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Dual-Modal Probe based on Polythiophene Derivative for Pre- and Intraoperative Mapping of Lymph Nodes by SPECT/Optical Imaging Bing Jia, Xin Zhang, Bing Wang, Muhua Chen, Fengting Lv, Shu Wang, and Fan Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01032 • Publication Date (Web): 26 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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ACS Applied Materials & Interfaces

Dual-Modal

Probe

based

on

Polythiophene

Derivative for Pre- and Intraoperative Mapping of Lymph Nodes by SPECT/Optical Imaging †‡§

Bing Jia, , ,

†§

§

†

Xin Zhang, , Bing Wang,#, Muhua Chen, Fengting Lv,# Shu Wang,*,# and Fan

Wang*,†,⊥

† Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, P. R. China ‡ Medical and Healthy Analytical Center, Peking University Health Science Center, Beijing 100191, P. R. China # Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ⊥ Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, P. R. China KEYWORDS：Polythiophene, Lymph nodes mapping, Dual-modality, SPECT imaging, Optical Imaging

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ABSTRACT.

The metastatic spread of primary tumors to regional lymph nodes is an important prognostic indicator for cancer staging and clinical therapy. Therefore, developing lymphatic mapping probes with improved accuracy and efficiency is of vital importance. Conjugated polymers have been established as useful optical probes for sensitive biological and chemical detection. As a member of CPs family, polythiophene derivatives have drawn increasing attraction due to their superior photo-stability, signal amplification ability and flexible structures for modification. And these excellent properties allow the promising in vivo application to real-time lymph nodes mapping. Here, we firstly reported radiolabeled dual-modal probe based on polythiophene derivative (99mTc-PTP) that was used for lymph nodes mapping with high sensitivity and specificity by preoperative single-photon emission computed tomography (SPECT) imaging and intraoperative optical guidance. 99mTc-PTP exhibits excellent radio-fluorescence guidance ability and remarkable biocompatibility, and holds great potential to be a powerful probe for noninvasive lymph nodes mapping.

INTRODUCTION Water soluble conjugated polymers (CPs) are characterized by large π-conjugated backbones and delocalized electronic structure which endow them excellent light-harvesting and amplifying properties.1-3 The polythiophene copolymer (PTP) is a member of CPs family and possess high photo-stability, superior fluorescence brightness, and lower biological toxicity.4,

5

These

properties make PTP a promising candidate for highly sensitive biological sensing and chemical detection, such as label-free biological imaging and diagnosis6-8. Recently, PTP have been applied for the applications of cell imaging, biomacromolecules detection and light-activated


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antibacterial activity.9-11 Previous studies have demonstrated the successful in vitro imaging applications, further explorations on the more important in vivo imaging of biological processes are in great expectation. Regional lymph nodes (LNs) status is one of the most powerful prognostic indicator for tumor theranostics.12, 13 Due to superior spatial visualization and high resolution, fluorescence imaging probes play important roles in intraoperative guidance.14 Combination of both fluorescence imaging and radio-guidance which possesses excellent tissue penetration permits assessment of LNs with both optimal spatial resolution and high sensitivity.12, 15 So far, the most commonly used method in the clinic for lymphatic mapping is a two-step injection.

99m

Tc-labeled colloids

was firstly injected followed by the injection of vital dyes (such as indocyanine green) several hours later.16,

17

This combined injection utilizes

99m

Tc-labeled colloids for sensitive pre-

operative localization of lymph nodes by gamma scintigraphy or single-photon emission computed tomography, and then vital dyes can be employed for intraoperative optical guidance. However, the applications of vital dyes are limited by their inherent toxicity and it requires separate administrations due to different rate of local migration of the radiocolloids and vital dyes.18, 19 In addition, blue dyes with small sizes may diffuse into surrounding anatomy and stain the surgical field blue, which could interfere with the clinical operation during the surgery.20 Consequently, new dual-modal lymphatic mapping probes are in great need to improve the efficiency and accuracy of lymph nodes mapping. With this in mind, in this work, we report a dual-modal probe for LNs imaging based on PTP backbone. Generally, PTP is composed of three essential components: (i) π-conjugated backbones, which endow PTP high fluorescence brightness and good photostability; (ii) the flexible backbones and terminal carboxyl groups of PTP can be easily modified by covalently


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linking with specific functional elements; (iii) the organic nature of chemical constitution endows PTP with excellent biosafety. Typically, PTP was conjugated with the chelator 6hydrazinonicotinyl (HYNIC) followed by radiolabeling with

99m

Tc. The resulting radiotracer

99m

Tc-PTP can be used for sensitive preoperative localization of lymph nodes by SPECT

imaging and intraoperative optical guidance through one-step injection (Scheme 1). This is the first polythiophene based dual-modal probe by special design and synthesis for in vivo imaging application. RESULTS AND DISCUSSION Firstly, we synthesized a chelator modified PTP derivative (HY-PTP) that can be radiolabeled by

99m

Tc for SPECT imaging. PTP and HYNIC-NHS were synthesized for preparing HY-PTP

complex according to the literature methods.10,

21

The synthesis procedures of HY-PTP are

summarized in Figure 1A. In generally, HY-PTP was synthesized in the following three steps. (i) PTP was coupled with BocNHCH2CH2NH2 through EDCI/sulfo-NHS coupling method followed by dissolution in NaOH aqueous solution and dialysis in water through a membrane with molecular weight cutoff of 3500 g•mol-1. PTP-NHBoc was obtained in a red solid with a yield of 81 %. (ii) PTP-NHBoc was dissolved in 4 M HCl aqueous solution and stirred at room temperature for 12 h to afford PTP-NH2 with a yield of 86 %. (iii) HYNIC-NHS was added to PTP-NH2 solution and incubating for 12 h. After purified by dialysis and freeze dried, HY-PTP was obtained with a yield of 89 %. The degree of conjugation (carboxyl group/HYNIC) for HYPTP was calculated to be 7.3 % based on 1H-NMR analyse (Figure S1). The hydrodynamic diameter (HD) of the as-synthesized HY-PTP measured by dynamic light scattering (DLS) is about 10.1 nm (Figure 1B and 1C) which can be rapid uptaken into the


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lymphatic system and used for LNs identification.15, 22, 23 The maximum absorption of HY-PTP was observed at 480 nm which coincided to π–π* transitions of the conjugated backbone structures. Upon excitation at 435–480/490 nm (band-pass/emission long-pass filter), the HYPTP solution exhibits yellow fluorescence and the maximum emission was observed at 574 nm. The fluorescence intensity was dependent on the concentrations of the HY-PTP nanoprobe which got highest at the concentration of 1.0 mg/mL, and the fluorescence intensity changed little as the concentration increased up to 2.0 mg/mL. This observation was further confirmed by in vivo imaging after abdominal subcutaneous injection into a nude mouse (Figure S2). After that, HY-PTP was radiolabeled with

99m

Tc. Tricine and trisodium triphenylphosphine-

3,3’,3’’-trisulfonate (TPPTS) were used as the coligands and the radiotracer was purified with a Vivaspin 30K ultra filtration device (Figure 1A)24. Radio-HPLC analysis was performed and the radiochemical purity of 99mTc-PTP was calculated to be 98%. (Figure S3). The solution stability of

99m

Tc-PTP in 1.0 mg/mL of excess cysteine (pH=7.4) and in saline was evaluated by radio-

HPLC, and 99mTc-PTP stayed stable in the cysteine and saline solution over 6 h (Figure S3). To evaluated the potential of

99m

Tc-PTP for in vivo lymphatic mapping through non-invasive

SPECT imaging, each nude mouse (n=6) was intradermally injected with 150 µCi of 99mTc-PTP (10 µg, 10 µL). Whole body SPECT/CT imaging was perfomed from 0.5 h to 24 h post injection (p.i.). As shown in Figure 2 and Figure S4, strong signals could be detected in both the draining axillary (sentinel lymph node, SLN) and lateral thoracic lymph nodes (2nd LN) at 1 h, while the signal in the secondary lymph node was much weaker. The lymph nodes detection efficiency as well as pharmacokinetics behavior of

99m

Tc-PTP could be better represented by Supplemental

Video of 3D-rendered SPECT/CT imaging at 1 h. With the help of CT imaging, we could clearly distinguish the SLN and 2nd LN by their spatial orientation. Only SLN could be detected under


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the same luminance coefficient at 24 h. According to the quantitative information got from the SPECT imaging, the lymph nodes uptake increased from 0.5 h to 4 h and remained declining until 24 h, which was consistent with the sharp decrease of the radioactivity in the injected paw from 0.5 h to 4 h. The uptake values of 99mTc-PTP in the SLN at 0.5, 4, and 24 h were calculated to be 3.2, 14.3 and 10.4 ID%. For lateral thoracic node (2nd LN), the uptake values were 0.7, 5.4, and 3.3 ID% at 0.5, 4, and 24 h, respectively. Next, we evaluated the potential of

99m

Tc-PTP for in vivo lymphatic mapping through optical

guidance. For this purpose, the mixture of

99m

Tc-PTP and ICG (10 µg of

99m

Tc-PTP and 2.5

µmol of ICG in 10 µL of water) was injected intradermally into the right paw of a nude mouse and imaged using a fluorescence imaging system. According to the SPECT/CT imaging and quantitative information in Figure 2 and Figure S4, the optimal imaging was obtained at 1 h. For this reason, the following intraoperative optical guidance was performed at this time-point. The fluorescence signals of 99mTc-PTP and ICG could be clearly detected from the in vitro image of the mixture (Figure 3). For lymphatic mapping, strong fluorescence signals of

99m

Tc-PTP could

be observed in axillary and lateral thoracic lymph nodes upon excitation at 435–480/490 nm (band-pass/emission long-pass filter) and removal of the skin 1 h p.i. Changed the excitation light into 684–729/745 nm, strong NIR fluorescence signals of ICG could be observed in the same areas. Since ICG has been approved by FDA to be used as a NIR fluorescence imageguided LNs tracer in oncologic surgeries22, 25, the areas that generated strong fluorescence signals of

99m

Tc-PTP were confirmed to be axillary and lateral thoracic lymph nodes. After that, lymph

nodes were dissected under the guidance of optical imaging. According to the ex vivo imaging, 99m

Tc-PTP preferential accumulated in LNs, since the fluorescence signals of

99m

Tc-PTP and

ICG associated with the LNs but not the adjacent fat tissue. The fluorescence signals of


99m

Tc-

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PTP highly coincided with ICG, indicating 99mTc-PTP performed high accuracy for lymph nodes detection and dissection. Previous studies have demonstrated that paticle sizes of the nanoprobes have great influence on their lymphatic trafficking behavior.26 Compared with ICG, background signals were much weaker since

99m

Tc-PTP is in an appropriate size (10.1 nm) to be rapidly

uptaken into the lymphatic system without diffusing into surrounding anatomy. For lymph nodes detection, it’s necessary to perform in vivo toxicity study to evaluate the biosafety of 99mTc-PTP. Fourteen female nude mice were equally divided into two groups. Each nude mouse of the experimental group (n=7) and control group (n=7) was intradermally injected with 150 µCi of 99mTc-PTP (10 µg of samples in 10 µL of water) or 10 µL of water, respectively. Blood routine analysis and body weight measurement were performed continuously for 7 days after administration. As shown in Figure S5, the parameters of important hematology markers, white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB) for the test group were in normal range without physiologically significant differences in comparison with the control group. No significant body weight fluctuation was observed compared to control group (Figure S7). We next evaluated the acute toxicity of

99m

Tc-PTP on Kunming mice which were

administrated at a high chemometry. Fourteen female Kunming mice were equally divided into two groups. Each animal in the experimental group was intravenously injected with 100 µg of 99m

Tc-PTP (150 µCi, in 100 µL of water). The dose given to each animal was up to 5 mg/kg

body weight (b.w.). Animals in the control group were injected with the same volume of water. Blood samples and body weight fluctuations were tested every other day. As shown in Figure S6, parameters for RBC, WBC, HGB, and PLT were also in normal range without physiologically significant differences in comparison with the control group. No significant changes in body


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weight were observed compared to the control group (Figure S7). Those results demonstrated that the in vivo toxicity of 99mTc-PTP is quite low. The excellent biocompatibility of 99mTc-PTP ensures its viability for further biological applications. To investigate the metabolic behavior of the radiotracer, whole body SPECT imaging of Kunming mice were performed. 150 µCi of 99mTc-PTP (10 µg in 100 µL of water) was injected into each animal via tail vein (n=5). SPECT/CT imaging was acquired at 1, 2, 4 and 8 h p.i. As shown in Figure 4, high liver and bladder uptake of 99mTc-PTP can be clearly visualized at 1 and 2 h p.i., and the accumulation declines rapidly through excretion in the following few hours. Urine samples were collected at 1 to 4 h p.i. to analyze the metabolic stability of 99mTc-PTP. The urine samples were centrifuged at 1500 rpm for 15 min and filtered through a 0.22 µm MillexLG syringe driven filter unit, then the supernatants were collected and analyzed by radio-HPLC. As shown in Figure S3, only about 20% metabolism can be observed during excretion from the renal route at 4 h. Those results demonstrated that

99m

Tc-PTP was mainly excreted from

hepatobiliary route and possessed a high metabolic stability in vivo. CONCLUSIONS In conclusion, we developed a radiolabeled polythiophene derivative

99m

Tc-PTP for

preoperative detection and intraoperative dissection of lymph nodes with high sensitivity and specificity. LNs can be located pre-operationally by SPECT imaging, and during surgery LNs can be visualized and dissected by optical guidance through one-step injection. Moreover, the dual-modal probe exhibits good biocompatibility in vivo even at a high dose. As a member of CPs family, PTPs have been prepared for label-free imaging of cancer cells due to the advantageous properties, such as excellent signal amplication effect and low toxicity.27


99m

Tc-

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PTP is the first PTP-based probe by special design and synthesis for in vivo imaging application. We believe that the reported

99m

Tc-PTP could be used as excellent imaging probe for LNs

mapping in human clinical settings. EXPERIMENTAL SECTION Synthesis of HY-PTP. Synthesis of PTP-NHBoc: 14 mg of PTP was dissolved in 1 mL of distilled water. Then the solution was mixed with 200 mM of EDCI aqueous solution (1mL) and 200 mM of NHS-SO3- aqueous solution (1 mL). BocNHCH2CH2NH2 aqueous solution (20 mM, 0.5 mL) was added 20 min later. After stirring at room temperature overnight, 40 mg NaOH was added to dissolve the precipitated polymers, and 2 M HCl aqueous solution was used to neutralize the excess base until the pH = 10. The mixture solution was purified by dialysis using a dialysis membrane (M = 3500 g•mol-1 for cutoff) in water for 3 days. The water was removed by freeze drying and a red solid was afforded (12 mg, 81%). 1H NMR (400 MHz, D2O, δ): 7.07.5 (br), 2.6-3.3 (br), 2.0-2.6 (br), 1.1-1.5(br). Synthesis of PTP-NH2: 12 mg of PTP-NHBoc was dissolved in HCl aqueous solution (4M, 6 mL) and stirred for 12 h at room temperature. 2 M NaOH aqueous solution was added to neutralize until pH = 10. The mixture solution was purified by dialysis using a dialysis membrane with a cutoff at M = 3500 g•mol-1 in water for 3 days. The water was removed by freeze drying to afford a red solid (10 mg, 86%). 1H NMR (300 MHz, D2O, δ): 7.0-7.5 (br), 3.04.5 (br), 2.0-3.0 (br). Synthesis of HY-PTP: To 3 mL of aqueous solution of PTP-NH2 (8 mg) was added HYNICNHS (5.6 mg, 0.012 mmol). After reacting for 12 h, the mixture solution was purified by dialysis for 3 days using a dialysis membrane (M = 3500 g•mol-1 for cutoff) in water. The water was


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removed by freeze drying to afford a red solid (8 mg, 89%).

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1

H NMR (300 MHz, DMSO,δ):

8.86 (s, 1H), 8.54 (s, 1H)，8.02 (d, 2H)，7.74 (d, 2H), 7.25-7.45 (m), 7.15 (d, 1H), 2.8-3.3 (br), 2.73 (s), 2.1-2.3(br). Radiochemistry and Solution Stability evaluation. Trisodium triphenylphosphine-3,3’,3’’trisulfonate (TPPTS) and tricine were used as coligands for the radiolabeling of HY-PTP. After radiolabeling, the solution was transferred to a Vivaspin 30K ultra filtration device (Mr cut-off 30 kDa; Sartorius Corp., Germany) and centrifuged at 12000×g for 10 min for three times to avoid interference from radioimpurities. Radio-HPLC analysis was performed and the radiochemical purity of

99m

Tc-PTP was calculated to be 98%. For the solution stability evaluation, 1.8 mCi of

99m

Tc-HYNIC-PTP (80 µL) was incubated in saline (0.9%, 0.2 mL) or L-cysteine (1.0 mg/mL in

water). Radio-HPLC analysis was performed to evaluate the solution stability of the samples after incubated for 1, 2, 4 ,6 and 12 h. In Vivo SPECT Imaging. Each nude mouse (n=6) was intradermally injected with 150 µCi of 99m

Tc-PTP (10 µg of samples in 10 µL of water). The mice were anesthetized by inhalation of

2% isoflurane in oxygen and SPECT/CT imaging was performed up to 24 h p.i. The SPECT and CT fusion images were obtained using the automatic fusion feature of the InVivoScope program. In Vivo Optical Imaging. Each nude mouse was intradermally injected with the mixture of 99m

Tc-PTP and ICG (10 µg of

99m

Tc-PTP and 2.5 µmol of ICG in 10 µL of water) and imaged

using a fluorescence imaging system (Maestro™, CRI Inc, Woburn, MA). Two excitation bandpass and emission long-pass filters of 684–729/745, 435–480/490 nm (excitation/emission) were consecutively used (500 msec for exposure). Maestro software was used to analyze the collected image. Lymphadenectomy was performed immediately after imaging. Upon ex vivo spectral


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fluorescence imaging using the same exposure time and wavelength, both the removed LNs were validated. Whole Body SPECT Imaging and Metabolic Stability evaluation. 150 µCi of

99m

Tc-PTP

(10 µg in 100 µL of water) was injected into each KM mouse via tail vein (n=3). At 1 h, 2 h, 4 h, 8 h p.i., the mice were anesthetized and imaged by NanoSPECT/CT camera. The SPECT and CT fusion images were obtained using the same procedure above. Urine samples were collected at 1, 2 and 4 h p.i. to investigate the metabolic stability of 99mTcPTP. Equal volume of ddH2O was added to the urine samples, then the solution was centrifuged for 15 min at the speed of 1500 rpm followed by filtering through a 0.22 µm Millex-LG syringe driven filter unit, the supernatants were collected and analyzed by radio-HPLC. In Vivo Toxicity evaluation. Fourteen female nude mice were equally divided into two groups. Each animal in the experimental group was intravenously injected with 150 µCi of

99m

Tc-PTP

(10 µg of samples in 10 µL of water). In the control group, each animal was intravenously injected with 10 µL of water instead of 99mTc-PTP. Body weight fluctuations were recorded and blood samples were harvested from orbital sinus at 1, 2, 3, 5, 7 days p.i. Blood samples were evaluated by the blood cell automatic analysis machinery for the following parameters: WBC, RBC, HGB and PLT. For acute toxicity study of

99m

Tc-PTP, Fourteen female Kunming mice were equally divided

into two groups. In the experimental group, each animal was intravenously injected with 100 µg of

99m

Tc-PTP (150 µCi, in 100 µL of water). In the control group, each animal was intravenously

injected with 100 µL of water instead of 99mTc-PTP. Blood samples and body weight fluctuations were tested every other day. Blood samples were evaluated by the blood cell automatic analysis


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machinery for the following parameters: WBC, RBC, HGB and PLT. The toxicity evaluation data are expressed as the mean ± SD (n=7), and analyzed by Two-tailed t test. The level of significance was set at p = 0.05.

Figure 1. A) Synthetic routes to HY-PTP and chemical structure of

99m

Tc-PTP. B) Size

distribution histogram of HY-PTP based on DLS measurement. C) The absorption and emission spectra of HY-PTP in aqueous solution. The maximum absorption was observed at 480 nm. Upon excitation at 435-480/490 nm (band-pass/emission long-pass filter), the maximum emission was observed at 574 nm.


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Figure 2. A) Whole body SPECT/CT imaging of a female BALB/c nude mouse at 1 h postinjection (p.i.). The axillary node (1st LN) and lateral thoracic node (2nd LN) could be clearly visualized. The “H” and “L” boundaries on the palette were defined to be the maximum and minimum uptake values. B) The uptake values analysis of the axillary node (1st LN), lateral thoracic node (2nd LN), paw for injection and whole body from 0.5 h to 24 h p.i. The uptake values were expressed as the percentage of the injected dose (%ID). The standard (Std.) accounted for 1 % of the injected dose. Results were expressed as mean ± SD (n = 6).


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Figure 3. A) In vitro fluorescence image (pseudocolor) of 99mTc-PTP (1 mg/mL, 125 mM), ICG (125 mM), the mixture of

99m

After intradermal injection of

Tc-PTP and ICG (125 mM for

99m

Tc-PTP and ICG) in water. B)

99m

Tc-PTP/ICG mixture for 1 h, fluorescence imaging of lymph

nodes was performed under excitation band-pass/emission long-pass filter of 435-480/490 nm (99mTc-PTP signal) and 684–729/745 nm (ICG signal). The signals detected under the paw indicated the axillary (white arrow) and lateral thoracic nodes (blue arrow).


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Figure 4. Whole body SPECT/CT images of Kunming mice at 1, 2, 4, and 8 h post intravenous injection of uptake of

99m

Tc-PTP (100 µg dissolved in 100 µL of ddH2O). High liver (L) and bladder (B)

99m

Tc-PTP could be detected at 1 h and 2 h p.i. The standard (Std.) accounted for 1%

of the injected dose. The “H” and “L” boundaries on the palette were defined to be the maximum and minimum uptake values.


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Scheme 1. Schematic representation of the design of

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99m

Tc-PTP and use it as dual-modal probe

for lymph nodes mapping by SPECT/Optical Imaging.


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ASSOCIATED CONTENT Supporting Information: General materials and methods; Animal preparation; 1H-NMR spectrum of HY-PTP; RadioHPLC analysis of 99mTc-PTP; Metabolic stability evaluation; 0.5 to 24 h SPECT/CT imaging of lymph nodes post intradermal injection; In Vivo toxicity evaluation (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Author Contributions §B.J., X.Z. and B.W. contributed equally to this work.

ACKNOWLEDGMENT This research was supported by the National Key R&D Program of China (2017YFA0205603), National Natural Science Foundation of China (NSFC) projects (81371614, 81630045, 81420108019, 81321003, 81427802), and Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12020216). ABBREVIATIONS PTP, Polythiophene derivative; SPECT, Single-photon emission computed tomography; SLN, Sentinel lymph node; HD, Hydrodynamic diameter; DLS, Dynamic light scattering.


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