- Email: [email protected]
Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection
Accepted Manuscript Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection Youbin Li, Xiaolong Li, Zhenluan Xue, Mingyang Jiang, Songjun Zeng, Jianhua Hao PII:
S0142-9612(18)30220-5
DOI:
10.1016/j.biomaterials.2018.03.041
Reference:
JBMT 18567
To appear in:
Biomaterials
Received Date: 25 October 2017 Revised Date:
22 March 2018
Accepted Date: 23 March 2018
Please cite this article as: Li Y, Li X, Xue Z, Jiang M, Zeng S, Hao J, Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection, Biomaterials (2018), doi: 10.1016/j.biomaterials.2018.03.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection
RI PT
Youbin Li, † Xiaolong Li, † Zhenluan Xue, † Mingyang Jiang, † Songjun Zeng †*, Jianhua Hao‡* †
College of Physics and Information Science and Key Laboratory of Low-dimensional Quantum Structures and Quantum Control of the Ministry of Education, and Synergetic Innovation Center for Quantum Effects and Applications, Hunan Normal University, Changsha 410081 , China
SC
Department of Applied Physics, The Hong Kong Polytechnic University, Hong Kong.
M AN U
‡
AC C
EP
TE D
* Address correspondence to [email protected], [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
In vivo optical imaging by using a new imaging window located at short-wavelength infrared region (1000-1700 nm, named as NIR II) presents an unprecedented improvement in imaging
RI PT
sensitivity and spatial resolution over the traditional visible and near-infrared light. However, the most developed NIR II-emitters are hardly excreted from live animals, leading to unknown
SC
long-term toxicity concerns, which hinder the widespread applications of this advanced imaging technology. Here, we developed a new generation molecular NIR II-emitting probe based on
M AN U
Nd-diethylene triamine pentacetate acid (DTPA) complex. The designed molecular Nd-DTPA probe with bright narrow band emission at 1330 nm is successfully used for highly sensitive in vivo NIR IIa bioimaging with rapid renal excretion and high biocompatibility and optical-guided tiny tumor (down to ~3 mm) detection for the first time. Moreover, the Nd-DPTA complex also
TE D
holds great promise as an X-ray contrast agent. These findings open up the possibility for designing a new generation of multi-modal small molecular probe for early tumor diagnosis and
EP
favor the clinic translation of the advanced NIR II imaging method.
AC C
KEYWORDS: molecule probe, NIR II bioimaging, renal clearance, tiny tumor diagnosis, X-ray bioimaging.
2
ACCEPTED MANUSCRIPT 1. Introduction Noninvasive fluorescent imaging by using optical probe is one of the most important imaging technique for the early detection of disease owing to the cellular or molecular level information
RI PT
and high imaging sensitivity.1,2 Although the traditional near infrared light (NIR I, 650–900 nm) emitting optical probes with low tissue-absorption nature are well-developed for deep-tissue bioimaging, some performance parameters (penetration depth, imaging sensitive and resolution
SC
etc.) are still limited by the large scattering losses of NIR I light.3-11 Recent optical simulations
M AN U
demonstrate a new advanced optical transparent window with short wavelength infrared light (SWIR, NIR II, 1000-1700 nm), which presents lower optical absorption and auto-fluorescence than NIR I window, but up to 1000-fold reduction in scattering losses, subsequently resulting in unprecedented improvements in detection depth and imaging resolution.2,12 Therefore, developing
TE D
the new advanced NIR II optical probe with high emission intensity, large optical penetration depth in biotissue, and high biocompatibility is vital important for sensitivity and high resolution optical bioimaging.
EP
In this context, some NIR II optical probes capable of producing NIR II emissions such as
AC C
quantum dots (QDs)13-21 and single-walled carbon nanotubes (SWNTs)22-29 are emerging. However, most of the reported NIR II-emitting QDs comprise the toxic elements such as lead or mercury, making them unsuitable for bioapplications.2,20 Single-walled carbon nanotubes (SWNTs) have demonstrated good performance in in vivo applications.22-29 However, SWNTs still face significant challenges including low emission intensity, broad emission peaks (band width: > 300 nm), and large size distributions, making them unattainable for the size-dependent biological barriers study.2,30 Recently, rare earth based nanoparticles with well controlled size and 3
ACCEPTED MANUSCRIPT narrow-band emission were emerged as promising probes for NIR II bioimaging owing to their advantages of high efficiency, low photo-bleaching and long luminescence lifetimes.2,31-35 However, most of the designed rare earth-based probes usually present unspecific liver/spleen
RI PT
uptake and are excreted slowly from live animals, leading to unknown long-term toxicity concerns, which remarkably limited their widespread application in NIR II bioimaging. As it is well known that the ability of rapid excretion of imaging contrast agent is vital for clinical use. Recently, Dai’s
SC
group demonstrated a pioneering study of designing small organic molecular probes with 1000 nm
M AN U
emission for NIR-II imaging.36-41 However, these small molecular probes still presented the broad-band emission and relative low photostability(>10% photobleaching degree in 1 h)38, which inhibited the practice use of nonoverlapping signals for multispectral imaging. Therefore, it is appealing to design novel NIR II imaging agent with narrow-band and bright NIR II emission,
TE D
rapid excretion, high biocompatibility for eventually overcoming the barrier of clinical use. Herein, we design a new type of molecular Nd-based complex (Nd-DTPA) with efficient narrow band NIR IIa emission centered at 1330 nm (band width: ~ 65 nm), high biocompatibility,
EP
and rapid excretion from the kidney. The NIR IIa emitting Nd-DTPA probe was successfully used
AC C
as in vivo tracking. More importantly, the optical-guided tiny tumor (< 3 mm) detection was demonstrated by using the designed Nd-DTPA probe for the first time. Apart from the excellent NIR IIa bioimaging nature, the designed Nd-DTPA can also be used as X-ray imaging agent owing to the large X-ray absorption coefficient of Nd element42-43.
2. Materials and methods 2.1. Materials 4
ACCEPTED MANUSCRIPT All chemical reagents were obtained from the commercial supply and used without further purification. Rare earth NdCl3•6H2O (99.99%) was purchased from QingDa elaborate Chemical Reagent
Co.
Ltd
(Shandong).
Diethylene
triamine
pentacetate
acid
(DTPA)
and
RI PT
N-Methyl-D-glutamines were obtained from the ScienMax. Xylenol orange (XO) was purchased form Sigma-Aldrich Trading Co, Ltd. (Shanghai, China). 2.2. Synthesis of Nd-DTPA
SC
The Nd-DTPA was were prepared using a modified literature protocol.44 For synthesis of the , then 1.1 mmol DTPA was
M AN U
Nd-based complex, 15 ml water in the beaker was heated to 50
added into the water and kept stirring for 20 min. 1 mmol NdCl3 solution (1 M) was then added and stirred until forming a transparent solution. The obtained Nd-DTPA solution was treated with N-Methyl-D-glutamines to adjust the pH value to 7.2.
TE D
2.3. Characterization
The photoluminescent spectrum of Nd-DTPA was detected with a NIR-II spectroscopy (NIRQuest512, Ocean Optics). The MDL-H 808 nm laser was used as an excitation light source at
EP
room temperature. UV-Vis absorption spectrum of Nd-DTPA in water solution was detected by
AC C
using a Spectrophotometer system (UV-1800, Hunan Sino-Jewell Electronics Co.Ltd.). The surface ligands of Nd-DTPA were analyzed via a Fourier transform infrared spectrum (FTIR) by using a Magna 760 spectrometer (Nicolet). The Nd content in the isolated organ was measured using an Agilent 7500a inductively coupled plasma mass spectrometry (ICP-MS). The Nd-DTPA was characterized using the nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was measured using a Bruker AV-400 spectrometer at room temperature. Chemical shifts were named as δ values (p.p.m), where D2O was used as an internal reference. 5
ACCEPTED MANUSCRIPT Splitting patterns were labeled as s (singlet), t (triplet), m (multiplet). The NMR spectra of Nd-DTPA are given as follows: 1
H NMR (400 MHz, D2O): δ 8.22 (s, 3H), 4.1 (t, 2H), 3.7 (m, 4h), 3.6 (m, 3H), 3.2 (m, 4H),
RI PT
2.8 (s, 5H). 2.4. Cytotoxicity evaluation
To evaluate the cell viability of Nd-DTPA, the viability of HeLa cells based on MTT
under 5% CO2. Then the cell culture medium in each
M AN U
(6000 cells per well) were kept at 37
SC
proliferation assay method was evaluated. In a typical process, HeLa cells in a 96-well micro plate
well was replaced by Dulbecco’s Modified Eagle Medium (DMEM) solution including 10% fetal bovine serum, 1% penicillin and streptomycin. The prepared Nd-DTPA solutions with different concentrations (0, 100, 200, 500, 1000 µL/mL) were added at 37 °C and with 5% CO2 for 24 h.
TE D
Finally, we measured the cell viability by using a typical MTT assay. 2.5. In vivo and ex vivo NIR-IIa optical bioimaging
A home-made small animal NIR-II bioimaging system equipped with a thermoelectric cooled
EP
InGaAs camera (Model: NIRvanaTM Camera System, operating temperature: -80
, Princeton
AC C
Instruments) and fiber-coupled MDL-H 808/980 lasers was used for NIR II bioimaging. The fluorescence signal was collected by using the system installed with 808 nm diode laser as light source and filtered by a (1200-1400 nm) band pass filter. For in vivo bioimaging, a Kunming mouse was intraperitoneally injected with pentobarbital sodium aqueous (150 µL/10 wt%). Then Nd-DTPA (125 µL, 0.05 M) was injected into the mouse by intravenous injection at tail vein. Finally, the fluorescence signal was collected by using the system under an excitation powder density of 100 mW/cm2. To further reveal the renal clearance, Kunming mice were intravenously 6
ACCEPTED MANUSCRIPT injected with Nd-DTPA (125 µL, 0.05 M), the corresponding isolated organs of the mice were collected after 20 min, 24 h, and 48 h injection for ex-vivo NIR-IIa imaging by using the same system. The experiment parameters were the same as above. All animal procedures comply with
RI PT
the institutional animal use and care regulations approved by the Laboratory Animal Center of Hunan Province. 2.6. Quantitative Biodistribution Measurement
SC
125 µL of Nd-DTPA (0.05 M) molecular probes were intravenously injected into the
M AN U
Kunming mouse. The urine and isolated organs (heart, liver, spleen, lung, and kidney) were collected after 20 min, 24 h, and 48 h post injection and used for ICP-MS. 2.7. Urine excretion rate evaluation
According to the previous report41, the renal excretion rate of Nd-DTPA from the mouse was
TE D
measured by intravenously injecting 125 µL (0.05 M) Nd-DTPA solution and the urine was collected for quantifying the amount of clearance. The mouse urines were collected in capillarity
EP
tubes at 20 min, 150 min, 3.5 h and 7.5 h injection, and the fluorescence intensity was measured with an InGaAs camera (Model: NIRvanaTM Camera System, Princeton Instruments). The injected
AC C
dose (%ID) was measured in a similar way to the previous report41:
%= ܦܫ m s ∑m s (I m − Icontrol ) ∗ Vm (I injected − I control ) ∗ Vm =
f
=
1
where s represents the number of the urine time-point from a mouse (S1, the first urine time-point, 7
ACCEPTED MANUSCRIPT Sf, the last urine time-point), I is the integrated luminescence intensity measured with a (1200-1400 nm) band-pass filter, Icontrol represents the luminescence intensity of control urine, Iinjected is the fluorescent intensity of the injected Nd-DTPA, Vm represents the volume of the urine,
2.8. NIR-IIa optical bioimaging of tiny tumor detection
RI PT
and Vinjected is the volume of the injected Nd-DTPA.
SC
Female BALB/c mice (18-21 g, 4 weeks old, purchased from Laboratory Animal Center of Hunan Province) were selected for establishing the mice model. 8 × 106 MAT-Ly-Lu-B-2 (Mat)
M AN U
cells in 100 µL of sodium chloride solution were subcutaneously injected into the female BALB/C mouseat the back for further culturing the tumor to about ≈ 3 mm in average size. We then carried out the in vivo tumor detection. Firstly, 150 µL of pentobarbital sodium aqueous solution (10 wt%) was intraperitoneally injected into the inoculated tumor mouse to anesthetize it. 125 µL of
TE D
Nd-DTPA solution was then injected into the mouse by intravenous injection. The fluorescence signal was captured at different period times by using the same bioimaging system equipped with
EP
808 nm laser as light source. The emission filter was set as 1330 nm, and the exposure time was set as 2000 ms. Ex-vivo bioimaging of the tumor was executed by the same parameters. The digital
AC C
picture of the tumor was taken by a Canon digital camera. 2.9. In vivo and in vitro X-ray bioimaging To demonstrate the feasibility of the as prepared Nd-DTPA molecules for in vivo X-ray bioimaging, a Kunming mouse was first anesthetized. And 125 µL of Nd-DTPA with concentration of 30 mg/mL was subcutaneously injected into the mouse. Then X-ray bioimaging was tested via an in vivo imaging system (Bruker In Vivo FX Pro). To assess the X-ray contrast efficacy, Nd-DTPA and Iobitridol were dispersed in water with different Nd and I concentrations 8
ACCEPTED MANUSCRIPT over the range from 0 to 60 mg/mL, respectively. In vitro phantom X-ray imaging based on these agents was collected. In vivo X-ray imaging was executed with the following parameters: voltage, 45 kVp; aluminum filter, 4 mm; and exposure time, 30 s.
RI PT
2.10. Quantum yield of Nd-DTPA The fluorescence quantum yield (QY) of Nd-DTPA in water was calculated in a similar
reference. The QY was calculated by the following formula:
M AN U
Φs = Φr (Fs/Fr)*(Ar/As)*(n2s/n2r)
SC
method to the previous report41 using a standard IR-26 dye (dissolved in DCE, QY = 0.5%) as a
where Φ represents QY, F denotes integrated photoluminescence emission intensity, A denotes absorbance at the maximum excitation wavelength and n is the refractive index of the solvent (n= 1.33 for water, n = 1.44 for DCE, respectively). The subscripts s and r represent the
2.11. Histology analysis
TE D
sample and reference solutions, respectively.
For histology analysis, Kunming mice were sacrificed at 3 and 7 day after intravenously
EP
injected with 125 µL (0.05 M) of Nd-DTPA solution. Major organs including heart, liver, spleen,
AC C
lung, and kidney were isolated from the mice. Then the tissue sections were stained with hematoxylin and eosin (H&E) and visualized by using an optical microscope. 3. Results and discussion
3.1. Synthesis and NIR II emitting property The Nd-DTPA was primarily synthesized through a chelation of the Nd3+ and DTPA, as presented in Scheme 1. The N-Methyl-D-glucamine was added into the Nd-DTPA solution to tune the pH value to 7.2. The surface ligands of the precursor and Nd-DTPA were analyzed by 9
ACCEPTED MANUSCRIPT Fourier-transform infrared (FTIR) spectroscopy (Fig. 1a) As demonstrated, for pure DTPA, the peaks at 1733 cm-1, 1700 cm-1, and 1634 cm-1 are ascribed to the stretching vibration of C=O (–COOH), C=O (–CONH–), and the deformation vibrations of N-H (–CONH–), respectively.
RI PT
While in Nd-DTPA sample, the stretching and deformation vibration bands of 1733/1700/1634 cm-1 were disappeared and a new vibration peaks at 1595 cm-1 was observed and ascribed to the vibration of O-C-O (carboxylate), unambiguously validating the coordination of COO- with
SC
Nd3+.44 To further reveal the formation of Nd-DTPA, we have performed 1H nuclear magnetic
M AN U
resonance (NMR) spectroscopy analysis. As shown in Supplementary Fig. S1, 1H NMR result further validate the formation of Nd-DTPA molecule. The ultraviolet (UV)–vis absorption spectrum of the Nd-DTPA solution in water presented several sharp absorption peaks of Nd3+ centered at 741 nm (4I9/2
4
F7/2), 801 nm (4I9/2
4
F5/2) and 872 nm (4I9/2
4
F3/2), indicating an
TE D
excellent absorption of NIR light of about 800 nm (Fig. 1b). To reveal the NIR II emitting properties, the NIR emission spectrum of Nd-DTPA was detected under the excitation of 808 nm laser. As shown in Fig. 1b and 1c, the Nd-DTPA presents the efficient NIR II emissions centered at
EP
1050 nm (band width: ~ 45 nm) and 1330 nm (band width: ~ 65 nm), which can be ascribed to the 4
I11/2 (1050 nm), and 4F3/2
4
I13/2 (1330 nm) of Nd3+, respectively. It is
AC C
electronic transitions 4F3/2
noted that the photo-stability is a key characteristic for molecular optical probes. Therefore, the photo-stability of Nd-DTPA molecule was tested with a continuous 808 nm laser excitation for 1 h at 1 w cm-2 (Fig. 1d). The photo-stability curve (Fig. 1e) and in vitro phantom bioimaging (Supplementary Fig. S2) reveal the designed Nd-DTPA present superior photo-stability with photobleaching degree of 3.4% in 1 h, which is relatively lower than the previous report40. To further prove the stability of Nd-DTPA, the leakage of Nd3+ in Nd-DTPA and NdCl3 were 10
ACCEPTED MANUSCRIPT measured using the XO as the indicator. As shown in Supplementary Fig. S3, free Nd3+ at a concentration as low as 0.125 mM and pH ranging from 3 to 5 can still react with the XO solution to form a red substance. On the contrary, the Nd-DTPA could not react with XO even at a
RI PT
concentration of 2 mM, which further demonstrated the strong chelation of Nd3+ and DTPA and high structure stability of Nd-DTPA. This result reveal the no leakage of Nd3+ or very limited leakage less than 0.125 mM from Nd-DTPA, which is stable and safe enough for the biological
SC
applications comparable with the standard of IC50 ~ 0.125 mM for free Gd3+.45 In addition, the QY
M AN U
of Nd-DTPA at 1050 nm is measured to be 0.63%, which exhibits higher QY than the SWNTs probes in the previous report.24-27 And the QY of Nd-DTPA at 1330 nm is measured to be 0.07%, which exhibits lower QY than Nd-DTPA at 1050 nm. These results suggest that the Nd-DTPA possesses remarkable narrow band emission in NIR II region (1050/1330 nm) and superior
TE D
photo-stability, making it an ideal agent for NIR-II bioimaging with high photostability. 3.2. In vivo NIR-IIa bioimaging
Prior to using Nd-DTPA for in vivo imaging, the cell toxicity of this designed molecular
EP
Nd-DTPA probe was evaluated via a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium
AC C
bromide (MTT) method (Supplementary Fig. S4). As demonstrated, the cell viability of Nd-DTPA in HeLa cells was 99% when treated with 100 µg mL-1 of Nd-DTPA. When further increasing the concentration of Nd-DTPA to 1000 µg mL-1, the cell viability was still remained at 90%, indicating the very low cell toxicity of the Nd-DTPA molecules. Encouraged by the low cytotoxicity and strong 1050 nm emission, we first performed in vivo NIR II bioimaging by detecting the 1050 nm emission via a home-made NIR II imaging system (Supplementary Fig. S5) installed with a fiber-coupled 808 nm laser as light source. As shown in 11
ACCEPTED MANUSCRIPT Fig. 2c and 2d, an obvious NIR II signal of mouse food was observed
by using a 1000-1100 nm
band-pass filter, while, the signal intensity of mouse food was almost disappeared by detecting the 1330 nm emission, indicating the efficiently diminishing the undesired interference signal caused
RI PT
by mouse food. Further in vivo bioimaging (Fig. 2e and 2f) also demonstrated that the undesired signal of stomach caused by mouse food was fully removed by measuring the 1330 nm emission via a band-pass filter (1200-1400 nm). It should be noted that imaging in the NIR-IIa window
SC
(1300 -1400 nm) can greatly minimize the scatter of rejection photons below 1300 nm, and avoid
M AN U
the light absorption from water vibration over 1400 nm.40 Based on the above analysis, our designed Nd-DTPA probe presents efficient NIR IIa emission located at 1330 nm, which is beneficial for bioimaging applications. Then, in vivo NIR IIa bioimaging of a Kunming mouse was performed by intravenous injection of Nd-DTPA. As demonstrated in Fig. 3a, within a few
TE D
second, a significant fluorescence signal in kidney was observed and the fluorescence signal in the kidney was gradually enhanced after 25 min injection and then tended to slight decrease after 35 min. The optical signal was finally disappeared within 120 min, indicting the rapidly excretion of
EP
the Nd-DTPA probe from the kidneys. In addition, NIR IIa bioimaging of the supine positioned
AC C
mouse was performed for further proof of the in vivo excretion mechanism of the Nd-DTPA molecules. As illustrated in Fig. 3b, strong signal was observed in bladder within 10 min, implying the rapid excretion of the Nd-DTPA probe from the kidney and vessels to the bladder. Moreover, the Nd-DTPA probe was fully excreted from the bladder within 11 h, indicating the fast renal clearance of the Nd-DTPA molecule. The urine sample collected after 2 h injection also showed a strong fluorescence signal, which further proved the renal clearance and high stability of Nd-DTPA in vivo. In general, renal excretion is a desirable pathway for the nanomaterial clearance, 12
ACCEPTED MANUSCRIPT owing to the rapid elimination of the nanoprobes to prevent the involved cellular internalization. The renal pathway is mainly dependent on glomerular filtration in the kidneys; thus, the probe size, and shape have key influence on the filtration efficiency.46 The filtration-size threshold of
RI PT
glomerular capillary walls is approximately 5 nm, and nanoprobes with a hydrodynamic diameter of less than 5 nm or 40 kDa are typically filtered by renal.46 Thus, our designed Nd-DTPA molecules possesses a lower molecular weight than 40 kDa should be excreted by renal clearance
SC
route from the living body, which is also consistent with the clinical used Gd-DTPA. To furhter
M AN U
reveal the renal excretion, the ex vivo biodistribution of Nd-DTPA in the living mouse was detected. As shown in Supplementary Fig. S6, the fluorescence signal from the kidney is stronger than that in the other organs after 20 min injection, indicating the main renal clearance of the small Nd-DTPA molecule. By using ICP-MS, we also studied the renal clearance kinetics of the small
TE D
molecules by measuring the Nd element content in the dissected organs and urines at different time points (20 min, 24 h and 48 h). As demonstrated in Supplementary Fig. S7 and S8, Nd-DTPA showed minimal accumulation in heart, liver, spleen, and lung. High accumulation of
EP
Nd-DTPA in the urine and kidney was observed after 20 min post injection. Moreover, the renal
AC C
excretion rate of Nd-DTPA from the mouse was evaluated by collecting urine at different time-points from the intravenously injected mice. As shown in Supplementary Fig. S9, the renal elimination rate constant was calculated to be about 0.13 h-1. These findings reveal that the Nd-DTPA with efficient emission at 1330 nm is an ideal probe for NIR IIa bioimaging with deep tissue penetration, low cytotoxicity and rapid excretion, paving the way for designing the new type of NIR IIa imaging agent and favoring the clinical translation of NIR II imaging technology.
13
ACCEPTED MANUSCRIPT 3.3. NIR IIa imaging-guided tiny tumor detection. In vivo highly sensitive tiny tumor detection has played a key role for early cancer diagnosis. However, tiny tumor (below 5 mm) detection was limited by the lower uptake of macro molecules
RI PT
drugs and dramatic comparable geometric resistances.47 Therefore, designing of nanoprobes with molecular functionality to be accumulated in the tumor site is a crucial step. It is noted that the photo-stability is a key characteristic for molecular optical probes. Therefore, the photo-stability
SC
of Nd-DTPA molecule in the acidic microenvironment (pH: 5~6) was also tested with a
M AN U
continuous 808 nm laser excitation for 1 h at 1 W cm-2. The photo-stability curve (Fig. S10) and in vitro phantom bioimaging (Fig. S11) reveal the designed Nd-DTPA present superior photo-stability with photobleaching degree of 6.0% in 1 h, which is relatively lower than the previous report41.
TE D
Compared to the traditional QDs and rare earth nanomaterials, the Nd-DTPA small molecule agents present a smaller size and excellent pharmacokinetics, making it efficient accumulation in the tumor site, as well as rapid excretion from living animals. Therefore, in vivo tiny tumor
EP
detection was performed by intravenous injection of 125 µL Nd-DTPA solution. As shown in Fig.
AC C
4a, after 60 s injection, obvious NIR IIa signal can be detected from the kidney and tumor site, indicating the NIR-IIa bioimaging based on Nd-DTPA molecule can afford the pinpointing location of tumor. The NIR-IIa fluorescence signal in the tumor site becomes distinguishable over time and is slightly decreased after 20 min injection, revealing a sensitive detection of tumor. To further demonstrate the sensitive detection of tiny tumor, ex-vivo NIR IIa imaging of the dissected tumor was performed. As demonstrated in Fig. 4c, strong signal was observed in the tumor site, further validating the accumulation of Nd-DTPA in tumor site. In addition, we further analyzed 14
ACCEPTED MANUSCRIPT time-dependent intensity change and maximal signal in the kidney and tumor site, as shown in Fig. 4d and 4e. The maximal signal intensity in the tumor site exhibit comparable intensity to that in kidney, demonstrating the efficient uptake of Nd-DTPA in tumor. To further reveal the uptake of
RI PT
Nd-DTPA in tumor site, the dissected tumor was used for ICP-MS assay to evaluate the quantitative Nd content in tumor. As shown in Supplementary Fig. S12, the content of Nd in tumor site is almost equal to the kidney, which is consistent with the in vivo results,
SC
unambiguously verifying the significant accumulation of Nd-DTPA molecular probe in tumor site.
M AN U
By putting together, we conclude that the designed Nd-DTPA molecule should be a suitable NIR II agent to achieve sensitive optical-guided tiny tumor diagnosis. 3.4. In vivo X-ray bioimaging
Recently, X-ray imaging has triggered considerable interests in diagnostic medicine owing to its
TE D
deep tissue penetration and high resolution.42,43 However, as X-ray imaging contrast agents, elements with high atomic number (high-Z), high biocompatibility and acceptable safety profiles are urgently needed.42 Currently, small iodinated molecules with effectively absorbed X-ray nature
EP
are used as X-ray contrast agent in clinical applications. Compared with the I-based X-ray
AC C
imaging agent, the Nd-based molecular probe may have superior X-ray imaging contrast because of the larger atomic number and K-edge value of Nd (43.5 keV) than I (33 keV).48 Therefore, apart from the excellent NIR IIa imaging, the designed Nd-DTPA can also be used as promising agent for X-ray bioimaging owing to the large K-edge value (43.5 keV). To validate this, X-ray imaging based on Nd-DTPA and iobitridol was performed. As shown in Fig. 5b and 5c, the in vitro phantom images of both agents exhibit obvious X-ray absorption enhancement when increasing their concentration. Moreover, the Nd-based agent presents a higher X-ray absorption 15
ACCEPTED MANUSCRIPT contrast than that of I at identical concentration. Hounsfield units (HU) values of Nd-DTPA and iobitridol were further tested. As demonstrated in supplementary Fig. 5d, the HU values of both agents increased linearly when increasing the concentration. More importantly, a higher HU value
RI PT
of Nd-DTPA was detected at an equivalent concentration. In order to further reveal the dual-modal imaging, 125 µL of Nd-DTPA molecular probe (0.05 M) was subcutaneously injected into a Kunming mouse, and both the NIR IIa and X-ray imaging of the same mouse were carried out. As
SC
demonstrated in Supplementary Fig. S13, both X-ray absorption contrast and fluorescence signal
M AN U
are observed at the same site of the mouse, indicating the successful dual-modal bioimaging. These results illustrate that our designed Nd-DTPA possesses superior X-ray imaging efficacy than that of I-based agent. We further carried out the in vivo X-ray bioimaging of a Kunming mouse (the left panel of Fig. 5e) without injection and with subcutaneous injection (the right panel
TE D
of supplementary Fig. 5e) of Nd-DTPA probe by using an in vivo imaging system (Bruker In Vivo FX Pro). As illustrated by the blue-dotted circles in Fig. 5e, the injection site exhibits high X-ray absorption contrast. However, no obvious signal is observed in the control mouse, revealing that
EP
the Nd-based contrast agent is an ideal probe for in vivo X-ray bioimaging.
AC C
3.5. Histological analysis
To evaluating the long-term in vivo toxicity, histological assessment based on Nd-DTPA was performed. As shown in Fig. 6, the mice were dissected in terms of the heart, liver, spleen, lung and kidney at 3 and 7 days after intravenously injected with Nd-DTPA. No tissue damage or any other signal of inflammatory lesion was observed, implying the high biocompatibility and low toxicity of the Nd-DTPA molecule. 4. Conclusions 16
ACCEPTED MANUSCRIPT In conclusion, we have developed a new type of Nd-DTPA molecule probe with fast excretion for NIR IIa bioimaging, which is an important counterpart of Gd-DTPA (a highly biocompatible magnetic resonance imaging agent for clinical use approved by the US Food and Drug
RI PT
Administration, FDA). Our designed Nd-DTPA possesses high photostability and narrow-band NIR IIa emission at 1330 nm. By combining bright narrow-band NIR-II emission and excellent biocompatibility, optical bioimaging-guided tiny tumor (down to ~3 mm) detection was
SC
successfully demonstrated for the first time. Except the excellent optical properties, the Nd-DTPA
M AN U
was also used for in vivo molecule X-ray imaging agent. These findings pave the way of designing a new dual-modal molecular probe for synergetic NIR II and X-ray bioimaging for highly sensitive tiny tumor (~ 3 mm) detection and promote the clinical translation of the next generation optical imaging technology by using NIR II region.
TE D
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21671064), Science and Technology Planning Project of Hunan Province (No. 2017RS3031) and
AC C
EP
the Hunan Provincial Innovation Foundation for Postgraduate (CX2017B223).
References
[1] Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature 2008; 452: 580-589. [2] Naczynski DJ, Tan MC, Tan M, Wall B, Wall J, Kulesa A, Chen S, Roth CM, Riman RE, Moghe PV. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 2013; 4: 2199. [3] Han SY, Deng RR, Xie XJ, Liu XG, Zhou J. Rare-earth-doped biological composites as in vivo 17
ACCEPTED MANUSCRIPT shortwave infrared reporters. Angew. Chem. Int. Ed. 2014; 53: 11702-11715. [4] Zhou J, Liu Z, Li FY. Upconversion nanophosphors for small-animal imaging. Chem. Soc. Rev. 2012; 41: 1323-1349.
RI PT
[5] Gai SL, Li CX, Yang PP, Lin J. Recent progress in rare earth micro/nanocrystals: soft chemical synthesis, luminescent properties, and biomedical applications. Chem. Rev. 2014; 114: 2343-2389.
SC
[6] Li XM, Zhang F, Zhao DY. Lab on upconversion nanoparticles: optical properties and
M AN U
applications engineering via designed nanostructure. Chem. Soc. Rev. 2012; 44: 1346-1378. [7] Chen GY, Yang CH, Prasad PN. Nanophotonics and nanochemistry: controlling the excitation dynamics for frequency up- and down-conversion in lanthanide-doped nanoparticles.Acc. Chem. Res. 2013; 46: 1474-1486.
TE D
[8] Zeng SJ, Yi ZG, Lu W, Qian C, Wang HB, Rao L, Zeng TM, Liu HR, Liu HJ, Fei B, Hao JH. Simultaneous realization of phase/size manipulation, upconversion luminescence enhancement, and blood vessel imaging in multifunctional nanoprobes through transition metal Mn2+ doping.
EP
Adv. Funct. Mater. 2014; 26: 4051-4059.
AC C
[9] Yi ZG, Li XL, Xue ZL, Liang X, Lu W, Peng H, Liu HR, Zeng SJ. and Hao, JH. Remarkable NIR enhancement of multifunctional nanoprobes for in vivo trimodal bioimaging and upconversion optical/T2 -weighted MRI-guided small tumor diagnosis. Adv. Funct. Mater. 2015; 25: 7119-7129.
[10] Pansare V, Hejazi S, Faenza W, Prudhomme RK. Review of long-wavelength optical and NIR imaging materials: contrast agents, fluorophores, and multifunctional nanocarriers. Chem. Mater. 2012; 24: 812-827. 18
ACCEPTED MANUSCRIPT [11] Altınoglu EI, Adair JH. Near infrared imaging with nanoparticles. WIREs Nanomedicine and Nanobiotechnology 2010; 2: 461-477. [12] Lim YT, Kim S, Nakayama A, Stott NE, Bawendi MG, Frangioni JV. Selection of quantum
RI PT
dot wavelengths for biomedical assays and imaging. Imaging. 2003; 2: 50-64. [13] Li CY, Zhang YJ, Chen GC, Hu F, Zhao K, Wang QB. Engineered Multifunctional Nanomedicine for Simultaneous Stereotactic Chemotherapy and Inhibited Osteolysis in an
SC
Orthotopic Model of Bone Metastasis. Adv. Mater. 2017; 29: 1605754.
M AN U
[14] Song CH, Zhang YJ, Li CY, Chen GC, Kang XF, Wang QB. Enhanced Nanodrug Delivery to Solid Tumors Based on a Tumor Vasculature-Targeted Strategy. Adv. Funct. Mater., 2016; 26: 4192-4200.
[15] Chen GC, Tian F, Zhang Y, Zhang YJ, Li CY, Wang QB. Tracking of Transplanted Human
TE D
Mesenchymal Stem Cells in Living Mice using Near-Infrared Ag2S Quantum Dots. Adv. Funct. Mater., 2014; 24: 2481-2488.
[16] Du YP, Xu B, Fu T, Cai M, Li F, Zhang Y, Wang QB. Near-Infrared Photoluminescent Ag2S
EP
Quantum Dots from a Single Source Precursor. J. Am. Chem. Soc. 2010; 132: 1470-1471.
AC C
[17] Li CY, Zhang YJ, Wang M, Zhang Y, Chen GC, Li L, Wu DM, Wang QB. In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window. Biomaterials. 2014; 35: 393-400. [18] Hong GS, Robinson JT, Zhang YJ, Diao S, Antaris AL, Antaris QB, Dai HJ. In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew. Chem. Int. Ed. 2012; 51: 9818-9821. [19] Zhang Y, Hong G, Zhang Y, Chen G, Li F, Dai HJ, Wang QB. In vivo fluorescence imaging 19
ACCEPTED MANUSCRIPT with Ag2S quantum dots in the second near-infrared region. ACS Nano 2012; 6: 3695-3702. [20] Chen J, Kong YF, Fang HW, Wo Y, Zhou DJ, Wu ZY, Li YX, Chen SY. Direct water-phase synthesis of lead sulfide quantum dots encapsulated by β-lactoglobulin for in vivo second near
RI PT
infrared window imaging with reduced toxicity. Chem. Commun. 2016; 52: 4025-4028. [21] Dong BH, Li CY, Chen CC, Zhang YJ, Zhang Y, Deng MJ, Wang QB. Facile synthesis of highly photoluminescent Ag2Se quantum dots as a new fluorescent probe in the second
SC
near-infrared window for in vivo imaging. Chem. Mater. 2013; 25: 2503-2509.
M AN U
[22] Welsher K, Liu Z, Sherlock SP, Robinson JT, Chen Z, Daranciang D, Dai HJ. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 2009; 4: 773-780.
[23] Robinson JT, Welsher K, Tabakman SM, Sherlock SP, Wang H, Luong R, Dai HJ. High
TE D
performance in vivo near-IR (>1 µm) imaging and photo thermal cancer therapy with carbon nanotubes. Nano Res. 2010; 3: 779-793.
[24] Welsher K, Sherlock SP, Dai HJ. Deep-tissue anatomical imaging of mice using carbon
AC C
8943-8948.
EP
nanotube fluorophores in the second near-infrared window. Proc. Natl. Acad. Sci. USA. 2011; 108:
[25] Yi H, Ghosh D, Ham MH, Qi J, Barone PW, Strano MS, Belcher AM. M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett. 2012; 12: 1176-1183. [26] Robinson JT, Hong G, Liang Y, Zhang B, Yaghi OK, Yaghi H. In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake. J. Am. Chem. Soc. 2012; 134: 10664-10669. 20
ACCEPTED MANUSCRIPT [27] Diao S, Hong GS, Robinson JT, Jiao LY, Antaris AL, Wu JZ, Choi CL, Dai HJ. Chirality enriched (12,1) and (11,3) single-walled carbon nanotubes for biological imaging. J. Am. Chem. Soc. 2012; 134: 16971-16974.
RI PT
[28] Hong GS, Lee JC, Robinson JT, Raaz U, Xie L, Huang NF, Cooke JP. & Dai HJ. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 2012; 18: 1841-1846.
nanotubes for simultaneous tumor imaging and photothermal
therapy. ACS Nano 2013; 7: 3644-3652.
M AN U
chirality sorted (6,5) carbon
SC
[29] Antaris AL, Robinson JT, Yaghi OK, Hong G, Diao S, Luong R, Dai HJ. Ultra-low doses of
[30] Perrault SD, Walkey C, Jennings T, Fischer HC, & Chan WCW. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett. 2009; 9: 1909-1915.
TE D
[31] Villa I, Vedda A, Cantarelli IX, Pedroni M, Piccinelli F, Bettinelli M, Speghini A, Quintanilla M, Vetrone F, Rocha U, Jacinto C, Carrasco E, Rodríguez FS, Juarranz A, Rosal BD, Ortgies DH, Gonzalez PH, Solé JG, and García DJ. 1.3 µm emitting SrF2:Nd3+ nanoparticles for high contrast
EP
in vivo imaging in the second biological window. Nano Res. 2015; 8: 649-665.
AC C
[32] Rui W, Li XM, Zhou L, Zhang F. Epitaxial seeded growth of rare-earth nanocrystals with efficient 800 nm near-infrared to 1525 nm short-wavelength infrared downconversion photoluminescence for in vivo bioimaging. Angew. Chem. Int. Ed. 2014; 53: 1-6. [33] Kamimura M, Matsumoto T, Suyari S, Umezawaab M, SogaRatiometric M. Near-infrared fluorescence nanothermometry in the OTN-NIR (NIR II/III) biological window based on rare-earth doped b-NaYF4 nanoparticles. J. Mater. Chem. B 2017; 5: 1917-1925. [34] Jiang XY, Cao C, Feng W, Li FY. Nd3+-doped LiYF4 nanocrystals for the bio-imaging in the 21
ACCEPTED MANUSCRIPT second near-infrared window. J. Mater. Chem. B 2016; 4: 87-95. [35] Zhong YT, Ma ZR, Zhu SJ, Yue JY, Zhang MX, Antaris AL, Yuan J, Cui R, Wan H, Zhou Y, Wang WZ, Huang NGF, Luo J, Hu ZY, Dai HJ. Boosting the down-shifting luminescence of rare-
RI PT
earth nanocrystals for biological imaging beyond 1500 nm. Nat. Commun. 2017; 8: 737. [36] Hong, GS, Antaris AL and Dai HJ. Near-infrared fluorophores for biomedical imaging. Nature Biomedical Engineering 2017; 10: 1-22.
SC
[37] Tao ZM, Hong GS, Ji CS, Chen CX, Diao S, Antaris AL, Zhang B, Zhou YP, Dai HJ.
M AN U
Biological imaging using nanoparticles of small organic molecules with fluorescence emission at wavelengths longer than 1000 nm. Angew. Chem. Int. Ed. 2013; 52: 13002-13006. [38] Hong GS, Zou YP, Antaris AL, Diao S, Wu D, Cheng K, Zhang XD, Chen X, Liu B, He YH, Wu JZ, Yuan J, Zhang B, Tao ZM, Fukunaga C & Dai HJ. Ultrafast fluorescence imaging in vivo
4206.
TE D
with conjugated polymer fluorophores in the second near-infrared window. Nat. Commun. 2014; 5:
[39] Antaris AL, Chen H, Diao S, Ma ZR, Zhang Z, Zhu SJ, Wang J, Lozano AX, Fan QL, Chew
EP
LL, Zhu M, Cheng K, Hong XC, Dai HJ & Cheng Z. A high quantum yield molecule-protein
AC C
complex fluorophore for near-infrared II imaging. Nat. Commun. 2017; 8: 15269. [40] Hong GS, Diao S, Chang JL, Antaris AL, Chen CX, Zhang B, Zhao S, Atochin DN, Huang PL, Andreasson KI, Kuo CJ, & Dai HJ. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nature Photonics. 2014; 8: 723-730. [41] Antairs AL, Chen H, Cheng K, Sun Y, Hong GS, Qu CR, Qu S, Deng ZX, Hu XM, Zhang B, Zhang XD, Yaghi OK, Alamparambil ZR, Hong XC, Cheng Z, Dai HJ. A small-molecule dye for NIR-II imaging. Nature Materials 2016; 15: 235-242. 22
ACCEPTED MANUSCRIPT [42] Liu YL, Ai KL, Liu JH, Yuan Q. H, He YY, Lu LH. A high-performance ytterbium based nanoparticulate contrast agent for in vivo X-ray computed tomography imaging. Angew. Chem. Int. Ed. 2012; 51: 1437-1442.
RI PT
[43] Yu SB, Watson AD. Metal-based X-ray contrast media. Chem. Rev. 1999; 99: 2353-2377. [44] Xu ZP, Kurniawan ND, Bartlett PF, Lu G. Enhancement of relaxivity rates of Gd–DTPA complexes by intercalation into layered double hydroxide nanoparticles. Chem. Eur. J. 2007; 13:
SC
2824-2830.
M AN U
[45] Lim CK, Singh A, Heo J, Kim D, Lee KE, Jeon H, Koh J, Kwon IC, Kim S, Gadolinium-coordinated elastic nanogels for in vivo tumor targeting and imaging. Biomaterials 2013; 34: 6846-6852.
[46] Yu MX, Zheng J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS
TE D
Nano, 2015; 9: 6655–6674.
[47] Liu CY, Gao ZY, Zeng JF, Hou Y, Fang F, Li YL, Qiao RR, Shen L, Lei H, Yang WS, Gao M. Y. Magnetic/upconversion fluorescent NaGdF4:Yb,Er nanoparticle-based dual-modal molecular
EP
probes for imaging tiny tumors in vivo. ACS Nano 2013; 7: 7227-7240.
AC C
[48 ] http://physics.nist.gov/PhysRefData/XrayMassCoef/.
23
ACCEPTED MANUSCRIPT Figure captions: Scheme 1. Schematic illustration of the molecular structure of the DTPA and Nd-DTPA, along with the synthesis process of Nd-DTPA and bio-application process in NIR-IIa window under the 808 nm excitation.
RI PT
Fig. 1. (a) FTIR spectra of Nd-DTPA and pure DTPA samples. (b) UV-vis absorption spectra (600-900 nm) and photoluminescent spectrum of the Nd-DTPA complex under the excitation of 808 nm laser, demonstrating the efficient narrow-band emissions centered at 1050 and 1330 nm. (c) Simplified energy level diagram. (d) A schematic diagram for the detection of the
SC
photostability. (e) Photostability curve of Nd-DTPA in water under continuous 808 nm illumination with power density of 1 w cm-2, indicating the high photostability of the Nd-DTPA
M AN U
even for 3600 s.
Fig. 2. (a) A schematic diagram of demonstrating the bright NIR-II emission (1000-1100 nm) from the mouse food in stomach under the excitation of 808 nm laser. (b) Digital photo of the food. (c) and (d) The in vitro imaging of mouse food with different band pass filters. In vivo NIR-II imaging of a mouse via intravenous injection of Nd-DTPA by using a 1000-1100 nm band pass
TE D
filter (e) and 1200-1400 nm band pass filter (f). Fluorescence interference signal from stomach was obviously detected in (e) by using a 1000-1100 nm band pass filter.
EP
Fig. 3. (a) In vivo NIR-IIa imaging of a mouse after intravenously injected with 125 µL Nd-DTPA solution. The prone positioned mouse was used to collect the kidney signals at various periods. (b)
AC C
NIR-IIa imaging of the same mouse in the supine position, revealing the rapid excretion of Nd-DTPA molecule via kidney. The lower right corner denotes the NIR-IIa imaging of the urine sample (digital photograph in the lower right corner) collected after 2 h injection.
Fig. 4. (a) NIR-IIa bioimaging of MAT-Ly-Lu-B-2 (Mat) cells tumor-bearing mouse after intravenously injected Nd-DTPA molecules at different periods. (b) In situ digital photography of the tumor-bearing mouse. (c) The digital photography of the tumor (left panel) and ex vivo NIR-IIa bioimaging (right panel), indicating the obvious fluorescence signal in the tumor site. (d) The time-dependent intensity changes of the kidney and tumor. (e) The maximal signal intensity in 24
ACCEPTED MANUSCRIPT the kidney and tumor site. Fig. 5. (a) A Schematic illustration of in vivo and in vitro X-ray imaging. (b) In vitro X-ray imaging of Nd-DTPA and iobitridol solution with different concentrations of Nd and I, respectively. (c) The corresponding pseudo-color images of (b). (d) X-ray absorption Hounsfield
RI PT
units (HU) value of Nd-DTPA (green lines) and iobitridol (red lines) as a function of the concentrations of Nd and I, respectively. (e) In vivo X-ray bioimaging of a Kunming mouse without injection (left) and subcutaneous injection (right) of Nd-DTPA. The injection site was marked with blue-dotted lines. The injected site with Nd-DTPA showed significant X-ray
SC
absorption contrast by using in vivo imaging system (Bruker In Vivo FX PRO) under 45 kVp voltage.
M AN U
Fig. 6. H&E stained heart, liver, spleen, lung and kidney collected from the mouse with injection of Nd-DTPA and control group without injection were analyzed. The isolated tissues present no
AC C
EP
TE D
obvious damage. All the scale bars are 100 µm.
25
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Scheme 1. Schematic illustration of the molecular structure of the DTPA and Nd-DTPA, along
EP
with the synthesis process of Nd-DTPA and bio-application process in NIR-IIa window under the
AC C
808 nm excitation.
26
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
TE D
Fig. 1. (a) FTIR spectra of Nd-DTPA and pure DTPA samples. (b) UV-vis absorption spectra (600-900 nm) and photoluminescent spectrum of the Nd-DTPA complex under the excitation of 808 nm laser, demonstrating the efficient narrow-band emissions centered at 1050 and 1330 nm.
EP
(c) Simplified energy level diagram. (d) A schematic diagram for the detection of the photostability. (e) Photostability curve of Nd-DTPA in water under continuous 808 nm
AC C
illumination with power density of 1 w cm-2, indicating the high photostability of the Nd-DTPA even for 3600 s.
27
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2. (a) A schematic diagram of demonstrating the bright NIR-II emission (1000-1100 nm) from the mouse food in stomach under the excitation of 808 nm laser. (b) Digital photo of the food.
EP
(c) and (d) The in vitro imaging of mouse food with different band pass filters. In vivo NIR-II imaging of a mouse via intravenous injection of Nd-DTPA by using a 1000-1100 nm band pass
AC C
filter (e) and 1200-1400 nm band pass filter (f). Fluorescence interference signal from stomach was obviously detected in (e) by using a 1000-1100 nm band pass filter.
28
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 3. (a) In vivo NIR-IIa imaging of a mouse after intravenously injected with 125 µL Nd-DTPA
EP
solution. The prone positioned mouse was used to collect the kidney signals at various periods. (b) NIR-IIa imaging of the same mouse in the supine position, revealing the rapid excretion of
AC C
Nd-DTPA molecule via kidney. The lower right corner denotes the NIR-IIa imaging of the urine sample (digital photograph in the lower right corner) collected after 2 h injection.
29
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. (a) NIR-IIa bioimaging of MAT-Ly-Lu-B-2 (Mat) cells tumor-bearing mouse after intravenously injected Nd-DTPA molecules at different periods. (b) In situ digital photography of the tumor-bearing mouse. (c) The digital photography of the tumor (left panel) and ex vivo NIR-IIa bioimaging (right panel), indicating the obvious fluorescence signal in the tumor site. (d) The time-dependent intensity changes of the kidney and tumor. (e) The maximal signal intensity in the kidney and tumor site.
30
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
Fig. 5. (a) A Schematic illustration of in vivo and in vitro X-ray imaging. (b) In vitro X-ray imaging of Nd-DTPA and iobitridol solution with different concentrations of Nd and I,
AC C
respectively. (c) The corresponding pseudo-color images of (b). (d) X-ray absorption Hounsfield units (HU) value of Nd-DTPA (green lines) and iobitridol (red lines) as a function of the concentrations of Nd and I, respectively. (e) In vivo X-ray bioimaging of a Kunming mouse without injection (left) and subcutaneous injection (right) of Nd-DTPA. The injection site was marked with blue-dotted lines. The injected site with Nd-DTPA showed significant X-ray absorption contrast by using in vivo imaging system (Bruker In Vivo FX PRO) under 45 kVp voltage.
31
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 6. H&E stained heart, liver, spleen, lung and kidney collected from the mouse with injection
TE D
of Nd-DTPA and control group without injection were analyzed. The isolated tissues present no
AC C
EP
obvious damage. All the scale bars are 100 µm.
32
S0142-9612(18)30220-5
DOI:
10.1016/j.biomaterials.2018.03.041
Reference:
JBMT 18567
To appear in:
Biomaterials
Received Date: 25 October 2017 Revised Date:
22 March 2018
Accepted Date: 23 March 2018
Please cite this article as: Li Y, Li X, Xue Z, Jiang M, Zeng S, Hao J, Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection, Biomaterials (2018), doi: 10.1016/j.biomaterials.2018.03.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Second near-infrared emissive lanthanide complex for fast renal-clearable in vivo optical bioimaging and tiny tumor detection
RI PT
Youbin Li, † Xiaolong Li, † Zhenluan Xue, † Mingyang Jiang, † Songjun Zeng †*, Jianhua Hao‡* †
College of Physics and Information Science and Key Laboratory of Low-dimensional Quantum Structures and Quantum Control of the Ministry of Education, and Synergetic Innovation Center for Quantum Effects and Applications, Hunan Normal University, Changsha 410081 , China
SC
Department of Applied Physics, The Hong Kong Polytechnic University, Hong Kong.
M AN U
‡
AC C
EP
TE D
* Address correspondence to [email protected], [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
In vivo optical imaging by using a new imaging window located at short-wavelength infrared region (1000-1700 nm, named as NIR II) presents an unprecedented improvement in imaging
RI PT
sensitivity and spatial resolution over the traditional visible and near-infrared light. However, the most developed NIR II-emitters are hardly excreted from live animals, leading to unknown
SC
long-term toxicity concerns, which hinder the widespread applications of this advanced imaging technology. Here, we developed a new generation molecular NIR II-emitting probe based on
M AN U
Nd-diethylene triamine pentacetate acid (DTPA) complex. The designed molecular Nd-DTPA probe with bright narrow band emission at 1330 nm is successfully used for highly sensitive in vivo NIR IIa bioimaging with rapid renal excretion and high biocompatibility and optical-guided tiny tumor (down to ~3 mm) detection for the first time. Moreover, the Nd-DPTA complex also
TE D
holds great promise as an X-ray contrast agent. These findings open up the possibility for designing a new generation of multi-modal small molecular probe for early tumor diagnosis and
EP
favor the clinic translation of the advanced NIR II imaging method.
AC C
KEYWORDS: molecule probe, NIR II bioimaging, renal clearance, tiny tumor diagnosis, X-ray bioimaging.
2
ACCEPTED MANUSCRIPT 1. Introduction Noninvasive fluorescent imaging by using optical probe is one of the most important imaging technique for the early detection of disease owing to the cellular or molecular level information
RI PT
and high imaging sensitivity.1,2 Although the traditional near infrared light (NIR I, 650–900 nm) emitting optical probes with low tissue-absorption nature are well-developed for deep-tissue bioimaging, some performance parameters (penetration depth, imaging sensitive and resolution
SC
etc.) are still limited by the large scattering losses of NIR I light.3-11 Recent optical simulations
M AN U
demonstrate a new advanced optical transparent window with short wavelength infrared light (SWIR, NIR II, 1000-1700 nm), which presents lower optical absorption and auto-fluorescence than NIR I window, but up to 1000-fold reduction in scattering losses, subsequently resulting in unprecedented improvements in detection depth and imaging resolution.2,12 Therefore, developing
TE D
the new advanced NIR II optical probe with high emission intensity, large optical penetration depth in biotissue, and high biocompatibility is vital important for sensitivity and high resolution optical bioimaging.
EP
In this context, some NIR II optical probes capable of producing NIR II emissions such as
AC C
quantum dots (QDs)13-21 and single-walled carbon nanotubes (SWNTs)22-29 are emerging. However, most of the reported NIR II-emitting QDs comprise the toxic elements such as lead or mercury, making them unsuitable for bioapplications.2,20 Single-walled carbon nanotubes (SWNTs) have demonstrated good performance in in vivo applications.22-29 However, SWNTs still face significant challenges including low emission intensity, broad emission peaks (band width: > 300 nm), and large size distributions, making them unattainable for the size-dependent biological barriers study.2,30 Recently, rare earth based nanoparticles with well controlled size and 3
ACCEPTED MANUSCRIPT narrow-band emission were emerged as promising probes for NIR II bioimaging owing to their advantages of high efficiency, low photo-bleaching and long luminescence lifetimes.2,31-35 However, most of the designed rare earth-based probes usually present unspecific liver/spleen
RI PT
uptake and are excreted slowly from live animals, leading to unknown long-term toxicity concerns, which remarkably limited their widespread application in NIR II bioimaging. As it is well known that the ability of rapid excretion of imaging contrast agent is vital for clinical use. Recently, Dai’s
SC
group demonstrated a pioneering study of designing small organic molecular probes with 1000 nm
M AN U
emission for NIR-II imaging.36-41 However, these small molecular probes still presented the broad-band emission and relative low photostability(>10% photobleaching degree in 1 h)38, which inhibited the practice use of nonoverlapping signals for multispectral imaging. Therefore, it is appealing to design novel NIR II imaging agent with narrow-band and bright NIR II emission,
TE D
rapid excretion, high biocompatibility for eventually overcoming the barrier of clinical use. Herein, we design a new type of molecular Nd-based complex (Nd-DTPA) with efficient narrow band NIR IIa emission centered at 1330 nm (band width: ~ 65 nm), high biocompatibility,
EP
and rapid excretion from the kidney. The NIR IIa emitting Nd-DTPA probe was successfully used
AC C
as in vivo tracking. More importantly, the optical-guided tiny tumor (< 3 mm) detection was demonstrated by using the designed Nd-DTPA probe for the first time. Apart from the excellent NIR IIa bioimaging nature, the designed Nd-DTPA can also be used as X-ray imaging agent owing to the large X-ray absorption coefficient of Nd element42-43.
2. Materials and methods 2.1. Materials 4
ACCEPTED MANUSCRIPT All chemical reagents were obtained from the commercial supply and used without further purification. Rare earth NdCl3•6H2O (99.99%) was purchased from QingDa elaborate Chemical Reagent
Co.
Ltd
(Shandong).
Diethylene
triamine
pentacetate
acid
(DTPA)
and
RI PT
N-Methyl-D-glutamines were obtained from the ScienMax. Xylenol orange (XO) was purchased form Sigma-Aldrich Trading Co, Ltd. (Shanghai, China). 2.2. Synthesis of Nd-DTPA
SC
The Nd-DTPA was were prepared using a modified literature protocol.44 For synthesis of the , then 1.1 mmol DTPA was
M AN U
Nd-based complex, 15 ml water in the beaker was heated to 50
added into the water and kept stirring for 20 min. 1 mmol NdCl3 solution (1 M) was then added and stirred until forming a transparent solution. The obtained Nd-DTPA solution was treated with N-Methyl-D-glutamines to adjust the pH value to 7.2.
TE D
2.3. Characterization
The photoluminescent spectrum of Nd-DTPA was detected with a NIR-II spectroscopy (NIRQuest512, Ocean Optics). The MDL-H 808 nm laser was used as an excitation light source at
EP
room temperature. UV-Vis absorption spectrum of Nd-DTPA in water solution was detected by
AC C
using a Spectrophotometer system (UV-1800, Hunan Sino-Jewell Electronics Co.Ltd.). The surface ligands of Nd-DTPA were analyzed via a Fourier transform infrared spectrum (FTIR) by using a Magna 760 spectrometer (Nicolet). The Nd content in the isolated organ was measured using an Agilent 7500a inductively coupled plasma mass spectrometry (ICP-MS). The Nd-DTPA was characterized using the nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was measured using a Bruker AV-400 spectrometer at room temperature. Chemical shifts were named as δ values (p.p.m), where D2O was used as an internal reference. 5
ACCEPTED MANUSCRIPT Splitting patterns were labeled as s (singlet), t (triplet), m (multiplet). The NMR spectra of Nd-DTPA are given as follows: 1
H NMR (400 MHz, D2O): δ 8.22 (s, 3H), 4.1 (t, 2H), 3.7 (m, 4h), 3.6 (m, 3H), 3.2 (m, 4H),
RI PT
2.8 (s, 5H). 2.4. Cytotoxicity evaluation
To evaluate the cell viability of Nd-DTPA, the viability of HeLa cells based on MTT
under 5% CO2. Then the cell culture medium in each
M AN U
(6000 cells per well) were kept at 37
SC
proliferation assay method was evaluated. In a typical process, HeLa cells in a 96-well micro plate
well was replaced by Dulbecco’s Modified Eagle Medium (DMEM) solution including 10% fetal bovine serum, 1% penicillin and streptomycin. The prepared Nd-DTPA solutions with different concentrations (0, 100, 200, 500, 1000 µL/mL) were added at 37 °C and with 5% CO2 for 24 h.
TE D
Finally, we measured the cell viability by using a typical MTT assay. 2.5. In vivo and ex vivo NIR-IIa optical bioimaging
A home-made small animal NIR-II bioimaging system equipped with a thermoelectric cooled
EP
InGaAs camera (Model: NIRvanaTM Camera System, operating temperature: -80
, Princeton
AC C
Instruments) and fiber-coupled MDL-H 808/980 lasers was used for NIR II bioimaging. The fluorescence signal was collected by using the system installed with 808 nm diode laser as light source and filtered by a (1200-1400 nm) band pass filter. For in vivo bioimaging, a Kunming mouse was intraperitoneally injected with pentobarbital sodium aqueous (150 µL/10 wt%). Then Nd-DTPA (125 µL, 0.05 M) was injected into the mouse by intravenous injection at tail vein. Finally, the fluorescence signal was collected by using the system under an excitation powder density of 100 mW/cm2. To further reveal the renal clearance, Kunming mice were intravenously 6
ACCEPTED MANUSCRIPT injected with Nd-DTPA (125 µL, 0.05 M), the corresponding isolated organs of the mice were collected after 20 min, 24 h, and 48 h injection for ex-vivo NIR-IIa imaging by using the same system. The experiment parameters were the same as above. All animal procedures comply with
RI PT
the institutional animal use and care regulations approved by the Laboratory Animal Center of Hunan Province. 2.6. Quantitative Biodistribution Measurement
SC
125 µL of Nd-DTPA (0.05 M) molecular probes were intravenously injected into the
M AN U
Kunming mouse. The urine and isolated organs (heart, liver, spleen, lung, and kidney) were collected after 20 min, 24 h, and 48 h post injection and used for ICP-MS. 2.7. Urine excretion rate evaluation
According to the previous report41, the renal excretion rate of Nd-DTPA from the mouse was
TE D
measured by intravenously injecting 125 µL (0.05 M) Nd-DTPA solution and the urine was collected for quantifying the amount of clearance. The mouse urines were collected in capillarity
EP
tubes at 20 min, 150 min, 3.5 h and 7.5 h injection, and the fluorescence intensity was measured with an InGaAs camera (Model: NIRvanaTM Camera System, Princeton Instruments). The injected
AC C
dose (%ID) was measured in a similar way to the previous report41:
%= ܦܫ m s ∑m s (I m − Icontrol ) ∗ Vm (I injected − I control ) ∗ Vm =
f
=
1
where s represents the number of the urine time-point from a mouse (S1, the first urine time-point, 7
ACCEPTED MANUSCRIPT Sf, the last urine time-point), I is the integrated luminescence intensity measured with a (1200-1400 nm) band-pass filter, Icontrol represents the luminescence intensity of control urine, Iinjected is the fluorescent intensity of the injected Nd-DTPA, Vm represents the volume of the urine,
2.8. NIR-IIa optical bioimaging of tiny tumor detection
RI PT
and Vinjected is the volume of the injected Nd-DTPA.
SC
Female BALB/c mice (18-21 g, 4 weeks old, purchased from Laboratory Animal Center of Hunan Province) were selected for establishing the mice model. 8 × 106 MAT-Ly-Lu-B-2 (Mat)
M AN U
cells in 100 µL of sodium chloride solution were subcutaneously injected into the female BALB/C mouseat the back for further culturing the tumor to about ≈ 3 mm in average size. We then carried out the in vivo tumor detection. Firstly, 150 µL of pentobarbital sodium aqueous solution (10 wt%) was intraperitoneally injected into the inoculated tumor mouse to anesthetize it. 125 µL of
TE D
Nd-DTPA solution was then injected into the mouse by intravenous injection. The fluorescence signal was captured at different period times by using the same bioimaging system equipped with
EP
808 nm laser as light source. The emission filter was set as 1330 nm, and the exposure time was set as 2000 ms. Ex-vivo bioimaging of the tumor was executed by the same parameters. The digital
AC C
picture of the tumor was taken by a Canon digital camera. 2.9. In vivo and in vitro X-ray bioimaging To demonstrate the feasibility of the as prepared Nd-DTPA molecules for in vivo X-ray bioimaging, a Kunming mouse was first anesthetized. And 125 µL of Nd-DTPA with concentration of 30 mg/mL was subcutaneously injected into the mouse. Then X-ray bioimaging was tested via an in vivo imaging system (Bruker In Vivo FX Pro). To assess the X-ray contrast efficacy, Nd-DTPA and Iobitridol were dispersed in water with different Nd and I concentrations 8
ACCEPTED MANUSCRIPT over the range from 0 to 60 mg/mL, respectively. In vitro phantom X-ray imaging based on these agents was collected. In vivo X-ray imaging was executed with the following parameters: voltage, 45 kVp; aluminum filter, 4 mm; and exposure time, 30 s.
RI PT
2.10. Quantum yield of Nd-DTPA The fluorescence quantum yield (QY) of Nd-DTPA in water was calculated in a similar
reference. The QY was calculated by the following formula:
M AN U
Φs = Φr (Fs/Fr)*(Ar/As)*(n2s/n2r)
SC
method to the previous report41 using a standard IR-26 dye (dissolved in DCE, QY = 0.5%) as a
where Φ represents QY, F denotes integrated photoluminescence emission intensity, A denotes absorbance at the maximum excitation wavelength and n is the refractive index of the solvent (n= 1.33 for water, n = 1.44 for DCE, respectively). The subscripts s and r represent the
2.11. Histology analysis
TE D
sample and reference solutions, respectively.
For histology analysis, Kunming mice were sacrificed at 3 and 7 day after intravenously
EP
injected with 125 µL (0.05 M) of Nd-DTPA solution. Major organs including heart, liver, spleen,
AC C
lung, and kidney were isolated from the mice. Then the tissue sections were stained with hematoxylin and eosin (H&E) and visualized by using an optical microscope. 3. Results and discussion
3.1. Synthesis and NIR II emitting property The Nd-DTPA was primarily synthesized through a chelation of the Nd3+ and DTPA, as presented in Scheme 1. The N-Methyl-D-glucamine was added into the Nd-DTPA solution to tune the pH value to 7.2. The surface ligands of the precursor and Nd-DTPA were analyzed by 9
ACCEPTED MANUSCRIPT Fourier-transform infrared (FTIR) spectroscopy (Fig. 1a) As demonstrated, for pure DTPA, the peaks at 1733 cm-1, 1700 cm-1, and 1634 cm-1 are ascribed to the stretching vibration of C=O (–COOH), C=O (–CONH–), and the deformation vibrations of N-H (–CONH–), respectively.
RI PT
While in Nd-DTPA sample, the stretching and deformation vibration bands of 1733/1700/1634 cm-1 were disappeared and a new vibration peaks at 1595 cm-1 was observed and ascribed to the vibration of O-C-O (carboxylate), unambiguously validating the coordination of COO- with
SC
Nd3+.44 To further reveal the formation of Nd-DTPA, we have performed 1H nuclear magnetic
M AN U
resonance (NMR) spectroscopy analysis. As shown in Supplementary Fig. S1, 1H NMR result further validate the formation of Nd-DTPA molecule. The ultraviolet (UV)–vis absorption spectrum of the Nd-DTPA solution in water presented several sharp absorption peaks of Nd3+ centered at 741 nm (4I9/2
4
F7/2), 801 nm (4I9/2
4
F5/2) and 872 nm (4I9/2
4
F3/2), indicating an
TE D
excellent absorption of NIR light of about 800 nm (Fig. 1b). To reveal the NIR II emitting properties, the NIR emission spectrum of Nd-DTPA was detected under the excitation of 808 nm laser. As shown in Fig. 1b and 1c, the Nd-DTPA presents the efficient NIR II emissions centered at
EP
1050 nm (band width: ~ 45 nm) and 1330 nm (band width: ~ 65 nm), which can be ascribed to the 4
I11/2 (1050 nm), and 4F3/2
4
I13/2 (1330 nm) of Nd3+, respectively. It is
AC C
electronic transitions 4F3/2
noted that the photo-stability is a key characteristic for molecular optical probes. Therefore, the photo-stability of Nd-DTPA molecule was tested with a continuous 808 nm laser excitation for 1 h at 1 w cm-2 (Fig. 1d). The photo-stability curve (Fig. 1e) and in vitro phantom bioimaging (Supplementary Fig. S2) reveal the designed Nd-DTPA present superior photo-stability with photobleaching degree of 3.4% in 1 h, which is relatively lower than the previous report40. To further prove the stability of Nd-DTPA, the leakage of Nd3+ in Nd-DTPA and NdCl3 were 10
ACCEPTED MANUSCRIPT measured using the XO as the indicator. As shown in Supplementary Fig. S3, free Nd3+ at a concentration as low as 0.125 mM and pH ranging from 3 to 5 can still react with the XO solution to form a red substance. On the contrary, the Nd-DTPA could not react with XO even at a
RI PT
concentration of 2 mM, which further demonstrated the strong chelation of Nd3+ and DTPA and high structure stability of Nd-DTPA. This result reveal the no leakage of Nd3+ or very limited leakage less than 0.125 mM from Nd-DTPA, which is stable and safe enough for the biological
SC
applications comparable with the standard of IC50 ~ 0.125 mM for free Gd3+.45 In addition, the QY
M AN U
of Nd-DTPA at 1050 nm is measured to be 0.63%, which exhibits higher QY than the SWNTs probes in the previous report.24-27 And the QY of Nd-DTPA at 1330 nm is measured to be 0.07%, which exhibits lower QY than Nd-DTPA at 1050 nm. These results suggest that the Nd-DTPA possesses remarkable narrow band emission in NIR II region (1050/1330 nm) and superior
TE D
photo-stability, making it an ideal agent for NIR-II bioimaging with high photostability. 3.2. In vivo NIR-IIa bioimaging
Prior to using Nd-DTPA for in vivo imaging, the cell toxicity of this designed molecular
EP
Nd-DTPA probe was evaluated via a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium
AC C
bromide (MTT) method (Supplementary Fig. S4). As demonstrated, the cell viability of Nd-DTPA in HeLa cells was 99% when treated with 100 µg mL-1 of Nd-DTPA. When further increasing the concentration of Nd-DTPA to 1000 µg mL-1, the cell viability was still remained at 90%, indicating the very low cell toxicity of the Nd-DTPA molecules. Encouraged by the low cytotoxicity and strong 1050 nm emission, we first performed in vivo NIR II bioimaging by detecting the 1050 nm emission via a home-made NIR II imaging system (Supplementary Fig. S5) installed with a fiber-coupled 808 nm laser as light source. As shown in 11
ACCEPTED MANUSCRIPT Fig. 2c and 2d, an obvious NIR II signal of mouse food was observed
by using a 1000-1100 nm
band-pass filter, while, the signal intensity of mouse food was almost disappeared by detecting the 1330 nm emission, indicating the efficiently diminishing the undesired interference signal caused
RI PT
by mouse food. Further in vivo bioimaging (Fig. 2e and 2f) also demonstrated that the undesired signal of stomach caused by mouse food was fully removed by measuring the 1330 nm emission via a band-pass filter (1200-1400 nm). It should be noted that imaging in the NIR-IIa window
SC
(1300 -1400 nm) can greatly minimize the scatter of rejection photons below 1300 nm, and avoid
M AN U
the light absorption from water vibration over 1400 nm.40 Based on the above analysis, our designed Nd-DTPA probe presents efficient NIR IIa emission located at 1330 nm, which is beneficial for bioimaging applications. Then, in vivo NIR IIa bioimaging of a Kunming mouse was performed by intravenous injection of Nd-DTPA. As demonstrated in Fig. 3a, within a few
TE D
second, a significant fluorescence signal in kidney was observed and the fluorescence signal in the kidney was gradually enhanced after 25 min injection and then tended to slight decrease after 35 min. The optical signal was finally disappeared within 120 min, indicting the rapidly excretion of
EP
the Nd-DTPA probe from the kidneys. In addition, NIR IIa bioimaging of the supine positioned
AC C
mouse was performed for further proof of the in vivo excretion mechanism of the Nd-DTPA molecules. As illustrated in Fig. 3b, strong signal was observed in bladder within 10 min, implying the rapid excretion of the Nd-DTPA probe from the kidney and vessels to the bladder. Moreover, the Nd-DTPA probe was fully excreted from the bladder within 11 h, indicating the fast renal clearance of the Nd-DTPA molecule. The urine sample collected after 2 h injection also showed a strong fluorescence signal, which further proved the renal clearance and high stability of Nd-DTPA in vivo. In general, renal excretion is a desirable pathway for the nanomaterial clearance, 12
ACCEPTED MANUSCRIPT owing to the rapid elimination of the nanoprobes to prevent the involved cellular internalization. The renal pathway is mainly dependent on glomerular filtration in the kidneys; thus, the probe size, and shape have key influence on the filtration efficiency.46 The filtration-size threshold of
RI PT
glomerular capillary walls is approximately 5 nm, and nanoprobes with a hydrodynamic diameter of less than 5 nm or 40 kDa are typically filtered by renal.46 Thus, our designed Nd-DTPA molecules possesses a lower molecular weight than 40 kDa should be excreted by renal clearance
SC
route from the living body, which is also consistent with the clinical used Gd-DTPA. To furhter
M AN U
reveal the renal excretion, the ex vivo biodistribution of Nd-DTPA in the living mouse was detected. As shown in Supplementary Fig. S6, the fluorescence signal from the kidney is stronger than that in the other organs after 20 min injection, indicating the main renal clearance of the small Nd-DTPA molecule. By using ICP-MS, we also studied the renal clearance kinetics of the small
TE D
molecules by measuring the Nd element content in the dissected organs and urines at different time points (20 min, 24 h and 48 h). As demonstrated in Supplementary Fig. S7 and S8, Nd-DTPA showed minimal accumulation in heart, liver, spleen, and lung. High accumulation of
EP
Nd-DTPA in the urine and kidney was observed after 20 min post injection. Moreover, the renal
AC C
excretion rate of Nd-DTPA from the mouse was evaluated by collecting urine at different time-points from the intravenously injected mice. As shown in Supplementary Fig. S9, the renal elimination rate constant was calculated to be about 0.13 h-1. These findings reveal that the Nd-DTPA with efficient emission at 1330 nm is an ideal probe for NIR IIa bioimaging with deep tissue penetration, low cytotoxicity and rapid excretion, paving the way for designing the new type of NIR IIa imaging agent and favoring the clinical translation of NIR II imaging technology.
13
ACCEPTED MANUSCRIPT 3.3. NIR IIa imaging-guided tiny tumor detection. In vivo highly sensitive tiny tumor detection has played a key role for early cancer diagnosis. However, tiny tumor (below 5 mm) detection was limited by the lower uptake of macro molecules
RI PT
drugs and dramatic comparable geometric resistances.47 Therefore, designing of nanoprobes with molecular functionality to be accumulated in the tumor site is a crucial step. It is noted that the photo-stability is a key characteristic for molecular optical probes. Therefore, the photo-stability
SC
of Nd-DTPA molecule in the acidic microenvironment (pH: 5~6) was also tested with a
M AN U
continuous 808 nm laser excitation for 1 h at 1 W cm-2. The photo-stability curve (Fig. S10) and in vitro phantom bioimaging (Fig. S11) reveal the designed Nd-DTPA present superior photo-stability with photobleaching degree of 6.0% in 1 h, which is relatively lower than the previous report41.
TE D
Compared to the traditional QDs and rare earth nanomaterials, the Nd-DTPA small molecule agents present a smaller size and excellent pharmacokinetics, making it efficient accumulation in the tumor site, as well as rapid excretion from living animals. Therefore, in vivo tiny tumor
EP
detection was performed by intravenous injection of 125 µL Nd-DTPA solution. As shown in Fig.
AC C
4a, after 60 s injection, obvious NIR IIa signal can be detected from the kidney and tumor site, indicating the NIR-IIa bioimaging based on Nd-DTPA molecule can afford the pinpointing location of tumor. The NIR-IIa fluorescence signal in the tumor site becomes distinguishable over time and is slightly decreased after 20 min injection, revealing a sensitive detection of tumor. To further demonstrate the sensitive detection of tiny tumor, ex-vivo NIR IIa imaging of the dissected tumor was performed. As demonstrated in Fig. 4c, strong signal was observed in the tumor site, further validating the accumulation of Nd-DTPA in tumor site. In addition, we further analyzed 14
ACCEPTED MANUSCRIPT time-dependent intensity change and maximal signal in the kidney and tumor site, as shown in Fig. 4d and 4e. The maximal signal intensity in the tumor site exhibit comparable intensity to that in kidney, demonstrating the efficient uptake of Nd-DTPA in tumor. To further reveal the uptake of
RI PT
Nd-DTPA in tumor site, the dissected tumor was used for ICP-MS assay to evaluate the quantitative Nd content in tumor. As shown in Supplementary Fig. S12, the content of Nd in tumor site is almost equal to the kidney, which is consistent with the in vivo results,
SC
unambiguously verifying the significant accumulation of Nd-DTPA molecular probe in tumor site.
M AN U
By putting together, we conclude that the designed Nd-DTPA molecule should be a suitable NIR II agent to achieve sensitive optical-guided tiny tumor diagnosis. 3.4. In vivo X-ray bioimaging
Recently, X-ray imaging has triggered considerable interests in diagnostic medicine owing to its
TE D
deep tissue penetration and high resolution.42,43 However, as X-ray imaging contrast agents, elements with high atomic number (high-Z), high biocompatibility and acceptable safety profiles are urgently needed.42 Currently, small iodinated molecules with effectively absorbed X-ray nature
EP
are used as X-ray contrast agent in clinical applications. Compared with the I-based X-ray
AC C
imaging agent, the Nd-based molecular probe may have superior X-ray imaging contrast because of the larger atomic number and K-edge value of Nd (43.5 keV) than I (33 keV).48 Therefore, apart from the excellent NIR IIa imaging, the designed Nd-DTPA can also be used as promising agent for X-ray bioimaging owing to the large K-edge value (43.5 keV). To validate this, X-ray imaging based on Nd-DTPA and iobitridol was performed. As shown in Fig. 5b and 5c, the in vitro phantom images of both agents exhibit obvious X-ray absorption enhancement when increasing their concentration. Moreover, the Nd-based agent presents a higher X-ray absorption 15
ACCEPTED MANUSCRIPT contrast than that of I at identical concentration. Hounsfield units (HU) values of Nd-DTPA and iobitridol were further tested. As demonstrated in supplementary Fig. 5d, the HU values of both agents increased linearly when increasing the concentration. More importantly, a higher HU value
RI PT
of Nd-DTPA was detected at an equivalent concentration. In order to further reveal the dual-modal imaging, 125 µL of Nd-DTPA molecular probe (0.05 M) was subcutaneously injected into a Kunming mouse, and both the NIR IIa and X-ray imaging of the same mouse were carried out. As
SC
demonstrated in Supplementary Fig. S13, both X-ray absorption contrast and fluorescence signal
M AN U
are observed at the same site of the mouse, indicating the successful dual-modal bioimaging. These results illustrate that our designed Nd-DTPA possesses superior X-ray imaging efficacy than that of I-based agent. We further carried out the in vivo X-ray bioimaging of a Kunming mouse (the left panel of Fig. 5e) without injection and with subcutaneous injection (the right panel
TE D
of supplementary Fig. 5e) of Nd-DTPA probe by using an in vivo imaging system (Bruker In Vivo FX Pro). As illustrated by the blue-dotted circles in Fig. 5e, the injection site exhibits high X-ray absorption contrast. However, no obvious signal is observed in the control mouse, revealing that
EP
the Nd-based contrast agent is an ideal probe for in vivo X-ray bioimaging.
AC C
3.5. Histological analysis
To evaluating the long-term in vivo toxicity, histological assessment based on Nd-DTPA was performed. As shown in Fig. 6, the mice were dissected in terms of the heart, liver, spleen, lung and kidney at 3 and 7 days after intravenously injected with Nd-DTPA. No tissue damage or any other signal of inflammatory lesion was observed, implying the high biocompatibility and low toxicity of the Nd-DTPA molecule. 4. Conclusions 16
ACCEPTED MANUSCRIPT In conclusion, we have developed a new type of Nd-DTPA molecule probe with fast excretion for NIR IIa bioimaging, which is an important counterpart of Gd-DTPA (a highly biocompatible magnetic resonance imaging agent for clinical use approved by the US Food and Drug
RI PT
Administration, FDA). Our designed Nd-DTPA possesses high photostability and narrow-band NIR IIa emission at 1330 nm. By combining bright narrow-band NIR-II emission and excellent biocompatibility, optical bioimaging-guided tiny tumor (down to ~3 mm) detection was
SC
successfully demonstrated for the first time. Except the excellent optical properties, the Nd-DTPA
M AN U
was also used for in vivo molecule X-ray imaging agent. These findings pave the way of designing a new dual-modal molecular probe for synergetic NIR II and X-ray bioimaging for highly sensitive tiny tumor (~ 3 mm) detection and promote the clinical translation of the next generation optical imaging technology by using NIR II region.
TE D
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21671064), Science and Technology Planning Project of Hunan Province (No. 2017RS3031) and
AC C
EP
the Hunan Provincial Innovation Foundation for Postgraduate (CX2017B223).
References
[1] Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature 2008; 452: 580-589. [2] Naczynski DJ, Tan MC, Tan M, Wall B, Wall J, Kulesa A, Chen S, Roth CM, Riman RE, Moghe PV. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 2013; 4: 2199. [3] Han SY, Deng RR, Xie XJ, Liu XG, Zhou J. Rare-earth-doped biological composites as in vivo 17
ACCEPTED MANUSCRIPT shortwave infrared reporters. Angew. Chem. Int. Ed. 2014; 53: 11702-11715. [4] Zhou J, Liu Z, Li FY. Upconversion nanophosphors for small-animal imaging. Chem. Soc. Rev. 2012; 41: 1323-1349.
RI PT
[5] Gai SL, Li CX, Yang PP, Lin J. Recent progress in rare earth micro/nanocrystals: soft chemical synthesis, luminescent properties, and biomedical applications. Chem. Rev. 2014; 114: 2343-2389.
SC
[6] Li XM, Zhang F, Zhao DY. Lab on upconversion nanoparticles: optical properties and
M AN U
applications engineering via designed nanostructure. Chem. Soc. Rev. 2012; 44: 1346-1378. [7] Chen GY, Yang CH, Prasad PN. Nanophotonics and nanochemistry: controlling the excitation dynamics for frequency up- and down-conversion in lanthanide-doped nanoparticles.Acc. Chem. Res. 2013; 46: 1474-1486.
TE D
[8] Zeng SJ, Yi ZG, Lu W, Qian C, Wang HB, Rao L, Zeng TM, Liu HR, Liu HJ, Fei B, Hao JH. Simultaneous realization of phase/size manipulation, upconversion luminescence enhancement, and blood vessel imaging in multifunctional nanoprobes through transition metal Mn2+ doping.
EP
Adv. Funct. Mater. 2014; 26: 4051-4059.
AC C
[9] Yi ZG, Li XL, Xue ZL, Liang X, Lu W, Peng H, Liu HR, Zeng SJ. and Hao, JH. Remarkable NIR enhancement of multifunctional nanoprobes for in vivo trimodal bioimaging and upconversion optical/T2 -weighted MRI-guided small tumor diagnosis. Adv. Funct. Mater. 2015; 25: 7119-7129.
[10] Pansare V, Hejazi S, Faenza W, Prudhomme RK. Review of long-wavelength optical and NIR imaging materials: contrast agents, fluorophores, and multifunctional nanocarriers. Chem. Mater. 2012; 24: 812-827. 18
ACCEPTED MANUSCRIPT [11] Altınoglu EI, Adair JH. Near infrared imaging with nanoparticles. WIREs Nanomedicine and Nanobiotechnology 2010; 2: 461-477. [12] Lim YT, Kim S, Nakayama A, Stott NE, Bawendi MG, Frangioni JV. Selection of quantum
RI PT
dot wavelengths for biomedical assays and imaging. Imaging. 2003; 2: 50-64. [13] Li CY, Zhang YJ, Chen GC, Hu F, Zhao K, Wang QB. Engineered Multifunctional Nanomedicine for Simultaneous Stereotactic Chemotherapy and Inhibited Osteolysis in an
SC
Orthotopic Model of Bone Metastasis. Adv. Mater. 2017; 29: 1605754.
M AN U
[14] Song CH, Zhang YJ, Li CY, Chen GC, Kang XF, Wang QB. Enhanced Nanodrug Delivery to Solid Tumors Based on a Tumor Vasculature-Targeted Strategy. Adv. Funct. Mater., 2016; 26: 4192-4200.
[15] Chen GC, Tian F, Zhang Y, Zhang YJ, Li CY, Wang QB. Tracking of Transplanted Human
TE D
Mesenchymal Stem Cells in Living Mice using Near-Infrared Ag2S Quantum Dots. Adv. Funct. Mater., 2014; 24: 2481-2488.
[16] Du YP, Xu B, Fu T, Cai M, Li F, Zhang Y, Wang QB. Near-Infrared Photoluminescent Ag2S
EP
Quantum Dots from a Single Source Precursor. J. Am. Chem. Soc. 2010; 132: 1470-1471.
AC C
[17] Li CY, Zhang YJ, Wang M, Zhang Y, Chen GC, Li L, Wu DM, Wang QB. In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window. Biomaterials. 2014; 35: 393-400. [18] Hong GS, Robinson JT, Zhang YJ, Diao S, Antaris AL, Antaris QB, Dai HJ. In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew. Chem. Int. Ed. 2012; 51: 9818-9821. [19] Zhang Y, Hong G, Zhang Y, Chen G, Li F, Dai HJ, Wang QB. In vivo fluorescence imaging 19
ACCEPTED MANUSCRIPT with Ag2S quantum dots in the second near-infrared region. ACS Nano 2012; 6: 3695-3702. [20] Chen J, Kong YF, Fang HW, Wo Y, Zhou DJ, Wu ZY, Li YX, Chen SY. Direct water-phase synthesis of lead sulfide quantum dots encapsulated by β-lactoglobulin for in vivo second near
RI PT
infrared window imaging with reduced toxicity. Chem. Commun. 2016; 52: 4025-4028. [21] Dong BH, Li CY, Chen CC, Zhang YJ, Zhang Y, Deng MJ, Wang QB. Facile synthesis of highly photoluminescent Ag2Se quantum dots as a new fluorescent probe in the second
SC
near-infrared window for in vivo imaging. Chem. Mater. 2013; 25: 2503-2509.
M AN U
[22] Welsher K, Liu Z, Sherlock SP, Robinson JT, Chen Z, Daranciang D, Dai HJ. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 2009; 4: 773-780.
[23] Robinson JT, Welsher K, Tabakman SM, Sherlock SP, Wang H, Luong R, Dai HJ. High
TE D
performance in vivo near-IR (>1 µm) imaging and photo thermal cancer therapy with carbon nanotubes. Nano Res. 2010; 3: 779-793.
[24] Welsher K, Sherlock SP, Dai HJ. Deep-tissue anatomical imaging of mice using carbon
AC C
8943-8948.
EP
nanotube fluorophores in the second near-infrared window. Proc. Natl. Acad. Sci. USA. 2011; 108:
[25] Yi H, Ghosh D, Ham MH, Qi J, Barone PW, Strano MS, Belcher AM. M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett. 2012; 12: 1176-1183. [26] Robinson JT, Hong G, Liang Y, Zhang B, Yaghi OK, Yaghi H. In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake. J. Am. Chem. Soc. 2012; 134: 10664-10669. 20
ACCEPTED MANUSCRIPT [27] Diao S, Hong GS, Robinson JT, Jiao LY, Antaris AL, Wu JZ, Choi CL, Dai HJ. Chirality enriched (12,1) and (11,3) single-walled carbon nanotubes for biological imaging. J. Am. Chem. Soc. 2012; 134: 16971-16974.
RI PT
[28] Hong GS, Lee JC, Robinson JT, Raaz U, Xie L, Huang NF, Cooke JP. & Dai HJ. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 2012; 18: 1841-1846.
nanotubes for simultaneous tumor imaging and photothermal
therapy. ACS Nano 2013; 7: 3644-3652.
M AN U
chirality sorted (6,5) carbon
SC
[29] Antaris AL, Robinson JT, Yaghi OK, Hong G, Diao S, Luong R, Dai HJ. Ultra-low doses of
[30] Perrault SD, Walkey C, Jennings T, Fischer HC, & Chan WCW. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett. 2009; 9: 1909-1915.
TE D
[31] Villa I, Vedda A, Cantarelli IX, Pedroni M, Piccinelli F, Bettinelli M, Speghini A, Quintanilla M, Vetrone F, Rocha U, Jacinto C, Carrasco E, Rodríguez FS, Juarranz A, Rosal BD, Ortgies DH, Gonzalez PH, Solé JG, and García DJ. 1.3 µm emitting SrF2:Nd3+ nanoparticles for high contrast
EP
in vivo imaging in the second biological window. Nano Res. 2015; 8: 649-665.
AC C
[32] Rui W, Li XM, Zhou L, Zhang F. Epitaxial seeded growth of rare-earth nanocrystals with efficient 800 nm near-infrared to 1525 nm short-wavelength infrared downconversion photoluminescence for in vivo bioimaging. Angew. Chem. Int. Ed. 2014; 53: 1-6. [33] Kamimura M, Matsumoto T, Suyari S, Umezawaab M, SogaRatiometric M. Near-infrared fluorescence nanothermometry in the OTN-NIR (NIR II/III) biological window based on rare-earth doped b-NaYF4 nanoparticles. J. Mater. Chem. B 2017; 5: 1917-1925. [34] Jiang XY, Cao C, Feng W, Li FY. Nd3+-doped LiYF4 nanocrystals for the bio-imaging in the 21
ACCEPTED MANUSCRIPT second near-infrared window. J. Mater. Chem. B 2016; 4: 87-95. [35] Zhong YT, Ma ZR, Zhu SJ, Yue JY, Zhang MX, Antaris AL, Yuan J, Cui R, Wan H, Zhou Y, Wang WZ, Huang NGF, Luo J, Hu ZY, Dai HJ. Boosting the down-shifting luminescence of rare-
RI PT
earth nanocrystals for biological imaging beyond 1500 nm. Nat. Commun. 2017; 8: 737. [36] Hong, GS, Antaris AL and Dai HJ. Near-infrared fluorophores for biomedical imaging. Nature Biomedical Engineering 2017; 10: 1-22.
SC
[37] Tao ZM, Hong GS, Ji CS, Chen CX, Diao S, Antaris AL, Zhang B, Zhou YP, Dai HJ.
M AN U
Biological imaging using nanoparticles of small organic molecules with fluorescence emission at wavelengths longer than 1000 nm. Angew. Chem. Int. Ed. 2013; 52: 13002-13006. [38] Hong GS, Zou YP, Antaris AL, Diao S, Wu D, Cheng K, Zhang XD, Chen X, Liu B, He YH, Wu JZ, Yuan J, Zhang B, Tao ZM, Fukunaga C & Dai HJ. Ultrafast fluorescence imaging in vivo
4206.
TE D
with conjugated polymer fluorophores in the second near-infrared window. Nat. Commun. 2014; 5:
[39] Antaris AL, Chen H, Diao S, Ma ZR, Zhang Z, Zhu SJ, Wang J, Lozano AX, Fan QL, Chew
EP
LL, Zhu M, Cheng K, Hong XC, Dai HJ & Cheng Z. A high quantum yield molecule-protein
AC C
complex fluorophore for near-infrared II imaging. Nat. Commun. 2017; 8: 15269. [40] Hong GS, Diao S, Chang JL, Antaris AL, Chen CX, Zhang B, Zhao S, Atochin DN, Huang PL, Andreasson KI, Kuo CJ, & Dai HJ. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nature Photonics. 2014; 8: 723-730. [41] Antairs AL, Chen H, Cheng K, Sun Y, Hong GS, Qu CR, Qu S, Deng ZX, Hu XM, Zhang B, Zhang XD, Yaghi OK, Alamparambil ZR, Hong XC, Cheng Z, Dai HJ. A small-molecule dye for NIR-II imaging. Nature Materials 2016; 15: 235-242. 22
ACCEPTED MANUSCRIPT [42] Liu YL, Ai KL, Liu JH, Yuan Q. H, He YY, Lu LH. A high-performance ytterbium based nanoparticulate contrast agent for in vivo X-ray computed tomography imaging. Angew. Chem. Int. Ed. 2012; 51: 1437-1442.
RI PT
[43] Yu SB, Watson AD. Metal-based X-ray contrast media. Chem. Rev. 1999; 99: 2353-2377. [44] Xu ZP, Kurniawan ND, Bartlett PF, Lu G. Enhancement of relaxivity rates of Gd–DTPA complexes by intercalation into layered double hydroxide nanoparticles. Chem. Eur. J. 2007; 13:
SC
2824-2830.
M AN U
[45] Lim CK, Singh A, Heo J, Kim D, Lee KE, Jeon H, Koh J, Kwon IC, Kim S, Gadolinium-coordinated elastic nanogels for in vivo tumor targeting and imaging. Biomaterials 2013; 34: 6846-6852.
[46] Yu MX, Zheng J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS
TE D
Nano, 2015; 9: 6655–6674.
[47] Liu CY, Gao ZY, Zeng JF, Hou Y, Fang F, Li YL, Qiao RR, Shen L, Lei H, Yang WS, Gao M. Y. Magnetic/upconversion fluorescent NaGdF4:Yb,Er nanoparticle-based dual-modal molecular
EP
probes for imaging tiny tumors in vivo. ACS Nano 2013; 7: 7227-7240.
AC C
[48 ] http://physics.nist.gov/PhysRefData/XrayMassCoef/.
23
ACCEPTED MANUSCRIPT Figure captions: Scheme 1. Schematic illustration of the molecular structure of the DTPA and Nd-DTPA, along with the synthesis process of Nd-DTPA and bio-application process in NIR-IIa window under the 808 nm excitation.
RI PT
Fig. 1. (a) FTIR spectra of Nd-DTPA and pure DTPA samples. (b) UV-vis absorption spectra (600-900 nm) and photoluminescent spectrum of the Nd-DTPA complex under the excitation of 808 nm laser, demonstrating the efficient narrow-band emissions centered at 1050 and 1330 nm. (c) Simplified energy level diagram. (d) A schematic diagram for the detection of the
SC
photostability. (e) Photostability curve of Nd-DTPA in water under continuous 808 nm illumination with power density of 1 w cm-2, indicating the high photostability of the Nd-DTPA
M AN U
even for 3600 s.
Fig. 2. (a) A schematic diagram of demonstrating the bright NIR-II emission (1000-1100 nm) from the mouse food in stomach under the excitation of 808 nm laser. (b) Digital photo of the food. (c) and (d) The in vitro imaging of mouse food with different band pass filters. In vivo NIR-II imaging of a mouse via intravenous injection of Nd-DTPA by using a 1000-1100 nm band pass
TE D
filter (e) and 1200-1400 nm band pass filter (f). Fluorescence interference signal from stomach was obviously detected in (e) by using a 1000-1100 nm band pass filter.
EP
Fig. 3. (a) In vivo NIR-IIa imaging of a mouse after intravenously injected with 125 µL Nd-DTPA solution. The prone positioned mouse was used to collect the kidney signals at various periods. (b)
AC C
NIR-IIa imaging of the same mouse in the supine position, revealing the rapid excretion of Nd-DTPA molecule via kidney. The lower right corner denotes the NIR-IIa imaging of the urine sample (digital photograph in the lower right corner) collected after 2 h injection.
Fig. 4. (a) NIR-IIa bioimaging of MAT-Ly-Lu-B-2 (Mat) cells tumor-bearing mouse after intravenously injected Nd-DTPA molecules at different periods. (b) In situ digital photography of the tumor-bearing mouse. (c) The digital photography of the tumor (left panel) and ex vivo NIR-IIa bioimaging (right panel), indicating the obvious fluorescence signal in the tumor site. (d) The time-dependent intensity changes of the kidney and tumor. (e) The maximal signal intensity in 24
ACCEPTED MANUSCRIPT the kidney and tumor site. Fig. 5. (a) A Schematic illustration of in vivo and in vitro X-ray imaging. (b) In vitro X-ray imaging of Nd-DTPA and iobitridol solution with different concentrations of Nd and I, respectively. (c) The corresponding pseudo-color images of (b). (d) X-ray absorption Hounsfield
RI PT
units (HU) value of Nd-DTPA (green lines) and iobitridol (red lines) as a function of the concentrations of Nd and I, respectively. (e) In vivo X-ray bioimaging of a Kunming mouse without injection (left) and subcutaneous injection (right) of Nd-DTPA. The injection site was marked with blue-dotted lines. The injected site with Nd-DTPA showed significant X-ray
SC
absorption contrast by using in vivo imaging system (Bruker In Vivo FX PRO) under 45 kVp voltage.
M AN U
Fig. 6. H&E stained heart, liver, spleen, lung and kidney collected from the mouse with injection of Nd-DTPA and control group without injection were analyzed. The isolated tissues present no
AC C
EP
TE D
obvious damage. All the scale bars are 100 µm.
25
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Scheme 1. Schematic illustration of the molecular structure of the DTPA and Nd-DTPA, along
EP
with the synthesis process of Nd-DTPA and bio-application process in NIR-IIa window under the
AC C
808 nm excitation.
26
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
TE D
Fig. 1. (a) FTIR spectra of Nd-DTPA and pure DTPA samples. (b) UV-vis absorption spectra (600-900 nm) and photoluminescent spectrum of the Nd-DTPA complex under the excitation of 808 nm laser, demonstrating the efficient narrow-band emissions centered at 1050 and 1330 nm.
EP
(c) Simplified energy level diagram. (d) A schematic diagram for the detection of the photostability. (e) Photostability curve of Nd-DTPA in water under continuous 808 nm
AC C
illumination with power density of 1 w cm-2, indicating the high photostability of the Nd-DTPA even for 3600 s.
27
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2. (a) A schematic diagram of demonstrating the bright NIR-II emission (1000-1100 nm) from the mouse food in stomach under the excitation of 808 nm laser. (b) Digital photo of the food.
EP
(c) and (d) The in vitro imaging of mouse food with different band pass filters. In vivo NIR-II imaging of a mouse via intravenous injection of Nd-DTPA by using a 1000-1100 nm band pass
AC C
filter (e) and 1200-1400 nm band pass filter (f). Fluorescence interference signal from stomach was obviously detected in (e) by using a 1000-1100 nm band pass filter.
28
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 3. (a) In vivo NIR-IIa imaging of a mouse after intravenously injected with 125 µL Nd-DTPA
EP
solution. The prone positioned mouse was used to collect the kidney signals at various periods. (b) NIR-IIa imaging of the same mouse in the supine position, revealing the rapid excretion of
AC C
Nd-DTPA molecule via kidney. The lower right corner denotes the NIR-IIa imaging of the urine sample (digital photograph in the lower right corner) collected after 2 h injection.
29
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 4. (a) NIR-IIa bioimaging of MAT-Ly-Lu-B-2 (Mat) cells tumor-bearing mouse after intravenously injected Nd-DTPA molecules at different periods. (b) In situ digital photography of the tumor-bearing mouse. (c) The digital photography of the tumor (left panel) and ex vivo NIR-IIa bioimaging (right panel), indicating the obvious fluorescence signal in the tumor site. (d) The time-dependent intensity changes of the kidney and tumor. (e) The maximal signal intensity in the kidney and tumor site.
30
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
Fig. 5. (a) A Schematic illustration of in vivo and in vitro X-ray imaging. (b) In vitro X-ray imaging of Nd-DTPA and iobitridol solution with different concentrations of Nd and I,
AC C
respectively. (c) The corresponding pseudo-color images of (b). (d) X-ray absorption Hounsfield units (HU) value of Nd-DTPA (green lines) and iobitridol (red lines) as a function of the concentrations of Nd and I, respectively. (e) In vivo X-ray bioimaging of a Kunming mouse without injection (left) and subcutaneous injection (right) of Nd-DTPA. The injection site was marked with blue-dotted lines. The injected site with Nd-DTPA showed significant X-ray absorption contrast by using in vivo imaging system (Bruker In Vivo FX PRO) under 45 kVp voltage.
31
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 6. H&E stained heart, liver, spleen, lung and kidney collected from the mouse with injection
TE D
of Nd-DTPA and control group without injection were analyzed. The isolated tissues present no
AC C
EP
obvious damage. All the scale bars are 100 µm.
32