peng, C.; Qin, J.; Zhou, B.; Chen, Q.; Shen, M.; Zhu, M.; Lu, X.; Shi, X. Targeted Tumor CT Imaging Using Folic Acid-Modified PEGylated Dendrimer-Entrapped Gold Nanoparticles. Polym. Chem. 2013, 4, 4412-4424. (36) Peng, C.; Zheng, L.; Chen, Q.; Shen, M.; Guo, R.; Wang, H.; Cao, X.; Zhang, G.; Shi, X. PEGylated Dendrimer-Entrapped Gold Nanoparticles for in vivo Blood Pool and Tumor Imaging by Computed Tomography. Biomaterials 2012, 33, 1107-1119. (37)Liu, H.; Wang, H.; Xu, Y.; Guo, R.; Wen, S.; Huang, Y.; Liu, W.; Shen, M.; Zhao, J.; Zhang, G.; Shi, X. Lactobionic Acid-Modified Dendrimer-Entrapped Gold Nanoparticles for Targeted Computed Tomography Imaging of Human Hepatocellular Carcinoma. ACS Appl. Mater. Interfaces 2014, 6, 6944-6953. (38) Chen, Q.; Li, K.; Wen, S.; Liu, H.; Peng, C.; Cai, H.; Shen, M.; Zhang, G.; Shi, X. Targeted CT/MR Dual Mode Imaging of Tumors Using Multifunctional Dendrimer-Entrapped Gold Nanoparticles. Biomaterials 2013, 34, 5200-5209. (39) Chen, Q.; Wang, H.; Liu, H.; Wen, S.; Peng, C.; Shen, M.; Zhang, G.; Shi, X. Multifunctional 20


Page 20 of 31

Page 21 of 31


Dendrimer-Entrapped Gold Nanoparticles Modified with RGD Peptide for Targeted Computed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tomography/Magnetic Resonance Dual-Modal Imaging of Tumors. Anal. Chem. 2015, 87, 3949-3956. (40)Wen, S.; Li, K.; Cai, H.; Chen, Q.; Shen, M.; Huang, Y.; Peng, C.; Hou, W.; Zhu, M.; Zhang, G.; Shi, X. Multifunctional Dendrimer-Entrapped Gold Nanoparticles for Dual Mode CT/MR Imaging Applications. Biomaterials 2013, 34, 1570-1580. (41) Zhang, Y.; Sun, Y.; Xu, X.; Zhang, X.; Zhu, H.; Huang, L.; Qi, Y.; Shen, Y. M. Synthesis, Biodistribution, and Microsingle Photon Emission Computed Tomography (SPECT) Imaging Study of Technetium-99m Labeled PEGylated Dendrimer Poly(amidoamine) (PAMAM)-Folic Acid Conjugates. J. Med. Chem. 2010, 53, 3262-3272. (42) Cao, Y.; He, Y.; Liu, H.; Luo, Y.; Shen, M.; Xia, J.; Shi, X. Targeted CT Imaging of Human Hepatocellular Carcinoma Using Low-Generation Dendrimer-Entrapped Gold Nanoparticles Modified with Lactobionic Acid. J. Mater. Chem. B 2015, 3, 286-295. (43) Liu, H.; Wang, H.; Xu, Y.; Shen, M.; Zhao, J.; Zhang, G.; Shi, X. Synthesis of PEGylated Low Generation Dendrimer-Entrapped Gold Nanoparticles for CT Imaging Applications. Nanoscale 2014, 6, 4521-4526. (44) Li, J.; Zheng, L.; Cai, H.; Sun, W.; Shen, M.; Zhang, G.; Shi, X. Polyethyleneimine-Mediated Synthesis of Folic Acid-Targeted Iron Oxide Nanoparticles for in vivo Tumor MR Imaging. Biomaterials 2013, 34, 8382-8392. (45) Motaleb,

M.

A.

Preparation

and

Biodistribution

of

Tc-99m-Lomefloxacin

and

Tc-99m-Ofloxacin Complexes. J. Radioanal. Nucl. Chem. 2007, 272, 95-99. (46) Zhu, J.; Peng, C.; Sun, W.; Yu, Z.; Zhou, B.; Li, D.; Luo, Y.; Ding, L.; Shen, M.; Shi, X. Formation of Iron Oxide Nanoparticle-Loaded Gamma-Polyglutamic Acid Nanogels for MR Imaging of Tumors. J. Mater. Chem. B 2015, 3, 8684-8693. (47) Ding, L.; Hu, Y.; Luo, Y.; Zhu, J.; Wu, Y.; Yu, Z.; Cao, X.; Peng, C.; Shi, X.; Guo, R. LAPONITE (R)-Stabilized Iron Oxide Nanoparticles for in vivo MR Imaging of Tumors. Biomater. Sci. 2016, 4, 474-482. 21



(48) Cheng, Y.; Zhu, J.; Zhao, L.; Xiong, Z.; Tang, Y.; Liu, C.; Guo, L.; Qiao, W.; Shi, X.; Zhao, J. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

131I-Labeled Multifunctional Dendrimers Modified with BmK CT for Targeted SPECT Imaging and Radiotherapy of Gliomas. Nanomedicine 2016, 11, 1253-1266. (49) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M. N.; Vezmar, I.; Whetten, R. L. Optical Absorption Spectra of Nanocrystal Gold Molecules. J. Phys. Chem. B 1997, 101, 3706-3712. (50) Zheng, L.; Zhu, J.; Shen, M.; Chen, X.; Baker, J. R., Jr.; Wang, S. H.; Zhang, G.; Shi, X. Targeted Cancer Cell Inhibition Using Multifunctional Dendrimer-Entrapped Gold Nanoparticles. Medchemcomm 2013, 4, 1001-1005. (51) Zhang, H.; Li, J.; Hu, Y.; Shen, M.; Shi, X.; Zhang, G. Folic Acid-Targeted Iron Oxide Nanoparticles as Contrast Agents for Magnetic Resonance Imaging of Human Ovarian Cancer. J. Ovarian Res. 2016, 9, 19. (52) Wang, H.; Zheng, L. F.; Peng, C.; Shen, M. W.; Shi, X.; Zhang, G. X. Folic Acid-Modified Dendrimer-Entrapped Gold Nanoparticles as Nanoprobes for Targeted CT Imaging of Human Lung Adenocarcinoma. Biomaterials 2013, 34, 470-480. (53) Zhu, J.; Zheng, L.; Wen, S.; Tang, Y.; Shen, M.; Zhang, G.; Shi, X. Targeted Cancer Theranostics Using Alpha-Tocopheryl Succinate-Conjugated Multifunctional Dendrimer-Entrapped Gold Nanoparticles. Biomaterials 2014, 35, 7635-7646.

22


Page 22 of 31

Page 23 of 31


Figure captions 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure

1.

(a)

Schematic

illustration

of

the

synthesis

of

FA-PEG-COOH

segment,

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs , and {(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs. (b) UV-Vis spectra of G2-DTPA-PEG-FA and {(Au0)6-G2-DTPA-PEG-FA} DENPs. (c) TEM image and (d) size distribution histogram of the {(Au0)6-G2-DTPA-PEG-FA} DENPs. Inset in (c) shows the high-resolution TEM image of the Au core particles. Figure 2. (a) MTT viability assay of HeLa cells treated with the {(Au0)6-G2-DTPA-mPEG} or {(Au0)6-G2-DTPA-PEG-FA} DENPs for 24 h. The cells treated with PBS were used as control and the data were expressed as mean ± S. D. (n=3). (b) Cellular uptake of Au in HeLa-HFAR and HeLa-LFAR cells treated with the {(Au0)6-G2-DTPA-mPEG} or {(Au0)6-G2-DTPA-PEG-FA} DENPs at different concentrations for 3 h. TEM images of HeLa cells after incubation with {(Au0)6-G2-DTPA-mPEG} (c) or {(Au0)6-G2-DTPA-PEG-FA} DENPs (d) for 3 h are also shown. Figure 3. (a) CT images and (b) X-ray attenuation intensity (HU) of HeLa-HFAR cells treated with PBS, {(Au0)6-G2-DTPA-PEG-FA} (1) and {(Au0)6-G2-DTPA-mPEG} (3), respectively, and HeLa-LFAR cells treated with the {(Au0)6-G2-DTPA-PEG-FA} (2) at different concentrations. (c) SPECT images and (d) SPECT signal intensity of HeLa-HFAR cells treated with PBS, {(Au0)6-G2-DTPA(99mTc)-PEG-FA} (1), and {(Au0)6-G2-DTPA(99mTc)-mPEG} (3), respectively, and HeLa-LFAR cells treated with the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} (2) at different doses (µCi). Figure 4. In vivo CT images (a, b) and signal intensity (c) of tumors after intravenous injection of the {(Au0)6-G2-DTPA(99mTc)-PEG-FA} (a) or {(Au0)6-G2-DTPA(99mTc)-mPEG} (b) DENPs ([99mTc] = 740 MBq·mL-1, [Au] = 0.08 M, in 100 µL PBS) at different time points postinjection. The dashed red cycle indicates the tumor site. Figure 5. In vivo SPECT/CT images of tumors (a, b), SPECT signal intensity of tumors (c), and SPECT

signal

ratio

(tumor/muscle)

(d)

{(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs (a) or

after

intravenous

injection

of

the

{(Au0)6-G2-DTPA(99mTc)-mPEG} DENPs (b)

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

([99mTc] = 740 MBq·mL-1, [Au] = 0.08 M, in 100 µL PBS) at different time points postinjection. The dashed red cycle indicates the tumor site. Figure 6. In vivo biodistribution of Au element in different organs and tumor at 24 h postinjection of the {(Au0)6-G2-DTPA(99mTc)-mPEG} or {(Au0)6-G2-DTPA(99mTc)-PEG-FA} DENPs ([99mTc] = 740 MBq·mL-1, [Au] = 0.08 M, in 100 µL PBS).

24


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 31

Figure 2

26


Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 31

Figure 4

28


Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5

29


Page 30 of 31

Control 0 99m {(Au )6-G2-DTPA( Tc)-mPEG}

500

0

99m

{(Au )6-G2-DTPA(

Tc)-PEG-FA}

400 300 200 100

***

or Tu m

ne y id K

Lu ng

n ee Sp l

er Li v

ea rt

0 H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Au mass per gram tissue (µg/g)


Figure 6

30


Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graphic

31


99mTc-Labeled Multifunctional Low-Generation ... - ACS Publications

Recommend Documents