The employment of nanoagents as platforms for drug delivery and contrast enhancement of bioimaging is an expanding area of nanotheranostics.1–8 Accordingly, it is indispensable to understand the pharmacokinetics of nanoagents, including the processes of uptake, transport, clearance, and metabolism.9–12 Since the vascular systems, including arterial, lymphatic, and venous vessels, are mainly responsible for the conduction and delivery of the nanoagents, acquiring the spatiotemporal details of nanoagents in the vasculatures has attracted intensive interest.13–15 However, to visualize the intravital vasculatures, it is crucial to overcome the problem of inherently low imaging contrast, so research efforts are directed toward optimizing the nanoagents and/or developing more effective bioimaging methods. Recently, noninvasive and real-time pharmacokinetics imaging of nanoagents in peripheral vasculatures such as thoracoepigastric veins (TVs)16–20 and peripheral arteries21–23 has received considerable attention. Such efforts facilitate the advancement of nanotheranostics related to peripheral vasculatures, e.g., drug delivery via lymphatic vessels24,25 and therapeutic treatment of portal hypertension induced by liver cirrhosis26 and peripheral arterial diseases (PADs),21 and so forth.
Current techniques employed for noninvasive imaging of in vivo vasculatures include x-ray computed tomography,27–30 magnetic resonance imaging,31,32 positron emission tomography,33,34 optical coherence tomography (OCT),35,36 and photoacoustic tomography,37–41 which are either clinically employed or of great preclinical potential. See Refs. 21, 42, and 43 for a comprehensive review. These bioimaging modalities, however, are limited in capturing enough dynamic details of blood vasculatures, owing to insufficient spatiotemporal resolutions.44–47 For instance, the recently developed microultrasonography offers a temporal resolution up to a millisecond; however, the spatial resolution deteriorates considerably with increasing imaging depth.48 The near-infrared OCT, featuring label-free, subcellular spatial resolution, millimeter penetration depth, and wide field-of-view (FOV), has been clinically adopted in ophthalmology, cardiology, and gastrointestinal cancer screening.49 In the case of OCT angiography, which is a promising OCT mode for intravital imaging of TVs, ear microvasculatures and tumor angiogenesis models of mice,49–52 the mice need to be immobilized, and the timescale for imaging a field of is on the order of a few minutes.49,50 As the majority of the conventional bioimaging methods rely on contrast agents, the emerging nanotheranostics utilizing the integrated functionalities of nanoagents offers a unique opportunity to optimize the aforementioned diagnostic modalities.
Regarding noninvasive visualization of the TVs of small model animals, fluorescence bioimaging has been shown to be advantageous in its high spatiotemporal resolutions.53,54 Particularly, the recent advances in synthetic nanophosphors capable of luminescence emission in the second near-infrared region (NIR-II, 1000 to 1400 nm), such as single-wall carbon nanotubes,55 inorganic quantum dots,56 and rare earth doped upconversion nanoparticles,57,58 provide new opportunities for real-time bioimaging with a spatial resolution of sub- and an imaging depth of millimeters. The NIR-II optical bioimaging, besides an impressive penetration depth, can minimize the interference from the autofluorescence of biological tissues, owing to the relatively low light absorption in this spectral window. In parallel to NIR-II bioimaging, two-photon excitation (TPE) fluorescence bioimaging can further improve the spatial resolution, owing to the spatially more confined optical excitation compared to the conventional one-photon excitation scheme.59–62
TPE bioimaging has been benefiting from the development of high-performance TPE chromophores as exogenous labels. In this relation, we have devised a number of highly efficient europium (Eu)-luminescent complexes exhibiting large two-photon absorption (TPA) cross sections at near-infrared photosensitization wavelengths63–67 and have succeeded in incorporating such complexes into specific polymeric nanocarriers for in vivo pharmacokinetics imaging.68 Based on these advances, we have recently built a TPE and time-resolved (TPE-TR) bioimaging apparatus to analyze the spatiotemporal evolution of the nanocarriers in small animals. Our TPE-TR imaging modality can effectively reject the interference of biological autofluorescence, i.e., the fluorescence originating from endogenous biological fluorophores, and is free from the effects of laser heating. In addition, it is characterized by an FOV of , a penetration depth of (in intralipid phantom) and a frame time of a few seconds.69
In this work, we have employed a Eu-luminescence-labeled polymeric nanocarrier as the angiographic contrast for TPE-TR imaging of the TV networks of intact and healthy mice. The polymeric nanoagent bears high two-photon sensitization efficiency, high photostability, and low biotoxicity. The TPE-TR apparatus has an FOV that can accommodate the thoracoabdominal region extending from axillary to subiliac domains, enabling an entire TV network to be visualized. The transport and clearance processes of the polymeric nanoagent in TV blood streams were monitored in a noninvasive and real-time manner for up to 2 h, which allowed the determination of the timescale, the clearance efficiency, and the major destination of the nanoagent. The spatiotemporal evolution of the nanoagent in TV networks revealed a clearance time constant of and an apparent clearance efficiency of 80% to 90% in both axial and lateral veins. In addition, liver is found to be the major organ for the sequestration of the polymeric nanoagent, and the apparent retention efficiency and the clearance time constant of the nanoagent in liver were found to be 32% and , respectively. Our TPE-TR bioimaging strategy for exploiting the pharmacokinetics of nanoagents in intravital microvasculatures may be applicable to the diagnosis of the peripheral vasculature-related diseases, as well as to the development of target-specific nanotherapy.
Materials and Methods
Synthesis and Characterization of the Eu-Luminescence-Labeled Nanoagent
The Eu-luminescent complex, , was synthesized as previously reported.67,70 Briefly, the benzene solutions of (51.9 mg, 0.05 mM) and dpbt (20.8 mg, 0.05 mM) were mixed under argon atmosphere and stirred for 3 h. The mixture was then evaporated to remove the solvents and further dried in vacuum to obtain the complexes. Poly(methyl methacrylate-co-methacrylic acid) (PMMA-MAA) was used as the matrix material for the fabrication of the Eu-luminescence-labeled polymeric nanoparticles. See Fig. 1 for the chemical structures of the Eu-complex and the polymer. The -doped polymeric nanoparticles in a colloidal solution were prepared by rapid mixing water with a solution of the Eu-complex and the polymer in acetone under reduced pressure in a microchannel mixer, which was followed by removing acetone via evaporation.68 Hereafter, the -doped PMMA-MAA nanoparticle will be referred to as Eu@PMMA-MAA [see the inset of Fig. 2(a) for the nanoparticle configuration].
The average hydrodynamic diameter of the Eu@PMMA-MAA colloidal nanoparticles () was 26.9 nm as determined by dynamics light scattering [DLS; Fig. 2(a)] at 25°C (Zeta potential and particle size analyzer; Zeta plus, Brookhaven Instruments). Figure 2(b) shows the UV–Vis absorption and the luminescence spectra of the colloidal solution of Eu@PMMA-MAA recorded, respectively, on a UV–Vis absorption spectrometer (UV3600PLUS; Shimadzu, Japan) and a fluorescence spectrophotometer (FLS-980; Edinburgh Instruments Ltd., United Kingdom). The action cross section of complexes, defined as the product of the TPA cross section () and the Eu-luminescence quantum efficiency at 613 nm (), , serves as a metric of the two-photon sensitization efficiency. With the colloidal solution of Eu@PMMA-MAA nanoparticles as the sample and a methanol solution of rhodamine B as the reference (, ),71 we determined the of the PMMA-MAA embedded complexes following the procedures described in Ref. 64. The cell viability test of the Eu@PMMA-MAA nanoagent was performed following the procedures described in Ref. 68. The data analysis was conducted by Student’s -test. The sampling number of each data point was six, and the difference with was considered to be statistically significant.
TPE-TR Bioimaging Apparatus
The instrumentation details of TPE-TR imaging have been reported elsewhere.69 Herein, we provide a brief description (Fig. 3). As shown in Fig. 3(a), the TPE-induced autofluorescence of endogenous fluorophores could be rejected by detecting the Eu-luminescence after the decay of autofluorescence. This was realized by setting an appropriate delay time for autofluorescence rejection (), which was longer than the autofluorescence duration () but much shorter than the lifetime of Eu-luminescence (0.84 ms). As shown in Fig. 3(b), the laser beam was shaped with aperture A in a way so that the beam after focusing by CL formed a focal line with homogeneous power distribution.69 The focal line was scanned over a horizontal plane by regulating the X-scanning unit, while the imaging optics and the intensified charge coupled device (ICCD) for image sensing remained static. The Z-height of imaging plane was adjustable by vertically moving the animal stage.
With delicate timing management among pulsed excitation, mechanical X-scanning, and image sensing, the push-broom type, line-by-line imaging mode was established [cf. Fig. 4(a)]. The average illuminating power of femtosecond laser with a repetition rate of 500 Hz was set at 350 mW. The time delay for autofluorescence rejection was , and the gate width for acquiring a line image was . The frame time was adjustable between 1 to 4 s depending on the repetition rate of pulsed excitation, and it was 4 s in this work. The TPE-TR apparatus has a lateral resolution of in air, and an axial resolution of in air or 4.6 to 6.6 mm in an intralipid phantom depending on the imaging depth.69
Mice Model and Pharmacokinetics Imaging
Immune-deficient balb/c nude mice (6 to 8 weeks, 20 to 30 g Charles River Laboratories, China) were used in this work, and they were treated in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996, National Research Council). Before experiment, a healthy mouse was kept in a box attached with a minitype anesthetic ventilator (Matrx VIP3000). After being anaesthetized with isoflurane vapor, the mouse was mounted on the animal stage equipped with a ventilation mask supplying anaesthetic vapor and oxygen and with an exhaust port connected to a cryogenic trap.69 The mouse was injected via caudal vein with colloidal solutions of Eu@PMMA-MAA nanoagent ( and ), and the real-time images were recorded every 5 min.
FOV Orientation and Time Origin of TPE-TR Imaging
A mouse was mounted on the stage at a desired Z-height [cf. Fig. 3(b)] with the help of an auxiliary optoelectrical positioning system, which was based on a laser triangulation scheme for distance sensing.72 The xiphoid of the mouse was taken as the referencing point, which was illuminated with a beam of weak continuous-wave laser and viewed with an auxiliary CCD camera. This auxiliary positioning system combined with the regulation of animal stage ensured the precise location of the TPE-TR imaging plane. To examine the superficial TVs, the imaging plane was placed 3 mm beneath the xiphoid [Fig. 4(a)]. As shown by the bright-field image recorded prior to the TPE-TR imaging, a single FOV could cover the thoracoabdominal area [Fig. 4(b)]. Accordingly, the TV network could be visualized clearly with the TPE-TR imaging [Fig. 4(c)]. To facilitate the subsequent kinetics analysis, we define the timing of caudal vein injection (duration, ) as a pseudotime origin.
Results and Discussion
Two-Photon Sensitization Efficiency, Photostability, and Cell Toxicity of the Nanoagent
Figure 5(a) plots the action cross section () of complexes doped in the nanoagent against the near-infrared TPE wavelengths. The is 34.9 GM at 800 nm, which is close to the maximal value of 42.5 at 840 nm. Since a Eu@PMMA-MAA nanoparticle accommodated about 400 molecules, the overall action cross section of the nanoagent was estimated to be at 800 nm. Figure 5(b) shows the time evolution profile of the Eu-luminescence from the Eu@PMMA-MAA nanoparticles sequestered in the liver tissues taken from a mouse, which before sacrifice was administered with the colloidal nanoagent via tail-vein injection. The luminescence intensity dropped by over the time course of 2 h, indicating an excellent photostability of the nanoagent, which is indispensable for the kinetics analysis based on luminescent labeling. Figure 5(c) shows the viability for three different lines of cells including the liver hepatocellular carcinoma (HepG-2), the Hela, and the human embryonic kidney 293 (HEK 293) cells, which were incubated with the colloidal nanoagent at different concentration. It is seen that, after 24 h, the cell viability remained 75% to 90% for different types of cells, indicating the rather low cell toxicity of the nanoagent.73 In addition, no abnormal physiological reaction was observed for the mice during the TPE-TR imaging experiment, proving the negligible biotoxicity of the polymeric nanoagent.
Autofluorescence Suppression and Imaging Depth
It is seen in Fig. 6 that, when the delay time for autofluorescence rejection was set as , the view field was overwhelmed by the autofluorescence. This implies that, under femtosecond laser excitation, the TPE-induced fluorescence from endogenous fluorophores may interfere seriously with the Eu-luminescence images. However, the autofluorescence was drastically suppressed by setting a of 5 to 20 ns. Further prolonging could completely reject the biological autofluorescence, e.g., setting at 50 to 500 ns essentially did not change the image brightness. The negligible decrease in Eu-luminescence intensity in to 500 ns is due to the long persistence of Eu-luminescence (lifetime, 0.84 ms) and the much shorter lived autofluorescence (lifetime, a few tens of nanoseconds). Besides the autofluorescence rejection, the TPE-TR bioimaging is nearly free from the effect of laser heating, which is benefited from the low dose of laser exposure, the spatially decentralized energy distribution, and the effective thermal dissipation in a living body.69 The heating free performance is crucial for deriving the pharmacokinetics information under normal physiological conditions.
In the case of living tissues, the imaging depth and spatial resolution are dependent on the light wavelength, the absorption, and scattering properties of tissues and the brightness of the labeled objects.43,47 As described below, we have estimated the TPE-TR imaging depth based on the anatomical structure and the model of subcutaneous tissue imaging. First, we measured the anatomical depth of the liver in proximity to the xiphoid [Fig. 4(d)]. In viewing the fact that the liver sequestrated with the nanoagent in a high concentration was distinctly visualized by TPE-TR imaging (vide infra), we roughly estimated the imaging depth to be slightly larger than 1 mm. To be more precise, we further prepared a bulky PMMA-MAA particle (projection section, ), which was doped with complexes at a concentration of . This concentration was one-tenth of that of the colloidal solution used for the caudal vein injection, so as to mimic the sequestration of nanoparticles in subcutaneous tissues. We then covered the bulky particle with individual pieces of mouse lash meat of different thicknesses and accordingly recorded the transdermal TPE-TR images [cf. Fig. 7(a)]. It is seen that the original morphology of the labeled particle (control) was retained at an imaging depth of . However, it became blurry yet distinctly recognizable with increasing up to 2.3 mm. Interestingly, the imaging quality does not alter appreciably upon varying the subcutaneous tissue thickness from 1.3 to 2.3 mm [Fig. 7(b)].
Characterization of the TVs as Visualized by In Vivo TPE-TR Imaging
Noninvasive and intravital TPE-TR images of three mice were recorded 5 min after the caudal vein injection of colloidal Eu@PMMA-MAA nanoagent [Figs. 8(a)–8(c), right column]. These images were obtained by placing the scanning plane 3 mm beneath the xiphoids of the mice. Despite the individual difference, each TV network consists of a pair of axial TVs (ATVs) along with the tributary and lateral TVs (LTVs) between the ATV pair. From the cross-sectional distribution profiles of Eu-luminescence intensity [cf. Fig. 8(b), inset], the ATV and LTV diameters, respectively, are determined to be 180 to and 130 to for the mice. These dimensions are in agreement with the TV diameters characterized by the use of anatomy-assisted optical microscopy.24,54 From the axillary to the femoral side, the ATVs become slightly thinner, whereas the spatial distribution of LTV thickness is heterogeneous. In Fig. 8, it is seen by comparing the TPE-TR images that some of the LTVs are interconnected, but the amount and patterns of interconnection are different among the mice. Here, we note that the LTVs are associated with collateral and more tenuous veins, which were not resolved with the present spatial resolution. The polymeric nanoagent in such higher-order veins, including venules and blood capillaries, was in substantially lower concentration compared to that in the principal LTVs, and hence appeared as a weak luminescent background.
For a healthy mouse, it takes about a minute for the tail-vein administered nanoagent to be circulated into the ATVs as demonstrated by a recent study using real-time NIR-II optical bioimaging.56 In this work, the TPE-TR scanning was started 2 to 5 min after tail-vein injection, a time when the concentration of nanoagent in TV bloods increased approximately to the maximum. Afterward, the blood clearance process began, which led to a systematic decay of the luminescent contrast of the TPE-TR images (vide infra). With the general geometric features of TV network in mind, we will come to examine the transport and clearance processes of the polymeric nanoagent.
Noninvasive and Real-Time TPE-TR Imaging of the TV Networks
To investigate the transport and clearance processes of the nanoagent in the peripheral TV blood streams, we placed the scanning plane 3 mm below the xiphoid of a mouse model. Figure 9 shows the real-time TPE-TR images at representative timing. The TPE-TR image at 2.5 min renders a distinct TV network along with a weak and broad Eu-luminescent background, which originated from the sparsely distributed nanoagent in the higher-order venules and blood capillaries. In addition, the femoral artery of the left hind limb was also visualized () despite optical defocusing. As time elapsed, the image of TV network became considerably dimmer at 35 min and eventually became unrecognizable after 60 min. In parallel, the Eu-luminescence in the region immediately beneath the xiphoid, , as well as in the abdominal region, , became gradually stronger. Note that the Eu-luminescent area was the reminiscence of nanoagent accumulation in the liver, because the upper layer of the liver was sensed by TPE-TR imaging with the present imaging depth [cf. Figs. 4(d) and 7]. The high capacity of liver sequestration is to be confirmed by the TPE-TR imaging analysis of dissected organs (vide infra). On the other hand, the relatively weak and long-lasting Eu-luminescence in area is presumably attributed to the retention of nanoagent in the milky spots, which are submillimeter lymphoid tissues consisting of macrophages and other lymphocytes on the omentum majus.74
Herein, we comment on the spatiotemporal evolution of the image patterns in the region (Fig. 9). From 2.5 to 20 min, the asymmetrical and diffusive luminescence image in was slightly upper shifted and transformed from vertical into horizontal orientation. Such spatiotemporal evolution is most likely due to the redistribution of the initially captured nanoagent among different lobes of the liver. We also would like to comment on the axial resolution of TPE-TR imaging. We have recorded, for the same mouse used for collecting the TPE-TR images as shown in Fig. 9, a complementary set of TPE-TR images by focusing on the femoral artery of the left hind limb. This was done in the same TPE-TR experiment by alternatively placing the scanning plane at 3 mm and at 10 mm, i.e., by targeting on the TV network and the femoral artery alternatively. The TPE-TR images focused on the femoral artery are shown in Fig. 10. It is seen that, with a 7-mm displacement of the focusing depth, the TV network visualized clearly in Fig. 9 became nearly invisible, implying that the axial resolution is substantially better than 7 mm. For the peripheral TVs at a submillimeter depth, the lateral resolution is as determined from the cross-sectional distribution of Eu-luminescence intensity of the finest LTVs (cf. Figs. 8 and 9). However, as mentioned in Sec. 3.2, the lateral resolution deteriorates considerably at an imaging depth exceeding 1 mm. To improve the imaging depth and the associated spatial resolution, the adoption of NIR-II luminescence labels and/or confocal imaging geometry would be helpful. At the present stage, our TPE-TR imaging modality with a 1 to 2 mm imaging depth is potentially useful for the detection of subcutaneous tumor models and peripheral vasculatures as OCT does.21,52
The aforementioned transport and clearance dynamics in regions are more clearly illustrated by the Eu-luminescence evolution profiles shown in Fig. 11(a). It is seen that the nanoagent concentration in both and domains increase in 2.5 to 10 min (cf. Fig. 9). This rising phase reflects the process of nanoagent uptake, which is followed by a clearance phase as indicated by the subsequent luminescence decay. The luminescence fluctuation in 20 to 80 min of the kinetics was due to the change in the postural and/or the physiological conditions of the mouse as corroborated by Video 1. On the other hand, the kinetics in Fig. 11(a) shows that the nanoagent concentration in the femoral artery decreased monotonically to a background level.
To quantify the spatiotemporal evolution of the nanoagent in the TV network, both ATVs and LTVs were divided into the segmental regions of interest (ROIs) as shown in Fig. 11(b), and the corresponding Eu-luminescence kinetics curves are shown in Fig. 11(c). Note that some of the luminescence kinetics in Fig. 11(c) as derived from the proximity of and regions exhibit higher background, which are due to apparent high retention efficiency in these regions. The time evolution profile of each local ROI could be well accounted for by a monoexponential decay function:
Figure 11(b) shows the spatial distribution of the clearance time constant along with the corresponding apparent clearance efficiency in parenthesis. It turns out that the ATVs and the LTVs exhibit a similar clearance time constant, 25 to 29 min, which is marginally longer than the clearance time constant of the femoral artery (). In addition, the apparent clearance efficiency, to 90%, is also similar for the entire TV network. Furthermore, the wide spread Eu-luminescence background exhibited a clearance time constant of and an apparent clearance efficiency of as derived from the Eu-luminescence kinetics in the ROIs in proximity of ATVs and LTVs (data not shown). Therefore, the TV network and the wide spread luminescent background all showed a rather high clearance efficiency. On the other hand, analysis of the and the kinetics in Fig. 11(a) derived the clearance time constant (apparent retention efficiency) of (32%) for the liver domain (), and of (38%) for the abdomen domain (). The relatively long clearance time constant and high retention rate of the liver domain () are to be attributed to the liver sequestration effect, whereas similar kinetics behavior of the abdominal domain () are presumably due to the retention of nanoagent in the milky spots on the omentum majus.74
Distribution of the Polymeric Nanoagent in the Internal Organs
The blood flow in the principal TVs and their tributary vessels plays an important role in the transport, clearance, and residence of polymeric nanoagents. To examine the distribution of the nanoagent in the internal organs accompanied the TV-blood clearance, the same mouse used for the in vivo pharmacokinetics analysis was sacrificed immediately after the TPE-TR imaging, and six different organs were accordingly dissected and subjected once again to TPE-TR imaging. The in vitro TPE-TR images are shown in Fig. 12(a). Judging from the Eu-luminescence brightness, the liver obviously exhibits the highest capacity of nanoagent deposition.
Figure 12(b) compares the total Eu-luminescence intensity of the internal organs displayed in Fig. 12(a). It is seen that the capacity of nanoagent sequestration of liver is of the spleen or the lung, of the kidney, and of the heart. Besides the absolute volumes, the vasculature characteristics are considered to be mainly responsible for the different retention capacities of the organs. The rather high uptake capacity of the liver, as well as the spleen despite a smaller volume, can be attributed to the abundant artery and venous vessels, and especially to the sinusoids at their vascular endings.75,76 The lung and the kidney are both rich in blood capillaries, and the difference in the retention capacities is likely attributed to the difference in their bulkiness. The much lower uptake capacity of the heart is obviously due to the much faster blood flow in the cardiac vessels and the relatively less tributary vasculatures. In contrast to the internal organs, no polymeric nanoagent was detected in the brain tissues, which is obviously due to the effective blood–brain barrier. Taken together, our results prove that the liver is the major organ for the nanoagent deposition during the 2-h circulation in blood vasculatures.
This work has made use of a Eu-luminescence-labeled polymeric nanoagent as an angiographic contrast, combined with the recently developed TPE-TR imaging technology, to constitute a powerful modality for noninvasive and real-time imaging analysis of nanoagent pharmacokinetics in the TV networks of living mice. The spatiotemporal evolution of the Eu@PMMA-MAA nanoagent was characterized by an average clearance time constant of in both axial and lateral veins of the TV network, which was comparable to that in the femoral artery. In addition, the real-time TPE-TR imaging analysis showed a clearance time constant of and an apparent retention efficiency of 32% of the nanoagent in the liver domain, which together with the TPE-TR imaging analysis of the dissected internal organs proved that the liver is mainly responsible for the retention of the polymeric nanoagent. This work demonstrates that the TPE-TR modality, which merits in capturing the real-time spatiotemporal details of the Eu-luminescence-labeled nanoagents, is of great potential in the pharmacokinetic analysis of nanoagents in intravital peripheral vasculatures, as well as in the development of target-specific nanocarriers for efficient drug delivery.
This work was supported by the National Natural Science Foundation of China (NSFC Grant Nos. 21227803, 21673288, 21373268, and 21573010). The authors are indebted to Professors Rong-Cheng Han and Yu-Qiang Jiang at the State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for their assistance in measuring the cell toxicity performance of the polymeric nanoagent.