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Synthesis, Characterization and Biomedical Applications of a Targeted DualModal Near Infrared-II Fluorescence and Photoacoustic Imaging Nanoprobe Kai Cheng, Hao Chen, Cesare H. Jenkins, Guanglei Zhang, Wei Zhao, Zhe Zhang, Fei Han, Jonathan Fung, Meng Yang, Yuxin Jiang, Lei Xing, and Zhen Cheng ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05966 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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Synthesis, Characterization and Biomedical Applications of a Targeted Dual-Modal Near Infrared-II Fluorescence and Photoacoustic Imaging Nanoprobe Kai Cheng†§‡, Hao Chen†‡, Cesare H. Jenkins§, Guanglei Zhangǁ§, Wei Zhao§, Zhe Zhang†, Fei Han§, Jonathan Fung†, Meng Yangδ, Yuxin Jiangδ, Lei Xing§*, Zhen Cheng†*

†

Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Department of Radiology,

Stanford University, California, 94305-5344. §Department of Radiation Oncology Stanford University School of Medicine Stanford, CA 94305-5847. ǁSchool of Computer and Information Technology, Beijing Jiaotong University, Beijing 100044, China. δChinese Academy of Medical Science, Peking Union Medical College Hospital, Department of Ultrasound, Beijing, 100730, China.

KEYWORDS: near-infrared window II, photoacoustic imaging, donor-acceptor chromophore, epidermal growth factor receptor (EGFR), thyroid carcinoma

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ABSTRACT. Our development of multifunctional dual-modal imaging probes aims to integrate the benefits from both the second near infrared (NIR-II) fluorescence (1000 - 1700 nm) and photoacoustic imaging with an ultimate goal of improving overall cancer diagnosis efficacy. Herein we designed a donor-acceptor chromophore based nanoparticle (DAP) as a dual-modal image contrast agent which has strong absorption in the NIR-I window and a strong fluorescence emission peak in the NIR-II region. The dual-modal DAPs composed of D-π-A-π-D type chromophores were PEGylated through nanoprecipitation. The multifunctional DAP surface was thus available for subsequent bioconjugation of EGFR Affibody (Ac-Cys-ZEGFR:1907) to target EGFR-positive cancers. The Affibody-conjugated DAPs appeared as highly monodisperse nanoparticles (~30 nm) with strong absorption in the NIR-I window (at c.a. 680 nm) and an extremely high fluorescence in the NIR-II region (maximum peak at 1000 nm). Consequently, the Affibody-DAPs show significantly enhanced photoacoustic and NIR-II fluorescence contrast effects in both in vitro and in vivo experiments. Moreover, the Affibody-DAPs have the capability to selectively target EGFR-positive tumors in an FTC-133 subcutaneous mouse model with relatively high photoacoustic and fluorescent signals. By taking advantage of high spatial resolution and excellent temporal resolution, photoacoustic/NIR-II fluorescence imaging with targeted dual-modal contrast agents allows us to specifically image and detect various cancers and diseases in an accurate manner.


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The field of molecular imaging and diagnosis has witnessed a significant growth in the past few years, largely due to the rapid development of various imaging modalities with excellent resolution and sensitivity, which hold great promises for dramatically improving patient medical outcomes and potentially reducing overall health-care costs.1-3 One of the most notable developments is the introduction of multi-modality molecular imaging, which is considered as the integration of two or more imaging modalities in order to take advantage of respective strengths while overcoming the weaknesses of each modality.4-6 Over the years, various multimodal approaches have been successfully explored to increase the diagnosis accuracy rate, shorten scanning and operation time, and simplify the workflow to improve efficiency.4-6 Recently, as one of the emerging hybrid imaging modalities that integrates optical excitation with ultrasonic detection based on the photoacoustic effect, photoacoustic imaging (PAI) intrinsically possesses high ultrasonic resolution and strong optical contrast.5, 7-8 By providing deep penetration beyond the optical diffusion limit while maintaining a high spatial resolution, PAI has great potential for noninvasive visualization of various superficial tissues, such as the thyroid, in clinical practices. When a high resolution is desired, however, the optical attenuation in the first near-infrared (NIR-I) window (650-950 nm) still limits the penetration of the photoacoustic imaging.9 Very recently, in vivo fluorescence imaging in the second NIR (NIR-II) window (1000-1700 nm) has attracted considerable attention due to its significant advantages over the traditional NIR-I window such as relatively high spatial resolution and temporal resolution, deep tissue penetration, largely reduced photon scattering, and negligible tissue


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autofluorescence.10-17 Recent successful applications in the NIR-II window for microvascular imaging and hemodynamic measurement confirmed the technique’s superior spatial and temporal resolution, which are comparable to or even better than those of computed tomography (CT) and ultrasound imaging.12 In particular, the NIR-II imaging has the ability to accurately delineate tumor positive margins based on preoperative/intraoperative images to improve diagnostic accuracy and confirm completeness of tumor resection.18 Because of its significant advantages over traditional NIR-I imaging, the NIR-II imaging provides many opportunities in preclinical and clinical applications. Considering the compatibility and flexibility of NIR-II fluorescence imaging and PAI, it is promising to combine these two modalities together to acquire complementary information and synergistically enhance the efficacy in cancer detection and diagnosis.

Recently, an emerging class of NIR-II organic compounds containing the donor-acceptor chromophores has been explored for NIR-II imaging because of their excellent optical properties such as tunable energy gaps, large Stokes shifts and acceptable quantum yields.11, 18-23 Previously we developed a water-soluble small-molecule NIR-II organic fluorophore (CH1055) for lymphatic imaging and targeted tumor imaging with excellent spatial resolution and superior in vivo behaviors.11 The electron donor (high-lying HOMO) and electron acceptor (low-lying LUMO) unit were linked by a conjugated π spacer in the architecture of CH1055, resulting in a donor-acceptor-donor (D- π-A-π-D) type of chromophore. To apply this chromophore for both


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PA and NIR-II imaging for living subjects, we modified its substituents to adjust the lipophility based on the original structure of CH1055. Although there was a slight blue-shift of the emission peak from 1055 nm to 1000 nm, the resultant donor-acceptor chromophore, referred as CH1000, showed very similar absorption and emission peaks as CH1055. To render CH1000 dye watersoluble and biocompatible for biomedical applications, we applied the nanoprecipitation process to encapsulate the CH1000 molecules with amphiphilic phospholipids to form a donor-acceptor chromophore based nanoparticle (DAP) which has an absorption in the NIR-I window and a fluorescence peak in the NIR-II region. Moreover, the corresponding physical, optical and chemical properties of the DAPs were characterized quantitatively and qualitatively. We further evaluated the potential of DAPs as dual modal contrast agents for both PA and NIR-II imaging in vitro and in vivo. To enhance tumor targeting efficiency, we conjugated DAP with targeting molecules, specifically the anti-epidermal growth factor receptor (EGFR) Affibody (Ac-CysZEGFR:1907).24 Since EGFR is a well-established tumor biomarker to differentiate between benign and malignant thyroid lesions,25 as a proof-of-concept, we evaluated the capability of AffibodyDAPs as dual-modal contrast agents for targeted PA and NIR-II imaging of follicular thyroid carcinoma in living subjects.

RESULTS


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Construction and Characterization of Affibody-DAPs. The construction of water-soluble DAPs involved two main-steps: the synthesis of a hydrophobic small-molecule organic NIR-II dye (CH1000, MW: 877.26 g/mol, Figure 1 and the experimental section in Supporting information (SI)) and the nanoprecipitation of CH1000 with the PEGylated phospholipid. First of all, the introduction of a strong electron donor (D, diphenylamino donor and phenylene spacer) and a strong electron acceptor (A, benzobis(1,2,5-thiadiazole)) in a small molecule results in a D-π-A-π-A type of chromophore core with a significantly low energy gap (Figure 1a, see detailed information about density functional theory calculations in the SI).11 The synthesis of Dπ-A-π-A chromophore core required multiple steps to complete (See SI for the detailed synthetic procedure and Scheme S1-S3). Different from the original version of CH1055 we reported recently,11 four carboxylic acid groups of CH1055 were replaced with two ethyl acrylate groups to improve the self-assembly capability of CH1000 with amphiphilic supramolecules in aqueous solutions. The resultant CH1000 showed excellent solubility and chemical stability in most of nonpolar and polar aprotic solvents (such as chloroform and tetrahydrofuran), but it was insoluble in aqueous solutions. On the other hand, in order to impart biological biocompatibility and improve targeting capability, the nanoprecipitation process was applied to generate watersoluble nanoparticles encapsulated with dyes (Figure 1b). Based on the transfer of the lipophilic CH1000 from a good solvent to a poor solvent condition, the nanoprecipitation led consequently to the nanoparticle formation via self-assembly of lipophilic CH1000 esters and amphiphilic PEGylated phospholipids (DSPE-mPEG5000 and DSPE-PEG5000-NH2) in an aqueous solution.


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The resultant dark-green water-soluble nanoparticles were referred as donor-acceptor nanoparticles (DAPs). The PEGylated phospholipid coating can efficiently reduce the nonspecific binding and prevent the aggregation of DAPs in physiological conditions.26 Transmission electron microscopy (TEM) showed that DAPs were monodispersed spheres with an average core size of 14.1 ± 2.1 nm (Figure 1c). At a high magnification (Figure S1b and c), the DAP negatively stained with phosphotungstic acid (PTA) was exhibited as a core/shell structure (the detailed characterization in SI): a hydrophobic core coated by a layer of an organic material with a different density (the thickness was approximately 4 nm), corresponding to a monolayer of phospholipid molecules.27 Moreover, the surface coating on the cores was further confirmed by the dynamic light scattering (DLS). The 9.4 nm difference between the average core size measured by TEM and the hydrodynamic size obtained by DLS (23.5 ± 1.2 nm, Figure 1d) was attributed to the PEGylated phospholipid surface coating. The thickness of the PEG layer was close to the calculated Flory radius of the PEG coil (MW ~5000 Da),8 suggesting that the core was coated with a monolayer of PEGylated phospholipid. The particle monodispersity of DAPs was further confirmed by their narrow size distribution with a polydispersity index (PDI) of 0.18 ± 0.04. As seen in Figure 1e, the zeta potential of DAPs was 2.77 ± 0.17 mV in the PBS. Although the amine-terminated phospholipids were incorporated into the DAPs, the majority of PEGylated phospholipids were terminated with methoxyl groups. The DAPs were thus considered approximately neutral, which can further minimize particle recognition by the immune system.28 In addition, the composition of DAPs was confirmed by Fourier-transform


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infrared spectroscopy (FTIR) (Figure S2). The determination of the CH1000 loading within DAPs is very important to characterize their chemical and optical properties. The incorporated CH1000 dyes within DAPs were extracted and measured spectroscopically according to a standard curve (see detailed information in SI). The loading of CH1000 molecules within DAP was 1.59 ± 0.24 × 10-4 mmol/g DAP. Based on the structural and morphological information of the DAP cores obtained by TEM, the total number of CH1000 dye molecules within a single DAP can be estimated accordingly (SI: Determination of CH1000 loading of DAPs and surface coating characterization).

Not only did the PEGylated phospholipid coating render the DAPs water-soluble and biocompatible, they also provided the functional groups for subsequent bioconjugation with biomarkers or targeting molecules. The amine groups on the surface of DAPs were quantitatively measured using a spectrophotometric method according to our previous publication.8,

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Quantification of free amine groups on the DAP surface is critical to stoichiometric control of bioconjugation of targeting molecules. The measurement involved two steps: conjugation of free amines with bifunctional linkers (N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)) and cleavage of the leaving groups from the terminus of the linkers (Scheme S4). The cleaved group (2-mercaptopyridine, MP) was used as an indicator for the amine measurement. An average of 0.021 ± 0.003 µmol amine /mg DAP (or 184 ± 28 per DAP) was measured. Considering a total number of PEGylated phospholipid molecules per NP (0.172 ± 0.026 µmol/mg DAP), only 12 %


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of PEG chains were reactive to bifunctional linkers, or 60 % of amine-terminated PEG chains were accessible to those linkers.

To enhance targeting specificity and efficiency, the DAPs were covalently grafted with an engineered small protein anti-EGFR Affibody which was an acetylated (Ac), cysteine (Cys)terminated three-helix Affibody monomer (Ac-Cys-ZEGFR:1907) (Figure S3-S4).30-31 Similar to the quantification process of free amine groups on the DAP surface, the covalent immobilization of Cys-terminated anti-EGFR Affibody required two consecutive steps: the activation of free amines with sulfhydryl-reactive cross-linkers and the thiol-maleimide coupling between the cross-linker and Cys-terminated Affibody (Scheme S5). To confirm the conjugation of DAPs with Affibody, the hydrodynamic sizes and zeta potentials of the resultant Affibody-DAPs were characterized by dynamic light scattering (DLS). An obvious increase in hydrodynamic size of Affibody-DAPs from 23.5 ± 1.2 nm to 31.4 ± 2.3 nm was attributed to the immobilization of Affibody on the DAP surface. A PDI of 0.20 ± 0.05 for Affibody-DAPs indicated that the surface modification did not significantly affect the particle monodispersity. A slight decrease in the zeta potential (-2.97 ± 0.16 mV for Affibody-DAPs) was due to the Affibody conjugation (Figure 1e). No significant size and zeta potential change were observed after incubation of Affibody-DAPs in the biological media for 24 hours (Figure S5 and Table S1), indicating excellent colloidal stability of Affibody-DAPs.


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It is important to quantify the amount Affibody bound to nanoparticles in order to optimize the targeting property of DAPs and improve their in vivo behavior. Although the amount of coupled Affibody can be estimated from the difference in spectrophotometric measurement of a total amount of initial Affibody and free Affibody in filtrates, this indirect measurement method often results in overestimation. An alternative spectrophotometric method, similar to quantification of free amines, was developed to provide the relatively accurate data after being validated by a reference standard. During the measurement, the Affibody molecules were immobilized on the DAPs using pyridyl disulfide reaction chemistry instead of maleimide reaction chemistry. Since the reaction is stoichiometric, the amount of the bound Affibody molecules was quantitatively determined by the spectrophotometric measurement of the released MP group. When an excess amount of Affibody (based on the number of amines on the surface) was used for surface immobilization, the maximum loading of Affibody per DAP was 1.32 ± 0.23 × 10-2 µmol /mg DAP (or 117 ± 21 per DAP). According to the number of the initial reactive amine groups, the surface coverage was calculated to 63.6 %, probably due to the limited accessibility of Affibody to functional PEG chains as well as the steric hindrance by each other. The corresponding footprint of Affibody on DAP surface was estimated to be 14.7 ± 0.5 nm2. Although the surface loading of the Affibody was directly determined using the disulfide exchange method rather than maleimide chemistry, the maximum loading of the Affibody mainly depended on the amount of free amine groups on the surface as well as the Affibody concentration in the solution. A saturated surface with a fully packed Affibody layer was finally obtained by either disulfide


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bonds or thioether linkages when a large access amount of Affibody was applied.30 Moreover, typical characteristic properties (such as sizes and zeta potentials) of the resultant AffibodyDAPs were very similar (Figure S6). Therefore, a thioether linkage was preferentially used to immobilize the Affibody molecule on a thiol-active DAP because it was irreversible and irreducible in the physiological condition and more stable than a disulfide bond.8

Optical Properties of Affibody-DAPs. Because of a high density packing of CH1000 in the core, the DAPs exhibited promising optical properties for biomedical imaging. Similar to CH1000, the DAPs in water exhibited two characteristic absorption peaks, one at c.a. 400 nm and the other with absorption maximum at 680 nm in the range of the first near-infrared window (NIR-I) (Figure 2a and Figure S7). A slight blue shift (20 nm) of the absorption maximum of DAPs at 680 nm with respect to the CH1000 at 700 nm in the monomeric form was attributed to the presence of strong inter-chromophore π-π stacking and H-aggregates of CH1000 in the DAP core.5 The extinction coefficient of CH1000 (εCH1000) in THF at 700 nm was calculated to be 8283 M-1cm-1, while the extinction coefficient of DAPs was 7400 M-1cm-1 in water (based on the molar concentration of CH1000 within DAPs) or 1.04 × 107 M-1cm-1 (based on the molar concentration of DAPs). Similar to the original version of CH-dye (CH1055), the CH1000 with two ethyl acrylate groups exhibited a large Stokes shift (~310 nm) and strong fluorescence with a maximum peak at 990 nm, due to its symmetrical D-π-A-π-A chromophore structure which


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significantly enhances the electronic interactions between donor and acceptor to reduce the HOMO-LUMO energy gap. Although replacing the carboxylic acid group of CH1055 with ethyl acrylate groups caused a blue shift of fluorescence emission from 1055 to 990 nm, the broad emission peak and its tail still extended into the NIR-II region (1000 - 1300 nm). The fluorescence spectra of both DAP and CH1000 were very similar; there was no obvious red/blue shift at the maximum fluorescence peak of DAPs. However, the fluorescence intensity dramatically decreased (47%, Figure S8) when the DAPs were formed via self-assembly of their constituent monomers (CH1000). Such fluorescence self-quenching was due to intermolecular ππ stacking of CH1000 and formation of aggregates within the core. As expected, the fluorescence signal of DAPs showed the excellent signal linearity with particle concentrations over a range of 0.7 - 141 µM based on CH1000 (or 0.5 - 100 nM DAP, Figure 2b). After being corrected with the laser energy at different wavelengths,32 the PA spectrum of DAPs was obtained and shown as a function of wavelength in Figure 2c. Similar to the extinction spectrum of DAPs, the PA spectrum exhibited a broad peak with the maximum PA intensity at 680 nm, extending over a wide NIR-I region (680 - 850 nm). Moreover, the PA amplitudes of DAPs as a function of molar concentration of nanoparticles showed an excellent linearity within a range from 8.8 to 282 µM based on CH1000 (or 6.25 - 200 nM DAP). The in vitro detection thresholds of the DAPs for both PA and fluorescence imaging were determined according to the background signal levels. The lowest detectable concentrations were 8.8 µM for PA imaging and 0.7 µM for NIR-II fluorescence imaging. Furthermore, the photoacoustic and fluorescence signals produced by the


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DAPs in vitro were highly linear and correlated with each other (R2 = 0.998, Figure S9). As seen in Figure 2e, photoluminescence excitation mapping (PLE) was performed on the DAPs and showed an excitation peak at c.a. 785 nm and a broad emission centered at 990 nm with a long tail covering the region between 1000 - 1300 nm, which was similar to the PLE mapping of CH1000 (Figure S10). To align well with the maximum excitation wavelength from PLE mapping, an 808-nm excitation laser was selected for in vitro and in vivo NIR-II fluorescence imaging not only because of the availability of tunable laser power but also because of the deeper penetration of the longer wavelength. The fluorescence lifetimes (τ) of both CH1000 and DAPs were measured using time-correlated single photon counting (TCSPC) technique. As seen in Figure 2f, there was an obvious decrease in the fluorescence lifetime when DAPs (τ = 0.86 ns) were self-assembled from CH1000 (τ = 1.33 ns), due to the dye aggregation and fluorescence quenching. A complete recovery of fluorescence lifetime (τ = 1.37 ns) was observed when freeze-dried DAPs were dispersed in an organic solvent, because the hydrophobic interaction between CH1000 and phospholipid molecules was disrupted by organic solvents, finally leading to DAP dissociation. By the linear-regression plot of integrated fluorescence intensities at the different concentrations on a molar basis, the quantum yield (QY) of the DAPs was determined to be 11.1% when using IR-1061 as a reference (QY = 1.8%) (Figure 2g).14 Compared to the original version of CH1055 (QY = 0.3%) in aqueous solutions,11 the DAPs showed a 37-fold increase in the quantum yield, which is significantly higher than most of previously reported NIR-II dye or quantum dots at the same condition.13-14 The relatively high QY makes it possible


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to acquire fluorescence images at extremely short exposure time (i.e. less than 50 ms), enabling fast dynamic scanning of microvasculature of tissues and organs with enhanced temporal resolution and spatial resolution. Furthermore, the photostability of DAPs under a long-term exposure of the laser radiation from NIR-II and PA imaging systems was investigated (Figure 2h). Under a continuous-wave (CW) 808 nm laser at a power density of 140 mW/cm2 for one hour, the DAPs showed a superior photostability in term of fluorescence intensity; there was only a less than 3% change during the laser exposure. Similarly, no photoacoustic amplitude was lost for DAPs after exposure to a tunable nanosecond pulsed laser (20-Hz pulse repetition frequency, wavelength-dependent laser power density, about 4~7 mJ/pulse on the sample surface), further confirming their superior photostability and suitability for a long-term in vivo PA molecular imaging. As importantly, Affibody-DAPs provided stable photoacoustic and fluorescence signal in various biological environments over the course of 48 hours (Figure S11).

Favorable In vivo Behavior of Affibody-DAPs. The in vivo biodistribution and pharmacokinetic of Affibody-DAPs were evaluated in the mice model (n = 4 per group) using positron emission tomography (PET). After conjugation with a copper chelator (1,4,7triazacyclononane-triacetic acid, NOTA), Affibody-DAPs were successfully labeled with PET radionuclide

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Cu at a specific activity of 0.562 GBq /mg NP (or 5 MBq /pmol NP) with a

radiolabeling yield of 60~70% (Scheme S6 and Figure S12). The coronal and transverse PET


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and computed tomography (CT) images of nu-nu mice were acquired at 1, 2, 4, 24, and 48 hours after a tail-vein injection of 3.7 MBq of 64Cu-Affibody-DAPs. As seen in Figure 3a, the majority of

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Cu-Affibody-DAPs accumulated in liver and spleen. The nonspecific uptakes of

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Cu-

Affibody-DAPs by major organs or tissues, delineated and localized by CT images, were quantitatively analyzed using PET images. The PET quantification data shown in Figure 3b revealed that the liver and spleen uptakes were prominent during the first four hours following injection, and gradually decreased over the next 48 hours, indicating that Affibody-DAPs mainly underwent clearance through the hepatobiliary pathway.33 Although the DAPs were sequestrated to some extent by the mononuclear phagocyte system (MPS), PEGylation dramatically improved the DAP in vivo biodistribution and prolonged their blood circulation time. The PET signal from the heart (as a cardiac blood pool) was used as an indicator to calculate the circulation time of the PET probes.8 The PEGylated DAPs showed a relatively long blood circulation half-life (τ1/2 = 9.8 h, Figure S13). Such a slow systemic clearance of circulating DAPs could potentially improve their bio-distribution and enhance their targeting capability in vivo. The intestine uptake reached a maximum value at 4 h post injection and then gradually decreased, which was consistent with the elimination of DAPs through hepatic excretion over time. The kidney retention of DAPs indicated that the renal excretion was also involved in the systemic clearance of DAPs from mouse body. It was worth noting that there were significant accumulation and retention of Affibody-DAPs in the draining lymph nodes (LNs) after 24-hour post-injection, especially in the brachial and axillary LNs (Figure 3a). Quantitative analysis showed the uptake


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of DAPs in LNs dramatically increased after 24-hour post-injection, reaching a plateau at 24hour time point and leveling off in the next 24 hours, which roughly matched their biological half-life. Such high LN accumulation was attributed to the amphiphilic phospholipid surface coating of DAPs as well as their particle size.7 Although the hydrophobic interaction between amphiphilic phospholipids and the cores shows excellent stability in the normal physiological conditions, it is sometime vulnerable to a lipid-rich environment such as lymphatics and LNs (often emulsified with lipids). Therefore, high LN accumulation of DAPs after intravenous administration suggested that the DAPs could be used as targeting imaging probes for LN mapping.

The biocompatibility of Affibody-DAPs was investigated in vitro and in vivo. In vitro cytotoxicity of Affibody-DAPs was evaluated via a standard colorimetric assay using murine fibroblast cell line NIR-3T3. The cells showed excellent viability in a large concentration range for 24 hours (Figure S18). Moreover, a pilot study was carried out to assess the in vivo cytotoxicity of Affibody-DAPs. After the mice were injected intravenously with a high dose of Affibody-DAPs (10 times than the imaging doses, 4 mg of CH1000 per kg mouse), the mice were euthanized and major organs were collected immediately for hematology analysis. As seen in Figure S19, histologic and microscopic examination revealed there were no common or uncommon toxic changes in the major organs.


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In Vivo LN Mapping Using Affibody-DAPs With Both Photoacoustic and NIR-II Fluorescence (NIR-II) Imaging. Since the LN mapping is clinically important for guiding surgical resection of tumors, there is an urgent need to engineer DAPs as multi-modality imaging probes for LN tracking with both photoacoustic and NIR-II fluorescence imaging. As a proof-ofconcept, the superficial LNs were selected for both PA and NIR-II imaging to validate the correlation between two imaging modalities. In a ventral view of the axillary region of a nude mouse, two LNs were clearly identified from both PA and NIR-II images (Figure 4). Although the field of view (FOV) of NIR-II was approximately 50 percent greater than that of PA imaging (20 × 20 mm), the axillary LN (indicated by a down arrow) was still differentiated from the brachial LN (indicated by an up arrow) in the NIR-II image, due to strong fluorescence signal and low light scattering in the NIR-II window. The high contrast effect provided by DAPs certainly allowed for accurate delineation and localization of LNs. Similar to NIR-II image, the PA image showed two LNs with a relatively high signal-to-noise ratio. After three-dimensional (3D) volume rendering of the PA data set, a coronal view of the axillary region matched to the NIR-II image. With an appropriate internal reference (i.e. blood vessel), it is possible to coregister the NIR-II images with a projection view from 3D PA data set (Figure S14). A 3D tomography image of LNs was reconstructed, and the morphology and homogeneity of LNs with a resolution of 280 µm were further evaluated. As seen in Figure 4e, the uptake or distribution of DAPs inside LNs was heterogeneous, suggesting that the DAPs entered the LNs via afferent lymphatic vessels to the subcapsular sinus and into the cortical sinuses. Moreover, the


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localization of LNs was further confirmed by the spectroscopic PA measurement. The PA spectra were acquired at multiple excitation wavelengths after being normalized with laser powers. The LN region showed a strong PA peak at 700 nm, which was in accordance with the DAP extinction peak (Figure 4f and Figure 2a). There was a small PA peak shift (from 680 nm to 700 nm) for DAP inside LNs, which was attributed to the wavelength-dependent attenuation of the excitation light fluence in biological tissues.5 Therefore, both PA and NIR-II imaging have similar detection depths and can be used to delineate and localize the LNs in vivo for intraoperative margin assessment.

PA and NIR-II Vascular Imaging Using DAPs. The ability to image and accurately quantify the modulation of vessel growth including the number and spacing of blood vessels, vascular permeability, and abnormalities in vessel walls, is very important to the detection and diagnosis of angiogenesis associated diseases such as cancers.13 To evaluate the spatial resolution acquired by PA and NIR-II imaging, the femoral artery and vein in the mouse hindlimb region were imaged with both imaging modalities after intravenous injection of DAPs. PA and NIR-II imaging were conducted subsequently 10 min after injection. A cross-sectional profile across the blood vessels in each modality image was used to analyze the spatial resolution. The PA intensity profile along the line perpendicular to the femoral vessel was shown in Figure 5a-b, while the corresponding fluorescence intensity was plotted with the physical location along the


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line as shown in Figure 5c-d. The peaks in the intensity profiles represented the femoral vessels; the calculation of the vessel widths was based on the corresponding full width at half maximum (FWHM) of the peaks.13 The vessel width obtained from PA image (300 µm) was very close to that from the NIR-II image (346 µm), suggesting that both imaging modalities were comparable in term of the spatial resolution for vascular imaging. A shoulder peak was found in the fluorescence profile of femoral vessels due to the existence of proximal femoral artery. To further distinguish the arteries from the veins, a time course of NIR-II fluorescence images focused on the femoral vessels in the hindlimb area was recorded immediately following intravenous injection of DAPs. Because there was a short time delay of fluorescence signals through the artery and vein in the first minute after tail vein injection of DAPs, principal component analysis (PCA) was used to analyze the dynamic contrast-enhanced images to distinguish the arteries from the veins.34 The high QY and brightness of DAPs in NIR-II allowed for high-frame-rate video imaging of mouse blood flow in the arteries and veins. The artery color-coded in red and the vein color-coded in yellow were successfully identified in the PCA overlaid image (Figure 5e). Two distinctive peaks were found in the fluorescence profile of the PCA overlaid image of femoral vessels. The vessel width extracted from the PCA overlaid image (359 µm) was consistent with the result from PAI.

It is also a challenge to differentiate the neovasculature from normal blood vessels and monitor the functional abnormalities of angiogenic blood vessels. Repeatedly and noninvasively imaging


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the same area over a period of hours or even days is very critical for minimizing the variation to detect the subtle changes in microvasculature. To demonstrate the capability to measure angiogenesis intravitally, the mouse xenograft model of thyroid carcinoma FTC-133 was used to conduct in vivo efficacy evaluation of dual PA and NIR-II modality imaging (Figure 5g-h and 5jk). The NIR-II images of the FTC-133 thyroid tumors in the NIR-II window were acquired at the predetermined time points after intravenous injection of DAPs. A time course of PA images of FTC-133 tumors was obtained immediately following the NIR-II imaging. The densities of blood vessels in the tumor visualized by both imaging modalities were very similar. It is possible to quantitate the blood vessel density in the tumor based on the PA or fluorescence intensity profile along the line across the tumor. More importantly, the multiple injections of DAPs did not significantly affect the intensity profiles. As seen in the Figure 5i and Figure 5l, although their peak widths slightly increased after 2nd injection of DAPs, their signal intensity profile was in agreement with that of the first injection, which makes it possible to monitor the subtle changes in the microvasculature by repeatedly imaging the same area over and over again. Moreover, the vessel widths extracted from NIR-II imaging were comparable with those from PAI, and the variation arising from different measurements could be minimized. By adjusting the angle of view, the PA image after the 3D volume rendering had a good colocalization with the NIR-II image. Therefore, as NIR-II and PAI contrast agents, the DAPs enabled both imaging modalities for angiogenesis diagnosis and monitoring.


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Targeted NIR-II and PA Imaging of FTC-133 Thyroid Tumor. The targeted NIR-II and PA imaging of FTC-133 thyroid tumor was achieved using Affibody-DAPs. First of all, the targeting capability of Affibody-DAPs to EGFR-positive FTC-133 cells were evaluated in vitro. Cellular uptake of Affibody-DAPs by FTC-133 cells with and without a blocking dose of Affibody were shown in Figure S15. Affibody-DAPs exhibited significantly higher uptake than the blocking group at the same condition, indicating that specific targeting ability of Affibody immobilized on DAPs contributed to the enhancement on the uptake of Affibody-DAPs by FTC-133 cells. Although the nanoparticle-based contrast agents can accumulate within the tumors via the enhanced permeability and retention (EPR) effect, the targeting moieties on the nanoparticles can improve their targeting specificity at the desired sites and minimize non-specific interactions of nanoparticles within normal tissues or organs. To evaluate the efficacy of Affibody-DAPs ability to detect EGFR-positive tumors in vivo, the FTC-133 tumor-bearing mice (n = 4 for each group) were intravenously injected with 100 µL of Affibody-DAPs (400 µg CH100 per kg mouse or 323 pmol per kg mouse body weight) in PBS, and the control mice for the receptor-blocking experiment (n = 4) were co-injected with the same amount of Affibody-DAP and 1.8 µmol of Affibody (Ac-Cys-ZEGFR:1907) per kg of mouse body weight. A time course of NIR-II fluorescence images of the mice at the lateral position was acquired by NIR-II camera with a FOV covering the whole mouse body in order to detect the tumor and monitor the Affibody-


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DAP uptake of major organs or tissues (Figure 6a). Immediately followed by the NIR-II imaging, the tumor areas were photoacoustically imaged at 14 different wavelengths in the range of 680 - 950 nm for spectroscopic analysis at 0.5, 1, 2, 4, 24, and 48-hour post-injection (Figure S16). After subtracting the pre-injection images, the images acquired at 700 nm were fused with the images at 850 nm to display both the probe signals and tumor vasculature simultaneously (Figure 6b).35 The tumors were clearly visualized after the injection for both PA and NIR-II imaging modalities. Although there were no significant differences in either PA or NIR-II signal intensities within the tumors between the targeting probe (Affibody-DAPs) group and the blocking group at early time points after injection, the signals within tumors of the targeting group increased dramatically after 4-hour injection, becoming significantly different than those of the blocking group at 24-hour post-injection. Quantitative analysis of the NIR-II signals from the tumor areas confirmed that the FTC-133 tumor uptake was efficiently inhibited by administration of a blocking dose of Affibody, suggesting that the Affibody-DAPs can selectively target the EGFR-positive tumor. The tumor uptake of Affibody-DAPs started to show significantly higher signal than that of the control group at the 4-hour post-injection, reaching their maximum at 24-hour injection, which was 2-fold higher than that of the control group (Figure 6c). Strong signals from livers of both the targeting group and control group were also observed (Figure 6d), indicating the majority of the injected Affibody-DAPs were sequestrated by the MPS. This result was consistent with the biodistribution study from previous PET quantification analysis. Over time, the liver uptake of Affibody-DAPs was roughly similar to that


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of the blocking group, but there was a significant difference at 4-hour post-injection, mainly due to the EGFR expression of murine liver.30 As seen in the Figure 6e, the tumor-to-liver signal ratios of the targeting group changed as a function of the time, becoming significantly different from the control group after 24-hour post-injection. Different from the quantitative analysis of the NIR-II signals from the 2D tumor images, the quantification of PA signal from the tumors needed to be done in 3D regions of interest around tumors. The PA signal from FTC-133 tumors increased steadily in the first four hours after injection, reaching a maximum value at 24-hour post-injection, which was a more than 2-fold higher signal than that of the blocking group. Although the PA signal gradually decreased in the next 24 hours, the FTC-133 tumors after postinjection of Affibody-DAPs still showed higher signal intensities than those from the control group, which was consistent with the result from NIR fluorescence quantification. Moreover, the photoacoustic spectra of tumor areas in the range of 680 - 940 nm can be extracted from a series of images at different wavelengths after laser intensity corrections (Figure S16). Compared to the blocking groups, the tumor areas showed a much stronger PA peak maximum at 700 nm 4 hours after injection of Affibody-DAPs. Since the background signals could be extracted from the spectra, the Affibody-DAPs within the tumors were clearly identified according to their characteristic spectra. When compared with one steady image, the spectroscopic photoacoustic analysis ultimately provides more accurate results for molecular targeting imaging of tumors.


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To confirm the in vivo imaging and quantification result, ex vivo imaging of tumors and other major organs or tissues was performed after 48-hour post-injection. Representative ex vivo NIRII fluorescence images of major organs, tissues and tumors was shown in Figure S17. Similar to the in vivo imaging result, the fluorescence signals in the FTC-133 tumors after injection of Affibody-DAP were much brighter than those of the blocking group. Quantitative analysis of the fluorescence signals showed that a significant enhancement (of 2.8-fold) in fluorescence intensity was observed from the FTC-133 tumors in the targeting group as compared to those treated with a blocking dose of Affibody, indicating excellent targeting specificity of the Affibody-DAPs to EGFR positive tumors. No significant difference was observed in other major organs and tissues between the targeting and blocking group. The organ and tissue uptakes of the Affibody-DAPs from ex vivo fluorescence biodistribution analysis were consistent with the PET quantification analysis results, further confirming the nonspecific uptake of the Affibody-DAPs by liver and spleen, but low accumulation in kidneys, muscle and other major organs. The relatively high uptake of NPs in LNs was also observed by fluorescence quantification analysis, in agreement with the PET quantification result. All these findings were consistent with the in vivo imaging results, demonstrating that Affibody-DAPs have favorable in vivo distribution, high tumor accumulation and hepatobiliary clearance. As importantly, they can be efficiently applied as both PA and NIR-II contrast agents for in vivo targeting imaging of EGFR positive tumors.


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DISCUSSION

In this study, we have successfully demonstrated the feasibility of using a donor-acceptor chromophore based nanoparticle to perform dual NIR-II and PA imaging both in vitro and in vivo. Although NIR-II and PA imaging modalities both rely on incident light, each modality has its own unique strengths in terms of sensitivity, spatial resolution, temporal resolution, and depth of tissue penetration for non-invasive imaging. Combining these two modalities together enables us to effectively acquire complementary functional and molecular information at targeted sites and synergistically enhance the efficacy and accuracy in cancer detection and diagnosis.

There are several important features that arise with the combination use of both PA and NIR-II imaging for the cancer detection and diagnosis. First of all, combining two imaging modalities can allow their strengths to complement each other and compensate individual weaknesses. Both PA and NIR-II imaging modalities have their inherent advantages and limitations. For example, PA imaging combines both strong optical contrast and high ultrasonic resolution in a single modality with a centimeter-scale penetration depth.9 Although recent development of image reconstruction algorithms makes it possible to provide real-time cross-sectional PA images, it is still tedious and time-consuming to process the volumetric images when high resolution threedimensional tomographic images are desired. According to the temporal resolution of currently available PA techniques, it remains a challenge to track rapid biological processes at a molecular level. On the other hand, the conventional fluorescence imaging at the NIR-I window (650 - 950


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nm) has impressive spatial and temporal resolution capabilities but it is still limited in terms of depth imaging (up to 0.2 mm depth) and suffer from poor axial resolution. Many efforts to combine both PA and conventional NIR-I imaging modalities have recently been made to produce co-registered images but still remains challenge for deep targeted sites due to the mismatch of their distinctive penetration depths. In contrast to conventional NIR-I imaging, in vivo fluorescence imaging in the NIR-II window can afford deep tissue penetration and high spatial resolution, due to reduced photon scattering, diminished tissue autofluorescence, and low levels of photon absorption.12 As importantly, the centimeter-scale imaging depth at a low resolution and micron-scale resolution (up to 3 mm depth) of anatomic and molecular features provided by in vivo NIR-II fluorescence imaging are compatible with those obtained by PA imaging, therefore offering significantly improved spatial co-registration of both imaging modalities.

Secondly, both PA and NIR-II imaging modalities have similar diagnostic capabilities in terms of spatial resolution and tissue penetration. Compared to the NIR-I imaging, NIR-II imaging offers higher imaging spatial resolution at up to centimeter tissue penetration depths, because of less photon scattering and minimal tissue autofluorescence in NIR-II window.12 Moreover, based on the optical contrast of tissues, PAI also provides high-resolution images with deep penetration depths beyond the optical diffusion limit.7 The results of PA and NIR-II vascular imaging using DAPs confirmed that both modalities were very similar in terms of the spatial resolution and


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tissue penetration; the number and spacing of blood vessels within normal tissues and tumors obtained by NIR-II imaging clearly matched those from PAI. It is of great clinical significance to provide the detailed information about microvascular density and branching patterns with high quality spatial resolution and deep tissue penetration, allowing us to monitor the tumor angiogenesis and further predict tumor aggressiveness and prognosis.

Thirdly, one of the most important motivations for developing hybrid contrast agents for both PA and NIR-II imaging modalities is to allow us to study the same target with the same imaging agent on different imaging platforms with different scales and visions. However, introducing two or more kinds of contrast agents into the multimodal imaging system may cause undesired side effect, and more importantly, the enhancement of one modality must not be at the expense of another. In order to develop a dual contrast agent for both PA and NIR-II imaging for the living subjects, we modified the original structure of CH1055 with different substituents to adjust its lipophility for in vivo use. The resultant CH1000 dyes not only remain strong absorption capability in the NIR-I window but also exhibit significantly enhanced fluorescence in the NIRII region when they are encapsulated within amphiphilic phospholipids to form water-soluble nanoparticles (DAPs). Their superior optical properties such as strong absorption, high quantum yield, and very large Stoke shift (more than 310 nm) gurantes the fluorescence/photoacoustic imaging acquisition with high signal-to-noise ratio, significantly reducing minimal interference between PA effect and NIR-II fluorescence when the same contrast agent is used simultaneously


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for both PA and NIR-II imaging. Moreover, the intensities of both PAI and NIR-II scale linearly with the amount of DAPs at a large range of concentration scales. As importantly, both imaging signals are highly correlated with each other (Figure S9). The excellent correlation between two imaging modalities was successfully confirmed by PA and NIR-II imaging of the blood vessels, LNs and tumors, making it possible to overlay both modal images in a co-registered fashion in presence of an appropriate reference. These engineered DAPs are very important for accurate delineation and localization of LNs for biopsy and evaluation of lymphatic diseases.

Fourthly, controlling the ratios of multiple doses of contrast agents for PA and NIR-II imaging is very important to reduce the interferences between two imaging modalities. Due to different sensitivities, the concentrations of contrast agents for PAI are normally higher than those for fluorescence imaging. There is an urgent need to develop multimodal contrast agents that require only one dose for both imaging modalities, therefore reducing side effects from having to use multiple doses. The DAPs we developed here are monodisperse so that each individual nanoparticle has nearly identical optical and chemical properties for biodistribution and contrast effects. Upon laser irradiation, the DAPs produce strong signals that can be caught by either PA or NIR-II imaging. The NIR-II imaging signal does not overwhelm PAI’s. The sensitivities of both PA and NIR-II imaging is still comparable so that a single dose of DAPs can facilitate both preoperative and intraoperative PA and NIR-II imaging.


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Finally, the use of dual PA and NIR-II imaging is of great importance in oncologic studies. The dual PA and NIR-II imaging has the ability to address biological processes or clinical questions with complementary information and synergistic advantages, which are hardly implemented using a single modality imaging alone. As expected, PAI provides high-resolution structural images of tumor angiogenesis, and NIR-II imaging has high sensitivity towards molecular probes for detecting tumor biomarkers. Compared to traditional NIR-I imaging, NIR-II imaging provides superior image quality and excellent spatial (~300 µm) and temporal resolution (100 ms) about the targets, and can permit precise discrimination between feeding arteries and draining veins (Figure 5).11 Importantly, high sensitivity and spatial resolution of NIR-II imaging allow us to noninvasively detect the tumors and delineate the margins of the tumors preoperatively and intraoperatively. The real-time intraoperative NIR-II imaging at a fast video frame rate makes it possible for highly sensitive and specific detection of tumor margins. However, the use of NIR-II imaging alone for tumor detection offers only two-dimensional (2D) visualization of the targets, and the loss of detailed spatial information may cause inaccurate target delineation. On the other hand, because of long acquisition and post-reconstruction times, PAI has relatively lower temporal resolution than NIR-II imaging (3 min vs. 100 ms). Considering its superior spatial resolution, targeting specificity and three-dimensional capabilities, however, PA imaging can provide preoperative volumetric images for a noninvasive tumor delineation and preoperative planning to guide stereotactic surgery. Therefore, both PA and NIR-II imaging modalities can be synergistically combined together using the DAPs as dual


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contrast agents to provide complementary information about the targets preoperatively and intraoperatively, and have ability to efficiently integrate multiple imaging information in a synergistic manner, potentially resulting in significant implications in preclinical and clinical research. Due to the availability of preclinical imaging system, potential clinical translation, and detection depths, current dual PA and NIR-II imaging technique is very valuable in the characterization of thyroid nodules and can confirm malignancy accurately and noninvasively. Moreover, dual PA and NIR-II imaging has a great potential to evolve into a clinically translatable platform to complement existing imaging techniques for the detection and treatment monitoring of various cancers.

CONCLUSION The donor-acceptor chromophore based nanoparticles displayed very promising optical properties and had an intrinsic ability to combine both PA and NIR-II fluorescence imaging modalities for biomedical imaging application including lymph node mapping, vascular imaging, and tumor detection. Affibody-conjugated nanoprobes have the capability to bind EGFR-positive tumors in FTC-133 subcutaneous mice model with relatively high photoacoustic and fluorescence signals. By using dual modal contrast agents, targeted photoacoustic/NIR-II fluorescence imaging allows us to specifically image and detect tumors in an accurate and quantitative manner.


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MATERIALS AND METHODS Materials.

N-hydroxysuccinimide

(NHS),

N-hydroxysulfosuccinimide

(sulfo-NHS),

N-

succinimidyl 3-(2-pyridyldithio) propionate (SPDP), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Pierce Biotechnology. DSPE-mPEG5000 and DSPEPEG5000-NH2 were ordered from Laysan Bio, Inc. The metal chelator, S-2-(4-Aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic

acid

(p-NH2-Bn-NOTA),

was

obtained

from

Macrocyclics. Unless otherwise mentioned, all other solvents and chemicals were purchased from Sigma /Aldrich without further purification.

General Methods. Mass spectra of synthetic polymers were recorded by a time-of-flight (TOF) mass spectrometer (AB SCIEX TOF/TOF 5800, Applied Biosystems) equipped with a matrixassisted laser desorption ionization (MALDI) ion source. FTIR spectra in a range of 400 - 4000 cm-1 were obtained using Thermo Scientific Nicolet iS50 FT-IR spectrometer. UV-vis-NIR spectra were acquired by Agilent Cary 6000i UV/Vis/NIR spectrophotometer. A CRC-15R PET dose calibrator (Capintec Inc., Ramsey, NJ) was used for all radioactivity measurements. The transmission electron microscope (TEM) images were recorded with a FEI Tecnai G2 F20 XTWIN transmission electron microscope operating at 120 kV. Samples were deposited and dried on copper grids covered with a Formvar/carbon support film after glow discharged. The


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characterization of sizes of NPs by TEM was performed according to the standard assay protocol (NIST - NCL Joint Assay Protocol, PCC-7), including sample preparation, measurement and result analysis. A minimum of 200 discrete particles was measured from each of at least two widely separated regions of the sample. Version V1.46 of NIH ImageJ was used for image processing, analysis and measurements.

Synthesis of CH1000. The detailed synthetic procedure of CH1000 was described in the Supporting information (SI). 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.6 Hz, 4H), 7.58 (d, J = 15.9 Hz, 2H), 7.37 (d, J = 8.5 Hz, 4H), 7.27 (t, J = 8.6 Hz, 8H), 7.20 (d, J = 0.9 Hz, 4H), 7.11 (dd, J = 12.2, 7.9 Hz, 6H), 6.26 (d, J = 15.9 Hz, 2H), 4.19 (q, J = 7.1 Hz, 4H), 1.27 (t, J = 7.1 Hz, 6H).

13

C NMR (101 MHz, CDCl3) δ 167.36, 152.75, 149.21, 147.56, 146.55, 144.05, 132.96,

129.74, 129.69, 129.31, 128.66, 126.30, 124.76, 123.32, 123.11, 120.23, 116.11, 60.39, 14.38. HRMS (ESI) Calcd. for: C52H41N6O4S2+ ([M+H]+ ): 877.2625. Found: 877.2622.

Synthesis of DAPs. A mixture of DSPE-mPEG5000 (1.0 mg) and DSPE-PEG5000-NH2 (0.20 mg) was dissolved in 1.0 mL of tetrahydrofuran (THF) containing 200 µg of CH1000. After thorough mixed, the solution was rapidly injected into cold deionized water (10 mL) with an ice bath under continuous sonication with a microtip-equipped probes sonicator (Branson, W-150) at a power output of 6 watts RMS for 60 seconds. The remaining THF in the mixture was evaporated at 40 °C under a nitrogen flow with rigorous stirring. The resultant aqueous solution was filtered through a polyethersulfone (PES) syringe driven filter (0.22 µm) (Millipore), and washed three


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times using a 30 kDa centrifugal filter units (Amicon centrifugal filter device, MWCO = 30 kDa) under centrifugation at 4000 rpm for 10 min at 4°C. The resultant products, DAPs, were finally concentrated and stored at 4°C. The concentrated DAPs were reconstituted in phosphate buffered saline (PBS) and filtered through a 0.22 µm syringe filter for cell and animal experiments. The hydrodynamic sizes and zeta potentials were measured using dynamic light scattering and zeta potential analyzer (Malvern, Zetasizer Nano ZS90).

TEM. About 5 µL of DAPs solution (28 nM based on CH1000) were placed on the glow discharged 400 mesh carbon/formvar coated Cu grids, followed by incubation in a drop of 1% phosphotungstic acid (PTA, pH = 7.5) for 3 min. The excess solution was drained off, and the sample was then air dried. TEM images were recorded on a Tecnai transmission electron microscope at an accelerating voltage of 120 kV. The core sizes of DAPs were analyzed using ImageJ software. The results were obtained by counting a minimum of 100 particles in three discrete areas on TEM images. The average size of DAPs was 14.1 ± 2.1 nm.

Quantification Analysis of Amine Groups of DAPs. The amine groups on the surface of DAPs were quantitatively measured according to the previous publication (Scheme S4).8, 29 About 300 µg DAPs (dry weight, 48 nmol CH1000) was dissolved in 200 µL of 10 mM PBS (239 nM CH1000, pH = 7.2). The solution was treated with 10 mM N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) in 50 µL of dimethyl sulfoxide (DMSO) solution under a nitrogen atmosphere and stirred for 4h at room temperature. After conjugation, the activated DAPs were


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purified by a centrifugal-filter (Amicon centrifugal filter device, MWCO = 10 kDa) and washed 3 times by repeating the centrifuge/dispersion step, to completely remove excess SPDP and byproduct. The purified DAPs were re-suspended in 0.2 ml of PBS and treated with 10 mM TCEP for 2 hours at room temperature. The thiol-reactive portion (2-pyridyldithio group) of SPDP reagents reacted with free sulfhydryls or TCEP, resulting in displacement of a 2mercaptopyridine group (MP), the concentration of which was determined by measuring the absorbance at 365 nm (the molar extinction coefficient of the MP was 7000 M-1cm-1 at 365 nm in the experimental condition). The filtrate was collected by a centrifugal-filter (MWCO = 10 kDa), and the absorption of the released MP was measured at 365 nm by a UV-vis spectrometer. The DAPs without coupling SPDP were treated in the same condition and used as control samples in the spectrophotometric analysis. The spectral measurement was done in triplicate. Assuming that all of the amine groups on the DAPs were accessible to the SPDP crosslinker, the number of amine groups should be equal to the number of cleaved MP groups. An average of 0.021 ± 0.003 µmol amine /mg DAP (or 184 ± 28 per DAP) was measured.

Synthesis of Cysteine-terminated Affibody ZEGFR:1904. The Affibody derivative, Ac-CysZEGFR:1907

(Ac-

CVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQA PK-NH2) with 59 amino acid residues and a cysteine at the N terminal, was synthesized and purified according to our previous publications.30-31 The calculated molecular weight of Ac-Cys-


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ZEGFR:1907 is 6688.5 g/mol, and the measured molecular weight was found to be 6688.7 g/mol (Figure S3). Synthesis of Affibody-DAPs. The conjugation of DAPs with Affibody ZEGFR:1904 was performed according to our previous publications.8,

30

Typically, the water-soluble DAPs (8.12 mg dry

weight, 1.30 µmol CH1000) pre-dissolved in 1 mL of degassed PBS (pH = 7.4) were incubated with the cross-linker solution (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate, sulfo-SMCC (1.5 mg, 3.4 µmol), which was freshly prepared in 50 µL of DMSO) for 2h at room temperature. After complete removal of excessive sulfo-SMCC and byproducts using a PD-10 column (GE Healthcare, Piscataway, NJ), the thiol-active DAPs were finally concentrated to the final volume of 1.0 mL with a centrifugal-filter (Amicon centrifugal filter device, MWCO = 10 kDa), and were then incubated with the Affibody (1.1 mg, 164 nmol) in the degassed PBS with stirring at 4°C under nitrogen protection. The final Affibody concentration in the mixture was 147 µM. The conjugation reaction proceeded for 24 h. After unconjugated Affibody and byproducts were removed via PD-10 column, the purified Affibody-DAPs were concentrated by a centrifugal-filter (MWCO = 30 kDa) and stored at 4°C for one month without any loss of targeting activity. The final Affibody-DAPs were reconstituted in PBS and filtered through a 0.22 µm filter for cell and animal uses. The concentrations of CH1000 within Affibody-DAPs were determined by a UV-vis spectrometer. A linear absorption versus concentration calibration curve was constructed by water-soluble DAPs with known amounts of CH1000 molecules (described in the previous section, extracted from lyophilized DAPs) using


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Agilent Cary 6000i UV-Vis-NIR spectroscopy. The absorption coefficient of CH1000 in DAPs in the water at 680 nm is 7150 M-1cm-1.

The average number of Affibody molecules on the surface of single DAP were first determined by indirect measurements. Briefly, the amount of coupled Affibody was calculated from the difference between the total amount of initial Affibody and free Affibody from filtrates, which were measured using HPLC according to the Affibody’s eluent time, peak position and absorption at 280 nm. The indirect measurements often overestimated the loading of Affibody (2.7 × 10-2 µmol /mg DAP). The bound Affibody on the surface of DAPs was quantitatively measured according to the previous publication.8, 29 Similar to the amine quantification process, as-synthesized DAPs were first activated using SPDP. The cysteine-terminated Affibody molecules were immobilized on the SPDP- activated DAPs in the same manner on the SMCC activated DAPs. The concentration of MP from filtrates after 24h conjugation was determined by measuring its absorption at 365 nm. The conjugation reaction is stoichiometric and the released MP groups were used as an indicator for measuring the amount of bound Affibody, so the loading of Affibody per DAP was 1.32 ± 0.23 × 10-2 µmol /mg DAP (or the bound Affibody per NP was calculated accordingly to be 117 ± 21 per NP).

Characterization of DAPs and Affibody-DAPs. The hydrodynamic sizes and zeta potentials of DAPs and Affibody-DAPs were characterized by dynamic light scattering (DLS) using a Malvern Zeta Sizer Nano S-90 DLS instrument (Malvern Instruments Ltd, UK). The Helium-


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Neon laser (4 mW) was operated at 633 nm with the scatter angle fixed at 90°, and the temperature was maintained at 25° during the measurement. Ultraviolet-visible-NIR (UV-visNIR) extinction spectra of CH1000 (in THF) and DAPs (in water) were recorded using Agilent Cary 6000i UV-Vis-NIR spectroscopy. FTIR spectra of the freeze-dried DAP and AffibodyDAPs in a range of 400 - 4000 cm-1 were obtained using Thermo Scientific Nicolet iS50 FT-IR spectrometer.

Fluorescence Properties of DAPs. Fluorescence spectra of CH1000 (in chloroform), DAPs (in water), and freeze-dried DAPs (in chloroform) were acquired using Horiba FluoroLog-3 Fluorimeter with both UV-visible detector (PPD.900, 250-900 nm) and an InGaAs IR detector (H10330B-75, 950-1700 nm). The fluorescence lifetime measurements of CH1000 (in chloroform), DAPs (in water), and freeze-dried DAPs (in chloroform) were measured by timecorrelated single photon counting (TCSPC). A high-power supercontinuum fiber laser series (SuperK EXTREME, NKT Photonics, Denmark) was used for fluorescence lifetime measurements.

Photoacoustic Imaging (PAI). The photoacoustic signals were recorded using a Nexus 128 photoacoustic instrument (Endra Inc., Boston, MA) with a series of laser wavelengths in the range of 680 - 950 nm using a continuous rotation mode (with a scan time of 12 s per wavelength, 240 views, 1 pulse/view).32 The spatial resolution of PA imaging is limited to be 280 µm. The PA data is reconstructed in volumes of 256 × 256 × 256 with 0.1 × 0.1 × 0.1 mm


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voxels. The system is equipped with a tunable nanosecond pulsed laser (7 ns pulses, 20-Hz pulse repetition frequency, wavelength-dependent laser power density, about 4~7 mJ/pulse on the animal surface) and 128 unfocused ultrasound transducers (with 5 MHz center frequency and 3 mm diameter) arranged in a hemispherical bowl filled with water (temperature is set to 38°C). The imaging data were analyzed using Amide's a Medical Image Data Examiner (AMIDE)36 and Osirix software (Pixmeo SARL, Bernex, Switzerland).37 Different concentrations of DAP aqueous solution ranging from 4 nM to 400 nM were filled into polyethylene capillaries (I.D. = 0.76 mm, O.D. = 1.22 mm, one-inch long, Becton Dickinson Co. MD). Both ends of the filled tubing were quickly sealed by a hot plate, and then immersed in the water bath (38°C) of the imaging tray for measurement of photoacoustic signals. For in vivo imaging, the tumor-bearing mouse anesthetized with 2% isoflurane in oxygen was placed in the imaging tray with an appropriate position within the focal field of view (20 mm diameter sphere). The PA signals within the FOV were recorded, and the average voxel intensities within the Region of Interests (ROIs) were quantitatively analyzed using AMIDE or Osirix.

NIR-II Fluorescence Imaging System. The Fluorescence NIR-II imaging system was designed and built for fluorescence in vivo and in vitro imaging in the second near-infrared fluorescence windows (950-1700 nm). As seen in the Figure S20, the instrument mainly includes a NIRvana InGaAs camera (Princeton Instruments, USA) with 640 × 512 focal plane array and 20 µm/pixel sensor, an integrated 808 nm laser diode system (Fiber coupling laser system, 0-10W,


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Changchun New Industries Optoelectronics Technology Co., Ltd.), and a customized imaging chamber. The NIR-II camera is equipped with a 50-mm lens set (f /1.4, Pentax) and a variety of longpass optical filters. At the output of the liquid light guide, a beam expander is used to expand the laser beam according to the field of view (FOV). The laser beam spots on the image stage were circles and can be adjusted from 3 cm to 10 cm in diameter accordingly. By changing the lens and the distance between NIR-II camera and imaging stage, the system provides multiple magnifications (FOVs from 2.5 × 2 cm2 to 10 × 8 cm2) with a minimum pixel size of 50 µm. The excitation laser power was set to 4 W. The laser power densities on different FOVs were measured using an optical power meter PM20A (Thorlabs, Inc.). The emission light was passed through a 1000 nm longpass filter (Thorlabs, FEL1000) and focused onto the InGaAs detector by a 50-mm lens (f /1.4, Pentax). For in vivo imaging, the mice bearing tumors (n = 4 per group) were anesthetized with 2% isoflurane in oxygen and placed with either the prone, lateral (sidelying), or supine positions. For dynamic imaging, a laser power density of 140 mW/cm2 was used to enable optimal temporal resolution. For steady state imaging, the laser power density was limited to 140 mW/cm2. The exposure time varied from 10 ms to 1000 ms or more depending on the brightness of the fluorescence and the speed of the camera. The images were acquired and analyzed by LightField software. The further analyses of the fluorescence images were performed by Matlab R2016a (MathWorks, Natick, MA, USA).


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Principal Component Analysis (PCA). The dynamic two-dimensional fluorescence contrastenhanced images were analyzed using principle component analysis (PCA).34, 38 The consecutive image frames were recorded in the first five minutes after tail-vein injection, and were then loaded into MATLAB R2016a. The built-in princomp function was used to perform PCA. PCA is a terrific tool for converting time correlation into spatial resolution, thus one of the powers of PCA is the ability to resolve features that cannot be seen from the raw images. By performing PCA, the faster flowing features showing up early in the frames were grouped in the positive fifth principal component, while the slower flowing features showing up later in the frames were grouped in the negative fourth principal component. Then, pixels selected by the positive fifth principal component were color-coded in red to represent arterial vessels, while pixels selected by the negative fourth principal component were color-coded in blue to represent venous vessels. Finally, the color-coded arterial and venous vessels were merged into one image to effectively delineate the spatial distribution of the vascular system.

Photostability Study. The photostability of Affibody-DAP in different buffers (28 µM based on CH1000 in PBS or 10%FBS) sealed in a polyethylene capillary tubing was analyzed with the Nexus 128 Endra photoacoustic instrument and scanned using the maximum absorption wavelength (c.a. 700 nm) for 30 min. The photostability and photodegradation were evaluated by the change in the photoacoustic intensity over time compared to the original value. Moreover, the photostability of Affibody-DAP was further determined with NIR-II fluorescence instrument.


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The fluorescent intensities of Affibody-DAP (28 µM based on CH1000) in PBS or 10% FBS sealed in a tubing were continuously monitored at the same laser wavelength (808 nm) and power density (140 mW/cm2) as for in vivo imaging. The photostability was quantitatively evaluated by the change in the fluorescent intensity over time compared to the original value. Radio-labeling (64Cu) of Affibody-DAPs. The

64

Cu radiolabeling procedure was conducted

according to our previous publications with a slight modification (Scheme S6).8,

39-40

To

conjugate the DAPs with both Affibody and the copper chelator (1,4,7-triazacyclononanetriacetic acid, NOTA), the DAPs need to be treated with a mixture of sulfo-SMCC and p-SCNBn-NOTA. Briefly, the sulfo-SMCC (0.5 mg, 1.1 µmol) dissolved in 15 µl of DMSO was mixed with 3.3 µl of p-SCN-Bn-NOTA solution in the water (33 mM, 0.11 µmol). The crosslinker stock solution was added into the DAPs (42 µM based on CH1000, 0.5 mL) in 10 mM PBS (pH = 7.4). The mixture was then stirred for 2 hours at room temperature. The activated DAP-NOTA NPs were purified by a size exclusion column (PD-10 column, GE Healthcare, Piscataway, NJ) and concentrated to the final volume of 0.5 mL with a centrifugal-filter (Amicon centrifugal filter device, MWCO = 30 kDa). A solution of Affibody (200 µg, 30 nmol) in a degassed PBS was added into the above thiol-active DAP-NOTA solution with a nitrogen protection. The mixture was stirred at 4°C under nitrogen protection for 24 hours. The Affibody modified DAP-NOTA NPs were purified by a centrifugal-filter (MWCO = 30 kDa). The 64Cu stock solution was diluted with 200 µL of 100 mM sodium acetate buffer (pH = 5.0) before making the final pH adjustment


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using 0.1 M sodium hydroxide (NaOH) solution. Twenty microliters of buffered

64

Cu stock

solution (1 mCi, 37 MBq) were then added to 150 µL of Affibody-DAP-NOTA (42 µM based on CH1000) with 150 µL of 100 mM sodium acetate buffer. The mixture was then incubated at 40°C for 1 h, and the radiolabeled DAPs were applied to a size-exclusion column (PD-10, GE) and eluted with PBS. The eluents containing

64

Cu-Affibody-DAPs were collected and

concentrated using a centrifugal-filter (MWCO = 30 kDa). The (abbreviated as

64

64

Cu-NOTA-Affibody-DAPs

Cu-Affibody-DAPs) were finally reconstituted in PBS and filtered through a

0.22 µm filter for cell and animal experiments. To test the stability of radio labeling,

64

Cu-

Affibody-DAPs were incubated in the physiological conditions. Aliquots of mixtures were filtered at different time points and the filtrates were collected and analyzed by PerkinElmer 1470 automatic gamma-counter for radioactivity measurement.

Cell Culture and Tumor Xenograft Model. Human thyroid FTC-133 cell line was obtained from European Collection of Cell Cultures (ECACC, Salisbury, UK), and derives from a lymph node metastasis of a follicular thyroid carcinoma with EGFR overexpression. FTC-133 cell lines were routinely maintained in DMEM/Ham’s F12 (Life Technology Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin (Life Technology-Invitrogen), and 2 mmol/L L-glutamine in a humidified atmosphere with 5% CO2 and 95% air at 37°C. The medium was changed every 24~48 h.


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Female nu/nu mice (5~7 weeks, Charles River Laboratories International, Inc., Wilmington, MA) were housed and maintained in accordance with federal and local institutional rules for the conduct of animal experimentation and received food and water ad libitum. All animal experiment’s protocols were approved by Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University, and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. The FTC-133 tumor model was prepared by subcutaneous implantation of 3 × 106 FTC-133 cells in 100 µL of PBS into the right shoulders of nude mice. Tumors were allowed to grow to a size of 500 ~ 700 mm3 (approximately 3~4 weeks) before the imaging experiments.

In Vivo Small-Animal PET/CT. In vivo small-animal PET/CT was carried out on SiemensInveon PET-CT (Siemens Medical Solutions USA, Inc., Knoxville, TN, USA) according to our previous publications.39-40 Five-minute static PET scans with a timing resolution of 3.5 ns and the energy window set to 350-650 keV were performed. For CT imaging, the radiographic tube was set to 80 kV with a tube current of 500 µA and exposure time of 255 ns. The radiolabeled 64

Cu-Affibody-DAPs (3.7 MBq (100 µCi) in 150 µL of PBS) were injected via tail veins into the

FTC-133 bearing mice (n = 4) at a dose of 37 µg CH1000 /kg (264 µg NP/kg or 30 pmol NP/kg) mouse weight. PET scans were performed at 1, 2, 4, 24, and 48 h post-injection (p.i.). Mice were anesthetized with isoflurane (3% for induction and 2% for maintenance) in 100% oxygen. Mice after injection were placed in the prone position and near the isocenter of the scanner, and 5 min


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static scans were acquired. All the small animal PET images were processed and analyzed using the Inveon Research Workplace (IRW). Region of interests (ROIs) for coronal and transaxial slices were drawn over the tumors and major organs on decay-corrected whole-body images. The PET images were reconstructed with IRW 4.0 software by two-dimensional ordered subsets expectation maximization (OSEM) algorithm. The radioactivity uptake of tumor and major organs were calculated using a region of interest (ROI) analysis over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue (%ID/g). Attenuation and decay correction were applied for the 64Cu isotope.

Lymph Node and Vascular Imaging. The fluorescence images of lymphatic basins were acquired on NIR-II imaging setup described in the previous section. Mice were placed in the lateral position 24 h after the injection of Affibody-DAPs (400 µg CH1000 /kg mouse weight or 323 pmol NP/kg mouse weight). A small FOV (3 × 2.4 cm2) covering the lymphatic basins was applied for a high magnification with pixel size of 47 µm. Immediately after NIR-II imaging, the PA images of lymphatic basins of mice were obtained on Endra system under the following parameters: under multiple laser wavelengths in the range of 680 - 950 nm using a continuous rotation mode (240 views, 1 pulse/view).

For dynamic contrast-enhanced fluorescent vascular imaging, mice (n = 4 per group) were placed in a supine position on the imaging stage, and the FOVs were adjusted to cover the left/right hindlimb. The laser power density at the imaging plane was 140 mW/cm2 (below the permissible


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exposure limit of 329 mW/cm2 at 808 nm guided by the International Commission on Nonionizing Radiation Protection).41 The NIR-II camera started recording image frames immediately after 5.8 pmol of Affibody-DAPs were intravenously injected via a tail vein. Since the readout time of 15 ms is required for the acquisition of each frame, the frame rate was 8.7 frame/second when the exposure time was set to 100 ms. The emission light was passed through a 1000 nm longpass filter (Thorlabs, FEL1000) and focused onto the InGaAs detector by a 50-mm lens (f /1.4, Pentax). For dynamic contrast-enhanced PA vascular imaging, the PA images of hindlimb blood vessels were obtained on Endra system under the following parameters: at a single laser wavelength (700 nm) using a continuous rotation mode (3 seconds for scanning, 100 views, 1 pulse/view).

FTC-133 Tumor Targeting In Living Mice. The fluorescence images of FTC-133 tumor bearing mice (n = 4 for each group) were acquired on NIR-II imaging instrument with the same setting described in the previous section. When the tumors reached a size of 500 ~ 700 mm3, the anesthetized mice were placed on the lateral position on the imaging stage, and the FOVs were adjusted to cover the whole mice body. The laser power density at the imaging plane was set to 140 mW/cm2 at 808 nm. A 1000 nm longpass filter (Thorlabs, FEL1000) was used for fluorescence image acquisition and the exposure time for images was set to 200 ms. The precontrast images were acquired for each mouse. The FTC-133 tumor bearing mouse was intravenously injected with 100 µL of Affibody-DAPs (400 µg CH1000 /kg mouse weight or


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323 pmol per kg mouse body weight) in PBS. For the receptor-blocking experiment, mice were co-injected with 1.8 µmol of Affibody (Ac-Cys-ZEGFR:1907) per kg of mouse body weight and 100 µL of Affibody-DAPs (400 µg CH1000 /kg mouse weight or 323 pmol per kg mouse body weight) in PBS. After injection, fluorescence images were acquired at 0.5, 1, 2, 4, 24, and 48-h post-injection. Quantitative analysis was conducted using ImageJ (1.46i).

The photoacoustic images of FTC-133 tumors were photoacoustically imaged using Endra photoacoustic computed tomographic (PACT) system as we described previously, and acquired immediately after fluorescence imaging at the pre-determined time intervals. The image protocol for living mice was optimized to obtain spectroscopic photoacoustic images of FTC133 tumors by using 240 views and 1 pulses/view for each wavelength in the range of 680 - 950 nm. The protocol normally takes three minutes to acquire the data at 14 different wavelengths in the range of 680 - 950 nm for spectroscopic analysis. The anesthetized mice were placed on the lateral position to allow the tumor to be immersed in the water at the tip of the dimple of the imaging tray. The pre-contrast images were acquired for each mouse at multiple wavelengths. After injection, photoacoustic images were spectroscopically acquired at 0.5, 1, 2, 4, 24, and 48-h postinjection. The laser energies at different wavelengths, recorded before and after each scan, were used to normalize the photoacoustic signals to correct the spectra. Normalization of the images by the laser energy was performed by Osirix. Quantification analysis was done via AMIDE and/or Osirix software using volumetric ellipsoid ROIs drawn based on the tumors.


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Ex Vivo NIR-II Fluorescence Imaging. The mice were sacrificed at 48 hours post-injection right after NIR-II fluorescence imaging and PA imaging. Tumors, organs and tissues (brain, heart, lung, liver, spleen, pancreas, stomach, intestine, kidneys, skin, muscle, and bone) were dissected and collected. After rinsed with PBS and drained with filter papers, the ex vivo images of tissues of interest were immediately acquired using the NIR-II fluorescence system with the same illumination setting as the in vivo imaging. The fluorescent images were processed with the same method as described previously.

Statistical Method. Measurement data were expressed as the mean ± s.d. Statistical analyses of the date was performed with GraphPad Prism 5.0 (GraphRad Software, Inc., San Diego, CA). A Student’s t-test was applied to identify significant differences between groups. A value of p < 0.05 was considered statistically significant.

ASSOCIATED CONTENT

The authors declare no competing financial interest.

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Details of the synthesis, characterization, and supporting Figures and Tables.


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AUTHOR INFORMATION Corresponding Authors *[email protected], *[email protected] Author Contributions ‡Kai Cheng and Hao Chen contributed equally.

ACKNOWLEDGMENTS

This work was partially supported by the Office of Science (BER), U.S. Department of Energy (DE-SC0008397), International S&T Cooperation Program of China (2015DFA30440); National Natural Science Foundation of China (81301268); Beijing Nova Program (Z131107000413063); National Natural Science Foundation of China (61601019); Beijing Natural Science Foundation (7164270); Fundamental Research Funds for Central Universities (2017RC025).

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(3) Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van de Velde, C. J.; Frangioni, J. V. Image-Guided Cancer Surgery Using Near-Infrared Fluorescence. Nat. Rev. Clin. Oncol. 2013, 10, 507-518. (4) Kircher, M. F.; de la Zerda, A.; Jokerst, J. V.; Zavaleta, C. L.; Kempen, P. J.; Mittra, E.; Pitter, K.; Huang, R.; Campos, C.; Habte, F.; Sinclair, R.; Brennan, C. W.; Mellinghoff, I. K.; Holland, E. C.; Gambhir, S. S. A Brain Tumor Molecular Imaging Strategy Using a New TripleModality MRI-Photoacoustic-Raman Nanoparticle. Nat. Med. 2012, 18, 829-834. (5) Fan, Q.; Cheng, K.; Yang, Z.; Zhang, R.; Yang, M.; Hu, X.; Ma, X.; Bu, L.; Lu, X.; Xiong, X.; Huang, W.; Zhao, H.; Cheng, Z. Perylene-Diimide-Based Nanoparticles as Highly Efficient Photoacoustic Agents for Deep Brain Tumor Imaging in Living Mice. Adv. Mater. 2015, 27, 843-847. (6) Qin, C.; Cheng, K.; Chen, K.; Hu, X.; Liu, Y.; Lan, X.; Zhang, Y.; Liu, H.; Xu, Y.; Bu, L.; Su, X.; Zhu, X.; Meng, S.; Cheng, Z. Tyrosinase as a Multifunctional Reporter Gene for Photoacoustic/MRI/PET Triple Modality Molecular Imaging. Sci. Rep. 2013, 3, 1490. (7) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotechnol. 2014, 9, 233-239. (8) Cheng, K.; Kothapalli, S. R.; Liu, H.; Koh, A. L.; Jokerst, J. V.; Jiang, H.; Yang, M.; Li, J.; Levi, J.; Wu, J. C.; Gambhir, S. S.; Cheng, Z. Construction and Validation of Nano Gold Tripods for Molecular Imaging of Living Subjects. J. Am. Chem. Soc. 2014, 136, 3560-3571. (9) Wang, L. V. Multiscale Photoacoustic Microscopy and Computed Tomography. Nat. Photon. 2009, 3, 503-509. (10) Yang, Q.; Ma, Z.; Wang, H.; Zhou, B.; Zhu, S.; Zhong, Y.; Wang, J.; Wan, H.; Antaris, A.; Ma, R.; Zhang, X.; Yang, J.; Zhang, X.; Sun, H.; Liu, W.; Liang, Y.; Dai, H. Rational Design of Molecular Fluorophores for Biological Imaging in the NIR-II Window. Adv. Mater. 2017, 29, 1605497-1605505. (11) Antaris, A. L.; Chen, H.; Cheng, K.; Sun, Y.; Hong, G.; Qu, C.; Diao, S.; Deng, Z.; Hu, X.; Zhang, B.; Zhang, X.; Yaghi, O. K.; Alamparambil, Z. R.; Hong, X.; Cheng, Z.; Dai, H. A Small-Molecule Dye for NIR-II Imaging. Nat. Mater. 2016, 15, 235-242. (12) Hong, G.; Antaris, A. L.; Dai, H. Near-Infrared Fluorophores for Biomedical Imaging. Nat. Biomed. Eng. 2017, 1, 0010. (13) Hong, G.; Lee, J. C.; Robinson, J. T.; Raaz, U.; Xie, L.; Huang, N. F.; Cooke, J. P.; Dai, H. Multifunctional in Vivo Vascular Imaging Using Near-Infrared II Fluorescence. Nat. Med. 2012, 18, 1841-1846. (14) Hong, G.; Zou, Y.; Antaris, A. L.; Diao, S.; Wu, D.; Cheng, K.; Zhang, X.; Chen, C.; Liu, B.; He, Y.; Wu, J. Z.; Yuan, J.; Zhang, B.; Tao, Z.; Fukunaga, C.; Dai, H. Ultrafast Fluorescence Imaging in Vivo with Conjugated Polymer Fluorophores in the Second Near-Infrared Window. Nat. Commun. 2014, 5, 4206.


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(15) Cosco, E. D.; Caram, J. R.; Bruns, O. T.; Franke, D.; Day, R. A.; Farr, E. P.; Bawendi, M. G.; Sletten, E. M. Flavylium Polymethine Fluorophores for Near- and Shortwave Infrared Imaging. Angew. Chem., Int. Ed. Engl. 2017, 56, 13126-13129. (16) Chen, Y.; Montana, D. M.; Wei, H.; Cordero, J. M.; Schneider, M.; Le Guevel, X.; Chen, O.; Bruns, O. T.; Bawendi, M. G. Shortwave Infrared in Vivo Imaging with Gold Nanoclusters. Nano Lett. 2017, 17, 6330-6334. (17) Wang, R.; Zhou, L.; Wang, W.; Li, X.; Zhang, F. In Vivo Gastrointestinal Drug-Release Monitoring through Second Near-Infrared Window Fluorescent Bioimaging with Orally Delivered Microcarriers. Nat. Commun. 2017, 8, 14702. (18) Shou, K.; Qu, C.; Sun, Y.; Chen, H.; Chen, S.; Zhang, L.; Xu, H.; Hong, X.; Yu, A.; Cheng, Z. Multifunctional Biomedical Imaging in Physiological and Pathological Conditions Using a NIR-II Probe. Adv. Funct. Mater. 2017, 27, 1700995. (19) Qian, G.; Wang, Z. Y. Near-Infrared Organic Compounds and Emerging Applications. Chem. Asian. J. 2010, 5, 1006-1029. (20) Sun, Y.; Qu, C.; Chen, H.; He, M.; Tang, C.; Shou, K.; Hong, S.; Yang, M.; Jiang, Y.; Ding, B.; Xiao, Y.; Xing, L.; Hong, X.; Cheng, Z. Novel Benzo-Bis(1,2,5-Thiadiazole) Fluorophores for in Vivo NIR-II Imaging of Cancer. Chem. Sci. 2016, 7, 6203-6207. (21) Sun, Y.; Ding, M.; Zeng, X.; Xiao, Y.; Wu, H.; Zhou, H.; Ding, B.; Qu, C.; Hou, W.; Er-bu, A. G. A.; Zhang, Y.; Cheng, Z.; Hong, X. Novel Bright-Emission Small-Molecule NIR-II Fluorophores for in Vivo Tumor Imaging and Image-Guided Surgery. Chem. Sci. 2017, 8, 34893493. (22) Feng, Y.; Zhu, S.; Antaris, A. L.; Chen, H.; Xiao, Y.; Lu, X.; Jiang, L.; Diao, S.; Yu, K.; Wang, Y.; Herraiz, S.; Yue, J.; Hong, X.; Hong, G.; Cheng, Z.; Dai, H.; Hsueh, A. J. Live Imaging of Follicle Stimulating Hormone Receptors in Gonads and Bones Using Near Infrared II Fluorophore. Chem. Sci. 2017, 8, 3703-3711. (23) Zhu, S.; Yang, Q.; Antaris, A. L.; Yue, J.; Ma, Z.; Wang, H.; Huang, W.; Wan, H.; Wang, J.; Diao, S.; Zhang, B.; Li, X.; Zhong, Y.; Yu, K.; Hong, G.; Luo, J.; Liang, Y.; Dai, H. Molecular Imaging of Biological Systems with a Clickable Dye in the Broad 800- to 1,700-nm Near-Infrared Window. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 962-967. (24) Miao, Z.; Ren, G.; Liu, H.; Qi, S.; Wu, S.; Cheng, Z. PET of EGFR Expression with an 18FLabeled Affibody Molecule. J. Nucl. Med. 2012, 53, 1110-1118. (25) Klutz, K.; Schaffert, D.; Willhauck, M. J.; Grünwald, G. K.; Haase, R.; Wunderlich, N.; Zach, C.; Gildehaus, F. J.; Senekowitsch-Schmidtke, R.; Göke, B.; Wagner, E.; Ogris, M.; Spitzweg, C. Epidermal Growth Factor Receptor-Targeted 131I-Therapy of Liver Cancer Following Systemic Delivery of the Sodium Iodide Symporter Gene. Mol. Ther. 2011, 19, 676685. (26) Jokerst, J. V.; Miao, Z.; Zavaleta, C.; Cheng, Z.; Gambhir, S. S. Affibody-Functionalized Gold-Silica Nanoparticles for Raman Molecular Imaging of the Epidermal Growth Factor Receptor. Small 2011, 7, 625-633.


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(27) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 2002, 298, 1759-1762. (28) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the NanoBio Interface. Nat. Mater. 2009, 8, 543-557. (29) Cheng, K.; Blumen, S. R.; MacPherson, M. B.; Steinbacher, J. L.; Mossman, B. T.; Landry, C. C. Enhanced Uptake of Porous Silica Microparticles by Bifunctional Surface Modification with a Targeting Antibody and a Biocompatible Polymer. ACS Appl. Mater. Interfaces 2010, 2, 2489-2495. (30) Yang, M.; Cheng, K.; Qi, S.; Liu, H.; Jiang, Y.; Jiang, H.; Li, J.; Chen, K.; Zhang, H.; Cheng, Z. Affibody Modified and Radiolabeled Gold-Iron Oxide Hetero-Nanostructures for Tumor PET, Optical and MR Imaging. Biomaterials 2013, 34, 2796-2806. (31) Su, X.; Cheng, K.; Jeon, J.; Shen, B.; Venturin, G. T.; Hu, X.; Rao, J.; Chin, F. T.; Wu, H.; Cheng, Z. Comparison of Two Site-Specifically (18)F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors. Mol. Pharm. 2014, 11, 3947-3956. (32) Bohndiek, S. E.; Bodapati, S.; Van De Sompel, D.; Kothapalli, S. R.; Gambhir, S. S. Development and Application of Stable Phantoms for the Evaluation of Photoacoustic Imaging Instruments. PLoS One 2013, 8, e75533. (33) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biomed. Eng. 2015, 33, 941-951. (34) Welsher, K.; Sherlock, S. P.; Dai, H. J. Deep-Tissue Anatomical Imaging of Mice Using Carbon Nanotube Fluorophores in the Second Near-Infrared Window. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 8943-8948. (35) Levi, J.; Kothapalli, S. R.; Bohndiek, S.; Yoon, J. K.; Dragulescu-Andrasi, A.; Nielsen, C.; Tisma, A.; Bodapati, S.; Gowrishankar, G.; Yan, X.; Chan, C.; Starcevic, D.; Gambhir, S. S. Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma. Clin. Cancer. Res. 2013, 19, 1494-1502. (36) Loening, A. M.; Gambhir, S. S. Amide: A Free Software Tool for Multimodality Medical Image Analysis. Mol. Imaging 2003, 2, 131-137. (37) Rosset, A.; Spadola, L.; Ratib, O. Osirix: An Open-Source Software for Navigating in Multidimensional DICOM Images. Journal of Digital Imaging 2004, 17, 205-216. (38) Hillman, E. M.; Moore, A. All-Optical Anatomical Co-Registration for Molecular Imaging of Small Animals Using Dynamic Contrast. Nat. Photon. 2007, 1, 526-530. (39) Cheng, K.; Yang, M.; Zhang, R.; Qin, C.; Su, X.; Cheng, Z. Hybrid Nanotrimers for Dual T1 and T2-Weighted Magnetic Resonance Imaging. ACS Nano 2014, 8, 9884-9896. (40) Zhang, R.; Cheng, K.; Antaris, A. L.; Ma, X.; Yang, M.; Ramakrishnan, S.; Liu, G.; Lu, A.; Dai, H.; Tian, M.; Cheng, Z. Hybrid Anisotropic Nanostructures for Dual-Modal Cancer Imaging and Image-Guided Chemo-Thermo Therapies. Biomaterials 2016, 103, 265-277.


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(41) Protection, I. C. o. N.-I. R. Revision of Guidelines on Limits of Exposure to Laser Radiation of Wavelengths between 400 nm and 1.4 mm. Health Phys. 2000, 79, 431-440.


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FIGURES.

Figure 1. Synthesis and characterization of Affibody-DAPs. (a) Optimized ground-state (S0) geometries, calculated HOMO and LUMO of CH1000 and CH1055 at the tuned-ωB97XD*/631G(d) level. The HOMO and LUMO energy levels as well as the energy gap were presented in the figures. (b) Schematic illustration of preparation of Affibody-DAPs. The DAPs were prepared through nanoprecipitation of the ester version of CH1000. The CH1000 molecule was represented as light green ovals. The phospholipid (DSPE-PEG-5000) has two hydrophobic tails and one hydrophilic PEG chain, and was illustrated as a purple ball with two dark gray tails and one light gray head. EGFR Affibody (Ac-Cys-ZEGFR:1907, three α-helices) were immobilized on


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the surface of DAPs via a bifunctional crosslinker. (c) Representative TEM image of negatively stained DAPs. Scale bar = 100 nm. (d) Hydrodynamic sizes of DAPs (black line and column) and Affibody-DAPs (green line and column). (e) Zeta potentials of DAPs and Affibody-DAPs.

Figure 2. Optical properties of DAPs. (a) Ultraviolet-visible-NIR (UV-vis-NIR) extinction (green lines) and fluorescence emission spectra (blue lines) of CH1000 (dot lines) and DAPs (solid lines). The fluorescent emission spectrum was obtained with an 808-nm excitation laser. (b),


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Linearity of fluorescence intensity of DAPs nm as a function of molar concentration of CH1000. (c) Photoacoustic spectrum of DAPs in the region of 680 - 940 nm. (d) Linearity of photoacoustic amplitudes of DAPs at 700 nm as a function of molar concentration of CH1000. (e) Photoluminescence excitation mapping of DAPs. (f) Fluorescence decay curves of CH1000 in chloroform (black circles), DAPs in water (red circles), and DAPs in chloroform (blue circles). The fluorescence decay profiles were fitted with the stretched exponential function to obtain the fluorescence lifetime τ of CH1000 (1.33 ns) and DAPs (0.86 ns in water and 1.37 ns in chloroform). (g) Plot of the integrated fluorescence spectra of both DAPs and IR-1061 NPs (a reference near-infrared fluorophore, quantum yield (QY) = 1.8%). The QYs were measured by the linear-regression plot of integrated fluorescent intensities at the different concentrations on a molar basis. The QY of DAPs was determined to be 11.1%, using IR-1061 as a reference. (h) Photostability of DAPs in terms of fluorescent intensities and photoacoustic amplitudes under continuous 808 nm exposure for 1 h at a power density of 140 mW/cm2.


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Figure 3. In vivo behavior of Affibody-DAPs. (a) Pharmacokinetic and bio-distribution in a mouse model after intravenous administration of 64Cu labeled Affibody-DAPs. Decay-corrected whole-body coronal PET/CT images of mice at 1, 2, 4, 24, and 48 h after tail-vein injection of 3.7 MBq of 64Cu labeled Affibody-DAPs (50 µg CH1000/kg mouse weight (or 40 pmol NP/kg)). (b) PET quantification of major organs and tissues after intravenous injection of 64Cu labeled Affibody-DAPs. (c) Uptakes of 64Cu labeled Affibody-DAPs in major organs and tissues (lymph node (LN), heart, lungs, liver, spleen, kidneys, intestine and muscle) for a time period up to 48 h after intravenous injection (data represent means ± s.d.).


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Figure 4. High-magnification fluorescent and photoacoustic imaging of lymph nodes of mice (n = 4 per group). (a)-(c) NIR-II fluorescence imaging of lymphatic basins in a mouse 24 h after the injection of Affibody-DAPs intravenously. (a) Ventral view of the axillary region of a nude mouse. (b) fluorescence image of axillary lymph nodes (proper axillary LN and accessory axillary LN indicated by white arrows) in a mouse in the NIR-II (excitation = 808 nm, 1000 nm long-pass filter, 100 ms expose time) window. (c) Merged fluorescence image of axillary lymph nodes. Photoacoustic imaging (at 700 nm) of axillary lymph nodes in a mouse before (d) and after (e) the injection of DAPs. (f) Photoacoustic amplitude spectra of lymphatic basins in a mouse before (black dots) and after (green circles) the injection.


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Figure 5. Dual fluorescent and photoacoustic imaging of blood vessels of mice using AffibodyDAPs. (a) photoacoustic imaging of mouse hindlimb vasculature in a mouse 30 min after the injection of Affibody-DAPs intravenously. Scale bar = 5 mm. (b) Photoacoustic amplitude profile along the line across the blood vessels. (c) fluorescence imaging of mouse hindlimb vasculature in the NIR-II (excitation = 808 nm, 1000 nm long-pass filter, 100 ms expose time) window. Scale bar = 5 mm. (d) Cross-sectional fluorescence intensity profiles along a white line of a mouse injected with DAPs. The shoulder peak was indicated by a red asterisk. (e) Principle


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component analysis (PCA) overlaid image based on the first 150 frames (30 seconds after injection). The arteries are color-coded in red and veins are color in yellow. (f) Cross-sectional PCA-weighted signal profiles along a white line. (g) Color-coded Fluorescence merged image of the FTC-133 thyroid tumor in the NIR-II (excitation = 808 nm, 1000 nm long-pass filter, 100 ms expose time) window. (h) High magnification image of thyroid tumor. (i) Cross-sectional fluorescence intensity profiles along a white line across FTC-133 tumor after the first injection of DAPs (black line) and the second injection of DAPs (dashed line) 24 hours after the first injection. (j)-(k) three-dimensional volume rendering of photoacoustic images (at 700 nm) of the thyroid tumor in a mouse before (j) and after (k) the injection of DAPs. Scale bar = 5 mm. (l) Cross-sectional PA intensity profiles along a white line across FTC-133 tumor.


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Figure 6. Targeted NIR-II fluorescent and photoacoustic imaging of FTC-133 thyroid tumor using Affibody-DAPs. (a) NIR-II fluorescence images of EGFT-positive FTC-133 tumor-bearing mice (n = 4) were obtained in the NIR-II (excitation = 808 nm, 1000 nm long-pass filter, 100 ms expose time) window before injection or at 1, 4, 24, and 48 hours after tail-vein injections of Affibody-DAPs (400 µg CH1000 /kg mouse weight (or 323 pmol NP/kg)). For a blocking group, a blocking dose of Affibody ZEGFR:1904 (12 mg/kg mouse) was co-injected with Affibody-DAPs. The NIR-II fluorescence images of nude mice bearing FTC-133 tumors were then acquired using identical acquisition settings. Tumors were delineated with white dotted lines and indicated by white arrows. (b) Coronal views of 3D volume rendering of photoacoustic images of FTC-133 tumors were acquired immediately after fluorescence imaging at the pre-determined time intervals. (c)-(e) Quantification analysis of fluorescence signals of tumor and liver regions in mice bearing FTC-133 tumors at 0, 0.03, 0.17, 0.5, 1, 1.5, 2, 3, 4, 24, and 48 hours after tail-vein injections of Affibody-DAPs. (c) The time-dependent fluorescence intensities in tumor regions


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were quantified and analyzed by using ImageJ (1.46i). Data were shown as mean ± S.D. (n = 4 per group. *p < 0.05). (d) The fluorescence intensities in the liver regions were changed in a duration dependent manner. (e) Tumor-to-liver ratios obtained for Affibody-DAPs over the course of 96 hours during targeting and blocking (n = 4 per group. *p < 0.05). (f) Quantification analysis of photoacoustic signals of tumor in mice bearing FTC-133 tumors at 0, 0.03, 0.17, 0.5, 1, 1.5, 2, 3, 4, 24, and 48 hours after tail-vein injections of Affibody-DAPs.


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Synthesis, Characterization, and Biomedical Applications of a

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