Bioluminescence Imaging of Inflammation in Vivo Based on

Subscriber access provided by AUT Library

Article

Bioluminescence Imaging of Inflammation in Vivo Based on Bioluminescence and Fluorescence Resonance Energy Transfer Using Nanobubbles Ultrasound Contrast Agent Renfa Liu, Jie Tang, Yunxue Xu, and Zhifei Dai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08359 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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

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

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

ACS Nano

Bioluminescence Imaging of Inflammation in Vivo

Based

on

Bioluminescence

and

Fluorescence Resonance Energy Transfer Using Nanobubbles Ultrasound Contrast Agent Renfa Liu#, Jie Tang#, Yunxue Xu, Zhifei Dai* Department of Biomedical Engineering College of Engineering Peking University Beijing 100871, China E-mail: [email protected] #

R. Liu and J. Tang contributed equally to this work.

ACS Paragon Plus Environment

1

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

Page 2 of 24

ABSTRACT Inflammation is an immunological response involved in various inflammatory disorders ranging from neurodegenerative diseases to cancers. Luminol has been reported to detect myeloperoxidase (MPO) activity in inflamed area through a lightemitting reaction. However, this method is limited by low tissue penetration and poor spatial resolution. Here, we fabricated a nanobubble

(NB) doped with two tandem

lipophilic dyes, red-shifting luminol-emitted blue light to near infrared region through a process

integrating

bioluminescence

resonance

energy

transfer

(BRET)

and

fluorescence resonance energy transfer (FRET). This BRET-FRET process caused a 24fold increase in detectable luminescence emission over luminol alone in an inflammation model induced by lipopolysaccharide. In addition, the echogenicity of the BRET-FRET NBs also enables perfused tissue microvasculature to be delineated by contrastenhanced ultrasound imaging with high spatial resolution. Compared with commerciallyavailable ultrasound contrast agent, the BRET-FRET NBs exhibited comparable contrastenhancing capability but much smaller size and higher concentration. This bioluminescence /ultrasound dual-modal contrast agent was then successfully applied for imaging of an animal model of breast cancer. Furthermore, biosafety experiments revealed that multi-injection of luminol and NBs didn’t induce any observable abnormality. By integrating the advantages of bioluminescence imaging and ultrasound imaging, this BRET-FRET system may have the potential to address a critical need of inflammation imaging. Keywords:

luminol, nanobubble, bioluminescence imaging, ultrasound imaging,

inflammation


2

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

ACS Nano

Inflammation is a fundamental aspect of various human diseases including atherosclerosis, and

1, 2

cancers.7-11

Alzheimer’s disease,3, In

the

inflammatory

4

Parkinson’s disease,5 bacterial infections6 process,

the

heme-containing

enzyme

myeloperoxidase (MPO) which is abundantly expressed in neutrophils, monocytes and macrophages plays a key role. MPO catalyzes the production of hypochlorous acid, tyrosyl radicals, aldehydes and hydroxyl radicals.12 These reactive species are cytotoxic, causing oxidative damage of host tissues and thus promoting inflammation. Therefore, accurately detecting and evaluating the MPO activity in vivo is important for the diagnosis and progress monitoring of these inflammatory disorders.13-15 Luminol has been reported to produce blue luminescence in vivo via a MPO-dependent process, thus making optical detection of MPO possible.12 Compared with other imaging modalities, the luminol-based bioluminescence imaging holds several advantages including low cost, high safety, no ionizing radiation and extremely high specificity. However, the short wavelength of light generated by luminol (λmax = 425 nm) makes the detection only feasible to superficial inflammatory foci. A previous research utilizing nearinfrared (NIR) quantum dots to red-shift luminol-emitted light to NIR region has successfully expanded the in vivo detectability of MPO to deep tissues.16, 17 However, the highly toxic metals involved in most quantum dots often lead to concerns of long-term biosafety.18-20 In addition, each acquisition of bioluminescence images usually requires several minutes and the spatial resolution is also very limited. Therefore, developing biocompatible agents to integrate the red-shifted bioluminescence imaging with other imaging modalities with high temporal and spatial resolution is a promising way for accurately detecting MPO activity in vivo. Compared with other diagnostic imaging techniques, ultrasound imaging is unique which is characterized with low cost, real-time, and high spatial resolution.21,22 With the use of ultrasound contrast agents, the ultrasound imaging can delicately delineate the microvasculature in deep tissues.23 However, the ultrasound imaging is not specific for inflammation imaging. Several groups have explored to combine ultrasound imaging with optical imaging to overcome the innate limitations of ultrasound imaging.24-29 However, conventional fluorescence imaging, when used for inflammation imaging, usually lacks of specificity and is characterized with high auto-fluorescence.30-35 By providing MPO-


3

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

dependent

imaging

with

superior

Page 4 of 24

specificity,

the

red-shifted

luminol-based

bioluminescence imaging is expected to be a promising candidate for combination with ultrasound imaging.26, 36-38 Herein, we sought to fabricate luminescing gas-encapsulated nanobubbles (NBs) as an energy transfer relay that integrates bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) to red-shift luminol-emitted blue light to NIR light, enabling MPO-dependent inflammation imaging in deep tissues. Luminol can freely penetrate into tissues to react with MPO, generating blue light and activing NBs through the BRET-FRET process (Figure 1). This BRET-FRET process makes

the

inflammation

detected

through

highly

sensitive

MPO-dependent

bioluminescence imaging in the NIR region. Furthermore, the echogenicity of NBs enables the inflamed region detected by optical imaging to be delineated by contrastenhanced ultrasound imaging with high spatial resolution. Therefore, this NB combined with luminol provides a non-invasive and highly sensitive imaging method for MPOassociated inflammation. RESULTS AND DISCUSSION To build up the BRET-FRET system, two tandem dyes were carefully chosen to allow for

efficient

energy

tetramethylindocarbocyanine

transfer

(Figure

perchlorate

(DiI)

2A).

1,1'-dioctadecyl-3,3,3',3'-

and

1,1'-dioctadecyl-3,3,3',3'-

tetramethylindodicarbocyanine perchlorate (DiD) were employed because of their lipophilic structure for efficient incorporation in the lipid membrane of NBs (Figure 1A). The NBs were fabricated using a modified mechanical vibration technique, which produces gas-filled bubbles by shaking the dye-doped lipid nanoparticles (LNPs) under inert C3F8 gas atmosphere. To make the bubbles produced mainly in the nano scale, not in the micro scale, the phospholipid concentration in LNPs was increased to as high as 10 μmol/mL. To obtain the highest fluorescence, the concentration of DiI alone in LNPs was firstly optimized. With the concentration of DiI increasing from 20 μM to 100 μM, the absorbance gradually increased while the fluorescence reached the maximum at the concentration of 60 μM (Figure 2B&S1A). Setting the total concentration of DiI and DiD as 60 μM, the optimal molar ratio of DiI/DiD was selected as 7:3 to get highest fluorescence intensity at 670 nm (Figure 2C&S1B). To further enhance the efficiency of


4

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

ACS Nano

energy transfer, the total concentration of DiI and DiD was varied from 60 μM to 180 μM and the optimal concentration was found to be 140 μM (Figure 2D&S1C). Therefore, the DiI-DiD LNPs with a concentration of DiI at 98 μM and DiD at 42 μM were used for further applications. The DiI-DiD NBs were then fabricated through mechanical agitation under atmosphere of C3F8. NBs doped with DiI or DiD alone at same concentration were also fabricated as control (Figure S1D&E). DiI-DiD NBs show a pink appearance in solution and flowed up as a white cake after standing for several minutes. After NBs formation, there was no significant changes in the fluorescence spectra (Figure 2E). Through the nanoparticle tracking analysis, the concentration of DiI-DiD NBs was measured to be 1.44 × 1013/mL and the mean diameter was ~178.8 nm while the size of DiI-DID LNP was ~112 nm (Figure 2F). The increased size is a result of gas encapsulation. The gas encapsulated in DiI-DiD NBs measured with the coulter counter was 72 μL/mL, which was nine times higher than that in Sonovue, a commercial ultrasound contrast agent that is widely used in clinic. Under the confocal fluorescence microscope, the fluorescence of DiI and DiD in DiI-DiD NBs perfectly merged together (Figure 2G), indicating DiI was located in the same NBs with DiD, which was beneficial for the optimal FRET between DiI and DiD. To determine whether the BRET-FRET process could occur between DiI-DiD NBs and luminol in a no-conjugated state, we set up in vitro MPO reactions with or without DiI-DiD NBs and detected the luminescence with the IVIS Spectrum imaging system. Upon stimulation with phorbol myristate acetate (PMA), neutrophils produce various reactive oxygen species and release MPO, which induce the luminol-dependent photoemission39. In the presence of DiI-DiD NBs, the total luminescence signal detected slightly decreased (Figure S2A&B), which is due to the energy loss during the energy transfer. Significantly higher luminescence signal was detected at 670 nm and 710 nm (Figure 2H&S2C) while the signal at 520 nm was reduced in presence of DiI-DiD NBs, supporting our hypothesis that the light generated by luminol could be red-shifted to NIR region through the BRETFRET process between DiI-DiD NBs and luminol. The depth of signal penetration of luminol + DiI-DiD NBs was assessed in vitro with a tissue-like phantom containing hemoglobin and gelatin

40, 41

in direct comparison with luminol alone. The luminescence


5

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

Page 6 of 24

signal of luminol + DiI-DiD NBs was observable at 9 mm of gel depth with signal intensities significantly greater than that of luminol alone (Figure 2I&S2D). With a high concentration of gas encapsulated and size in nanoscale, the DiI-DiD NB is expected to be an excellent ultrasound contrast agent. The contrast-enhancing capability of DiI-DiD NBs was evaluated with a clinically-applicable ultrasound imaging machine. The DiI-DiD NB shows comparable contrast enhancement compared with Sonovue (Figure 2J). At a gas concentration as low as 0.5 μL/L, the DiI-DiD NBs provided a signal of 28.89±1.52 dB, while the Sonovue provided 20.59±3.72 dB (Figure S3). When increasing the gas concentration to 10 μL/L, the DiI-DiD NBs generated a signal of 40.59±1.59 dB in contrast to 37.41±0.45 dB by Sonovue. After intravenous injection, the fluorescence signal and ultrasound signal generated by DiI-DiD NBs in blood didn’t change significantly in 30 min (Figure 2K&S4&S5), providing enough time to serve as energy transfer relay for BRET-FRET bioluminescence imaging of MPO. The capability of luminol + DiI-DiD NBs for MPO imaging was further evaluated in an inflammation model induced by intraperitoneal injection of lipopolysaccharide (LPS), which increases MPO activity in several organs, including heart, lung, spleen, etc. (Figure S6). The parameters involved in the bioluminescence imaging by luminol + DiI-DiD NBs were optimized. Setting the dose of DiI-DiD NBs NBs as 100 μL/mouse, the luminol at the dose of 4 mg/mouse was found to get the highest luminescence signal (Figure 3A&S7A). The optimal dose of DiI-DiD NBs was also found to be 100 μL/mouse when keeping the dose of luminol at 4 mg/mouse (Figure 3B&S7B). After co-injecting 4 mg luminol and 100 μL DiI-DiD NBs per mouse, the bioluminescence images were obtained every 2 min. As shown in Figure 3C&S8, the highest luminescence signal was detected at 6 min post luminol + DiI-DiD NBs injection. Compared with injecting DiI-DiD NBs and luminol separately (NBs was injected 5 min post luminol injection) or injecting DiI-DiD LNPs and luminol together, injecting luminol and DiI-DiD NBs together get the highest signal (Figure S9). Taken together, the optimal luminescence images can be obtained at ~6 min after co-injecting 4 mg luminol and 100 μL DiI-DiD NBs per mouse. We further determined whether the luminescence imaging by luminol + DiI-DiD NBs occurred via BRET-FRET process in a MPO-dependent manner. As expected, minimal luminescence was detected when injecting luminol alone, while luminol + DiI-DiD NBs


6

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

ACS Nano

caused a nearly 24-fold increase in detectable luminescence emission over luminol alone (Figure 3D&E), which is comparable to bioluminescence imaging obtained by luminol combined with NIR quantum dots.16 On the contrary, luminol + DiI NBs and luminol + DiD NBs resulted in only 2-fold and 7-fold increase in luminescence emission, respectively. In addition, pretreating the animal with the irreversible MPO inhibitor 4-ABAH resulted in significantly reduced luminescence emission, demonstrating that the luminescence emission by luminol + DiI-DiD NBs was dependent on the MPO activity. In accordance with the in vitro results, a very high luminescence signal was detected around 670 nm and 710 nm (Figure 3F&S10), indicating that the enhanced luminescence was the result of red-shifted signal by the BRET-FRET process of luminol and DiI-DiD NBs. To further evaluate if the bioluminescence signal through the BRET-FRET process could reflect the degree of inflammation, we established an inflammation model by subcutaneously injecting the potent protein kinase C activator PMA. The degree of inflammation is related to the amount of PMA injected in one site. 10 μL PMA solution is locally injected to the leg of mice. After 3 h, luminol and DiI-DiD NBs were utilized to evaluate the inflammation. As shown in Figure S11, the luminescence signal is linearly related to the concentration of PMA in the range from 0.2 mg/mL to 1 mg/mL. Some malignant tumors have been shown to attract infiltration of neutrophils which secret large quantities of active MPO during their progression.42-44 We further evaluated the feasibility of DiI-DiD NBs for dual-mode imaging of MPO activity in triple-negative breast cancer. The 4T1-luc xenograft model was established through intramuscular injection of 4T1-luc cells into the right leg of Balb/c nude mice. Two weeks post tumorcell injection, luminescence signal can be detected in the right leg with luminol + DiI-DiD NBs (Figure 4A), indicating the MPO activity in these lesions. Using the luciferase gene engineered into the tumor cells, the bioluminescence imaging with luciferin also show the development of tumor in the same site(Figure 4B). Contrast-enhanced ultrasound imaging was further conducted to the selected region indicated by the luminescence imaging. The results clearly show the perfusion of NBs in the leg and a circular area with higher vasculature density were delineated (Figure 4C), which is further confirmed to be tumor with H&E staining (Figure 4D).


7

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

Page 8 of 24

Furthermore, we evaluated the biosafety of luminol and DiI-DiD NBs to mice in detail. 20 healthy Balb/c mice were randomly divided into two groups. The mice were injected with luminol + DiI-DiD NBs at the optimized dose or equal volume of saline every 3 days for five injections per mice in total. Throughout this process, all the mice survived in good condition. No body weight change was observed during the treatment (Figure 5A). At the 18th day post the first injection, blood samples were collected for a comprehensive analysis of blood chemistry and whole blood count. As shown in Figure 5B&5C&Table S1, no statistic difference was observed in any of the parameters that we studied. Subsequently, major organs including heart, liver, spleen, lung, kidney and thymus were collected and weighed. The organ weight didn’t show any significant difference between these two groups (Figure 5D). The histological examination revealed normal appearance and no noticeable abnormality including inflammation, cell necrosis, and apoptosis in all the organs studied (Figure 5E). In this BRET-FRET system, luminol react with MPO to generate blue light to activate DiI-DiD NBs through BRET. Considering that the Forster distance between luminol and DiI is not likely to exceed 10 nm, luminol, MPO and DiI-DiD NBs should be in the same place to make BRET-FRET happen. As shown in the immunohistochemistry images of major organs after LPS challenging (Figure S5), MPO exists in both intravascular and extravascular tissues. Intravenously injected NBs and luminol could interact with intravascular MPO very easily. For extravascular MPO, although luminol could penetrate the blood vessels, it is not clear if DiI-DiD NBs could leak out of the vessels to make extravascular BRET-FRET proceed. 10 μL PMA solution at the concentration of 1 mg/mL was injected into the right leg of Balb/c mice to induce inflammation. As expected, higher luminescence signal could be detected with luminol + DiI-DiD NBs in the PMA-treated side (Figure S12A). The mice were then sacrificed and blood was removed by saline perfusion. With the fluorescence imaging, significant higher fluorescence signal can be detected in the PMA-treated side (Figure S12B), indicating that DiI-DiD NBs could accumulate in the extravascular space and the extravascular BRET-FRET probably also occurred. Considering the average diameter of DiI-DiD NBs is ~178.8 nm, which is difficult to leak out of the vasculature purely relying on the intrinsic enhanced permeability and retention (EPR) effect, the nanomaterials-induced endothelial leakiness (NanoEL) effect


8

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

ACS Nano

might be involved in this process. With NanoEL, nanomaterials can lead to the formation of micrometer-sized gaps in the blood vessel endothelial walls, which is a phenomenon found in a lot of nanomaterials as reported by David et al.45-47 Therefore, with luminol and DiI-DiD NBs, both intravascular and extravascular BRET-FRET were utilized for MPO detection. Admittedly, in this system, using luminol and fluorescent dyes in a nonconjugated state will reduce the BRET-FRET efficiency because BRET-FRET only occurs when randomly-distributed luminol and fluorescent dyes meet with MPO. A more efficient way might be conjugating luminol with fluorescent dyes as reported by Xu et al.48 However, conjugating luminol with organic dyes is involved with complicated chemical synthesis and might cause some additional unexpected safety issues, such as producing a high level of ROS.48 In fact, using luminol and organic dyes in nonconjugated state as in our system is enough for sensitive MPO imaging, but more facile to prepare and safer. CONCLUSIONS In conclusion, we demonstrated a luminescing NB employing an energy transfer relay that integrates BRET and FRET to red-shift luminol-emitted blue light to red light, enabling MPO-dependent inflammation imaging in deep tissues. The DiI-DiD NBs caused a 24fold increase in detectable luminescence emission over luminol alone in mice challenged by LPS. In addition, the NBs can also be utilized as an ultrasound contrast agent. Compared with commercially-available microbubbles, the DiI-DiD NBs shows comparable ultrasound contrast-enhancing capability but smaller size and higher concentration. In an animal model of breast cancer, the DiI-DiD NBs combined with luminol clearly shows the MPO activity in the tumor, while the contrast-enhanced ultrasound imaging delineates the anatomical structure and vasculature development of the inflamed region indicated by bioluminescence imaging. In addition, multi-injection of luminol and the NBs didn’t cause any observable toxicity. This methodology provides a non-invasive and highly sensitive means for accurate visualization of inflammatory diseases. MATERIALS AND METHODS Materials Luminol sodium salt, 4-aminobenzoic hydrazide (4-ABAH), gelatin, agar, intralipid glycerol and propylene glycol were obtained from Sigma Aldrich. HSPC and DSPE-PEG


9

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

Page 10 of 24

2000 were obtained from A.V.T. (Shanghai) Pharmaceutical Co., Ltd. DiI and DiD were purchased from Fanbo Biochemicals. Lipopolysaccharide (LPS) was bought from Shanghai Yuanye Biotechnology Co., Ltd. C3F8 gas was obtained from Zhengzhou Xingdao Chemical Technnology Co., Ltd. All chemicals are of analytical grade and used directly without further purification. Millipore quality deionized water (DI water) (resistivity is 18.2 MΩ·cm) was utilized in all experiments. Preparation and characterization of NBs The dye-doped lipid nanoparticles were first prepared. HSPC and DSPE-PEG2000 were dissolved in ethanol. Then HSPC and DSPE-PEG2000 were mixed at the molar ratio of 95:5. Certain amount of DiI and/or DiD in ethanol were also added. The ethanol was then removed by the rotary evaporator. Then aqueous solution (PBS: glycerol: propylene glycol = 8:1:1) was added resulting into the final lipid concentration at 10 μmol/mL. After heated at 70 °C, the solution was sonicated with a sonicator (Misonix Sonicator 4000, 50% power, 3s on and 2s off) for 5 min to get the dye-doped LNPs. To get the dye-doped NBs, 1 mL lipid nanoparticles solution was added to a 2-mL vial and the headspace of the vial was filled with C3F8 gas. After agitated with the amalgamator for 45s, the dye-doped NBs were obtained. The size and concentration of NBs was measured with Coulter counter (Multisizer 4e) and the Malvern Panalytical NanoSight NS300 Instrument. The absorption spectra were recorded using an UV-vis-NIR spectrometer (Evolution 220, Thermo Scientific), while the fluorescence spectra were determined on a Shimadzu RF6000 fluorescence spectrometer. The confocal images were taken on a Nikon A1RSi+ confocal microscope. The bioluminescence images were obtained with an IVIS Spectrum system (Perkin Elmer). The contrast-enhanced ultrasound imaging was conducted using a DC-8 ultrasound system equipped with a L12-3E linear-array probe (Mindray) (Frequency: 4.4 MHz; Mechanical index: 0.1). Phantom experiments To evaluate the echogenicity of the NBs, tissue-like phantom was prepared using a gel made from distilled water containing agar

for jellification (1.3% w/w) and intralipid

emulsion for light diffusion (6% v/v). The shape of the phantom is shown in Figure S13A.


10

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

ACS Nano

Each sample was repeated for at least four times. The saline was used as a negative control. To evaluate the optical penetration of the bioluminescence imaging, a hemoglobincontaining phantom was prepared. In brief, gelatin was suspended at the concentration of 10% (w/v) in tris-buffered saline (TBS) containing 50 mM Tris HCl (pH 7.4), 150 mM NaCl, and 0.1% w/v NaN3. The mixture is then warmed to 40°C with constant shaking until completely dissolved. The solution was then cooled to 25°C and the desired amounts of bovine hemoglobin (170 μM) and intralipid (1%v/v) were added with constant stirring. The solution was then poured into rectangular molds with a thickness of 1.5 mm and let set overnight at 4 °C. In vitro MPO assay Fresh blood was collected from a healthy New Zealand rabbit. The neutrophils were then separated from blood with a neutrophil separation kit (Solarbio, P4180) according to the manufacturer’s instructions. The obtained neutrophils were suspended in DMEM cell culture media and the concentration is quantified with a cytometry. In a 96-well black plate, an MPO activity assay was set up as follows: 1×107 neutrophils in 110 µl DMEM media, 20 µl of 40mg/ml luminol and 10 µl of NBs were added in to each well. 20 μl PMA (40 µM) is then added to start the reaction. The photoemission reaches the maximum at ~30 min and remains stable for over 30 min. So the plate was assayed using the IVIS Spectrum instrument with a 5-min exposure time at 30-60min post PMA stimulation. For the optical penetration assay, 2, 4 and 6 sheets of hemoglobin-containing phantom were placed on the top of the plate to get the penetration depth of 3 mm, 6 mm and 9 mm, separately (Figure S13B) Animals Balb/c mice were purchased from Beijing Vital River Laboratories and all animal experiments were conducted under protocols approved by Peking University Laboratory Animal Center. Bioluminescence imaging in LPS-challenged mice The Balb/c mice were intraperitoneally injected with 100 μL LPS (1mg/mL) 3h before the experiments. During the experiments, the mice were injected retro-orbitally with a


11

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

Page 12 of 24

mixture of luminol and DiI-DiD NBs. The bioluminescent imaging were then started at certain time points with a 1-min exposure time. For MPO-inhibition experiments, 4-ABAH (100 μL/mouse, 13 mM) was injected 15 min before the experiments. Immunohistochemistry Fresh tissue was embedded in optimal cutting temperature (OCT) compound (TissueTek), frozen on liquid nitrogen and stored at -80 °C until used. Sections were fixed in methanol/acetone and then blocked (2 h, at RT) using 10% (v/v) goat serum and 1% BSA in TBS. Immunohistochemical staining was carried out using following antibodies: AntiMyeloperoxidase (Abcam, ab9535, 1:1000) overnight at 4 °C. The sample was then incubated with a secondary antibody (Abcam, ab6721, 1:1000) (1 h, at RT). The antibody was then detected with a DAB staining kit (Solarbio, DA1010). The nucleus was stained with hematoxylin. Samples were imaged using a ZEISS Axio Scan.Z1 slider scanner. Dual-modal imaging of tumor The tumor model was established through intramuscular injection of tumor cells (5×105 4T1-luc cells/mouse) into the left leg of Blab/c mice. 2 weeks post tumor-cell injection, bioluminescence imaging with luminol + DiI-DiD NBs was conducted. After the luminescence emission disappeared, luciferin (0.75 mg/mouse) was injected to determine the development of tumor metastatic foci. After that, the contrast-enhanced ultrasound imaging was used to determine vasculature changes in selected area indicated by luminescence imaging. The dual-modal imaging was repeated with 4 mice. Biosafety assay 20 Balb/c mice were randomly divided into two groups. The mice were injected with luminol + DiI-DiD NBs (100 μL 40mg/mL luminol and 100 μL DiI-DiD NBs) or 200 μL saline at the 0, 3, 6, 10, 14 day. At 18th days, blood samples were collected from the orbit for whole blood count and blood chemistry assay. The mice were then sacrificed. Major organs including heart, liver, spleen, lung, kidney and thymus were collected, weighed and stained using standard hematoxylin & eosin (H&E) staining protocol. ASSOCIATED CONTENT Supporting Information. The Supporting information is available free of charge.


12

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

ACS Nano

Figure S1-S13 showing absorption spectra, luminescence images of in vitro MPO assay, quantitative results of phantom experiments of ultrasound imaging, luminescence images of blood samples after NBs injection, immunohistochemistry images of major organs after LPS challenging, luminescence images of LPS- and PMA- challenged mice, and whole blood count results of blood samples for biosafety assay. The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID Renfa Liu: 0000-0001-6426-4126 Zhifei Dai: 0000-0002-8283-3190 Author Contributions R. Liu and J. Tang contributed equally to this work. ACKNOWLEDGMENT This contribution was ﬁnancially supported by National Key Research and Development Program of China (No. 2016YFA0201400), Beijing Natural Science Foundation - Haidian original innovation joint fund (No. 17L20170), National project for research and development of major scientific instruments (No. 81727803) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421004). REFERENCES 1.

Daugherty, A.; Dunn, J. L.; Rateri, D. L.; Heinecke, J. W., Myeloperoxidase, A

Catalyst for Lipoprotein Oxidation, is Expressed in Human Atherosclerotic Lesions. J. Clin. Invest. 1994, 94, 437-444. 2.

Aanei, I. L.; Huynh, T.; Seo, Y.; Francis, M. B., Vascular Cell Adhesion Molecule-

Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques. Bioconjug. Chem. 2018, 29, 2526-2530. 3.

Reynolds, W. F.; Rhees, J.; Maciejewski, D.; Paladino, T.; Sieburg, H.; Maki, R.

A.; Masliah, E., Myeloperoxidase Polymorphism is Associated with Gender Specific Risk for Alzheimer's Disease. Exp. Neurol. 1999, 155, 31-41.


13

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

4.

Page 14 of 24

Sharma, R.; Kim, S.-Y.; Sharma, A.; Zhang, Z.; Kambhampati, S. P.; Kannan, S.;

Kannan, R. M., Activated Microglia Targeting Dendrimer–Minocycline Conjugate as Therapeutics for Neuroinflammation. Bioconjug. Chem. 2017, 28, 2874-2886. 5.

Choi, D.-K.; Pennathur, S.; Perier, C.; Tieu, K.; Teismann, P.; Wu, D.-C.; Jackson-

Lewis, V.; Vila, M.; Vonsattel, J.-P.; Heinecke, J. W., Ablation of the Inflammatory Enzyme Myeloperoxidase Mitigates Features of Parkinson's Disease in Mice. J. Neurosci. 2005, 25, 6594-6600. 6.

Pagoto, A.; Stefania, R.; Garello, F.; Arena, F.; Digilio, G.; Aime, S.; Terreno, E.,

Paramagnetic Phospholipid-Based Micelles Targeting VCAM-1 Receptors for MRI Visualization of Inflammation. Bioconjug. Chem. 2016, 27, 1921-1930. 7.

Ikeda, A.; Doi, Y.; Nishiguchi, K.; Kitamura, K.; Hashizume, M.; Kikuchi, J.-i.; Yogo,

K.; Ogawa, T.; Takeya, T., Induction of Cell Death by Photodynamic Therapy with WaterSoluble Lipid-Membrane-Incorporated [60] Fullerene. Org. Biomol. Chem. 2007, 5, 11581160. 8.

Fletcher, N.; Memaj, I.; Morris, R.; Saed, G., Targeting Myeloperoxidase Enhances

Apoptosis in Chemoresistant Epithelial Ovarian Cancer Cells by Reversing SNitrosylation of Caspase-3. Gynecol. Oncol. 2018, 149, 73. 9.

Chu, D.; Dong, X.; Shi, X.; Zhang, C.; Wang, Z., Neutrophil-Based Drug Delivery

Systems. Adv. Mater. 2018, 30, 1706245. 10.

Yang, Z.; Dai, Y.; Yin, C.; Fan, Q.; Zhang, W.; Song, J.; Yu, G.; Tang, W.; Fan, W.;

Yung, B. C., Activatable Semiconducting Theranostics: Simultaneous Generation and Ratiometric Photoacoustic Imaging of Reactive Oxygen Species in Vivo. Adv. Mater. 2018, 30, 1707509. 11.

Xiao, D.; Jia, H. Z.; Zhang, J.; Liu, C. W.; Zhuo, R. X.; Zhang, X. Z., A Dual-

Responsive Mesoporous Silica Nanoparticle for Tumor-Triggered Targeting Drug Delivery. Small 2014, 10, 591-598. 12.

Gross, S.; Gammon, S. T.; Moss, B. L.; Rauch, D.; Harding, J.; Heinecke, J. W.;

Ratner, L.; Piwnica-Worms, D., Bioluminescence Imaging of Myeloperoxidase Activity in Vivo. Nat. Med. 2009, 15, 455-461.


14

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

ACS Nano

13.

Chen, J. W.; Querol Sans, M.; Bogdanov Jr, A.; Weissleder, R., Imaging of

Myeloperoxidase in Mice by Using Novel Amplifiable Paramagnetic Substrates. Radiology 2006, 240, 473-481. 14.

Chen, J. W.; Breckwoldt, M. O.; Aikawa, E.; Chiang, G.; Weissleder, R.,

Myeloperoxidase-Targeted Imaging of Active Inflammatory Lesions in Murine Experimental Autoimmune Encephalomyelitis. Brain 2008, 131, 1123-1133. 15.

Rodríguez, E.; Nilges, M.; Weissleder, R.; Chen, J. W., Activatable Magnetic

Resonance Imaging Agents for Myeloperoxidase Sensing: Mechanism of Activation, Stability, and Toxicity. J. Am. Chem. Soc. 2009, 132, 168-177. 16.

Zhang, N.; Francis, K. P.; Prakash, A.; Ansaldi, D., Enhanced Detection of

Myeloperoxidase Activity in Deep Tissues through Luminescent Excitation of NearInfrared Nanoparticles. Nat. Med. 2013, 19, 500-505. 17.

Miao, Q.; Pu, K., Organic Semiconducting Agents for Deep-Tissue Molecular

Imaging: Second Near-Infrared Fluorescence, Self-Luminescence, and Photoacoustics. Adv. Mater. 2018, 1801778. 18.

Zhang, Y.; Hong, G.; Zhang, Y.; Chen, G.; Li, F.; Dai, H.; Wang, Q., Ag2S Quantum

Dot: A Bright and Biocompatible Fluorescent Nanoprobe in the Second Near-Infrared Window. ACS Nano 2012, 6, 3695-3702. 19.

Popow-Stellmaszyk, J.; Bajorowicz, B.; Malankowska, A.; Wysocka, M.; Klimczuk,

T.; Zaleska-Medynska, A.; Lesner, A., Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity. Bioconjug. Chem. 2018, 29, 1576-1583. 20.

Hu, L.; Sun, Y.; Li, S.; Wang, X.; Hu, K.; Wang, L.; Liang, X.-j.; Wu, Y.,

Multifunctional Carbon Dots with High Quantum Yield for Imaging and Gene Delivery. Carbon 2014, 67, 508-513. 21.

Ke, H.; Wang, J.; Dai, Z.; Jin, Y.; Qu, E.; Xing, Z.; Guo, C.; Yue, X.; Liu, J., Gold-

Nanoshelled Microcapsules: A Theranostic Agent for Ultrasound Contrast Imaging and Photothermal Therapy. Angew. Chem. Int. Ed. 2011, 123, 3073-3077. 22.

Slagle, C. J.; Thamm, D. H.; Randall, E. K.; Borden, M. A., Click Conjugation of

Cloaked Peptide Ligands to Microbubbles. Bioconjug. Chem. 2018, 29, 1534-1543.


15

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

23.

Page 16 of 24

Errico, C.; Pierre, J.; Pezet, S.; Desailly, Y.; Lenkei, Z.; Couture, O.; Tanter, M.,

Ultrafast Ultrasound Localization Microscopy for Deep Super-Resolution vascular imaging. Nature 2015, 527, 499-502. 24. Z.,

Ke, H.; Xing, Z.; Zhao, B.; Wang, J.; Liu, J.; Guo, C.; Yue, X.; Liu, S.; Tang, Z.; Dai, Quantum-Dot-Modified

Microbubbles

with

Bi-Mode

Imaging

Capabilities.

Nanotechnology 2009, 20, 425105. 25.

Wang, Y. M.; Judkewitz, B.; DiMarzio, C. A.; Yang, C., Deep-Tissue Focal

Fluorescence Imaging with Digitally Time-Reversed Ultrasound-Encoded Light. Nat. Commun. 2012, 3, 928. 26.

Bozhko, D.; Osborn, E. A.; Rosenthal, A.; Verjans, J. W.; Hara, T.; Kellnberger, S.;

Wissmeyer, G.; Ovsepian, S. V.; McCarthy, J. R.; Mauskapf, A., Quantitative Intravascular Biological Fluorescence-Ultrasound Imaging of Coronary and Peripheral Arteries in Vivo. Eur. Heart. J. Cardiovasc. Imaging. 2016, 18, 1253-1261. 27.

Bosca, F.; Bielecki, P. A.; Exner, A. A.; Barge, A., Porphyrin-Loaded Pluronic

Nanobubbles: A New US-Activated Agent for Future Theranostic Applications. Bioconjug. Chem. 2018, 29, 234-240. 28.

Wilson, K.; Homan, K.; Emelianov, S., Biomedical Photoacoustics beyond Thermal

Expansion Using Triggered Nanodroplet Vaporization for Contrast-Enhanced Imaging. Nat. Commun. 2012, 3, 618. 29.

Wang, J. X.; Lin, C. Y.; Moore, C.; Jhunjhunwala, A.; Jokerst, J. V., Switchable

Photoacoustic Intensity of Methylene Blue via Sodium Dodecyl Sulfate Micellization. Langmuir 2018, 34, 359-365. 30.

Guo, M.; Huang, J.; Deng, Y.; Shen, H.; Ma, Y.; Zhang, M.; Zhu, A.; Li, Y.; Hui, H.;

Wang, Y., pH-Responsive Cyanine-Grafted Graphene Oxide for Fluorescence Resonance Energy Transfer-Enhanced Photothermal Therapy. Adv. Funct. Mater. 2015, 25, 59-67. 31.

Fan, W.; Shen, B.; Bu, W.; Chen, F.; Zhao, K.; Zhang, S.; Zhou, L.; Peng, W.; Xiao,

Q.; Xing, H., Rattle-Structured Multifunctional Nanotheranostics for Synergetic Chemo/Radiotherapy and Simultaneous Magnetic/Luminescent Dual-Mode Imaging. J. Am. Chem. Soc. 2013, 135, 6494-6503.


16

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

ACS Nano

32.

Chen, Y.; Qiao, L.; Ji, L.; Chao, H., Phosphorescent Iridium (III) Complexes as

Multicolor Probes for Specific Mitochondrial Imaging and Tracking. Biomaterials 2014, 35, 2-13. 33.

Zhu, A.; Miao, K.; Deng, Y.; Ke, H.; He, H.; Yang, T.; Guo, M.; Li, Y.; Guo, Z.;

Wang, Y., Dually pH/Reduction-Responsive Vesicles for Ultrahigh-Contrast Fluorescence Imaging and Thermo-Chemotherapy-Synergized Tumor Ablation. ACS Nano 2015, 9, 7874-7885. 34.

Lin, L.; Liang, X.; Xu, Y.; Yang, Y.; Li, X.; Dai, Z., Doxorubicin and Indocyanine

Green Loaded Hybrid Bicelles for Fluorescence Imaging Guided Synergetic Chemo/Photothermal Therapy. Bioconjug. Chem. 2017, 28, 2410-2419. 35.

Hameed, S.; Chen, H.; Irfan, M.; Bajwa, S. Z.; Khan, W. S.; Baig, S. M.; Dai, Z.,

Fluorescence Guided Sentinel Lymph Node Mapping: From Current Molecular Probes to Future Multimodal Nanoprobes. Bioconjug. Chem. 2018, 30, 13-28. 36.

Liao, A.-H.; Li, Y.-K.; Lee, W.-J.; Wu, M.-F.; Liu, H.-L.; Kuo, M.-L., Estimating the

Delivery Efficiency of Drug-Loaded Microbubbles in Cancer Cells with Ultrasound and Bioluminescence Imaging. Ultrasound. Med. Biol. 2012, 38, 1938-1948. 37.

Bal, G.; Schotland, J. C., Ultrasound-Modulated Bioluminescence Tomography.

Phys. Rev. E. 2014, 89, 031201. 38.

Huynh, N. T.; Hayes-Gill, B. R.; Zhang, F.; Morgan, S. P., Ultrasound Modulated

Imaging of Luminescence Generated within A Scattering Medium. J. Biomed. Opt. 2013, 18, 020505. 39.

Suematsu, M.; Oshio, C.; Miura, S.; Suzuki, M.; Houzawa, S.; Tsuchiya, M.,

Luminol-Dependent Photoemission from Single Neutrophil Stimulated by Phorbol Ester and Calcium Ionophore: Role of Degranulation and Myeloperoxidase. Biochem. Biophys. Res. Commun. 1988, 155, 106-111. 40.

Degrand, A. M.; Lomnes, S. J.; Lee, D. S.; Pietrzykowski, M.; Ohnishi, S.; Morgan,

T.; Gogbashian, A.; Laurence, R. G.; Frangioni, J. V., Tissue-Like Phantoms for NearInfrared Fluorescence Imaging System Assessment and the Training of Surgeons. J. Biomed. Opt. 2006, 11, 014007.


17

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

41.

Page 18 of 24

Xiong, L.; Shuhendler, A. J.; Rao, J., Self-Luminescing BRET-FRET Near-Infrared

Dots for in Vivo Lymph-Node Mapping and Tumour Imaging. Nat. Commun. 2012, 3, 1193. 42.

Tao, K.; Fang, M.; Alroy, J.; Sahagian, G. G., Imagable 4T1 Model for the Study of

Late Stage Breast Cancer. BMC Cancer 2008, 8, 228. 43.

Wculek, S. K.; Malanchi, I., Neutrophils Support Lung Colonization of Metastasis-

Initiating Breast Cancer Cells. Nature 2015, 528, 413-417. 44.

Coffelt, S. B.; Wellenstein, M. D.; de Visser, K. E., Neutrophils in Cancer: Neutral

No More. Nat. Rev. Cancer. 2016, 16, 431-446. 45.

Setyawati, M.; Tay, C. Y.; Chia, S.; Goh, S.; Fang, W.; Neo, M.; Chong, H. C.; Tan,

S.; Loo, S. C. J.; Ng, K., Titanium Dioxide Nanomaterials Cause Endothelial Cell Leakiness by Disrupting the Homophilic Interaction of VE–Cadherin. Nat. Commun. 2013, 4, 1673. 46.

Wang, J.; Zhang, L.; Peng, F.; Shi, X.; Leong, D. T., Targeting Endothelial Cell

Junctions with Negatively Charged Gold Nanoparticles. Chem. Mater. 2018, 30, 37593767. 47.

Peng, F.; Setyawati, M. I.; Tee, J. K.; Ding, X.; Wang, J.; Nga, M. E.; Ho, H. K.;

Leong, D. T., Nanoparticles Promote in Vivo Breast Cancer Cell Intravasation and Extravasation by Inducing Endothelial Leakiness. Nat. Nanotechnol. 2019, 14, 279-286. 48.

Xu, X.; An, H.; Zhang, D.; Tao, H.; Dou, Y.; Li, X.; Huang, J.; Zhang, J., A Self-

Illuminating Nanoparticle for Inflammation Imaging and Cancer Therapy. Sci. Adv. 2019, 5, eaat2953.


18

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

ACS Nano

Figure 1. Schematic illustration of luminol + DiI-DiD NBs for dual-modal imaging of MPO activity. (A) DiI and DiD were co-incorporated in the lipid layer of the NBs. MPO mainly expressed in neutrophils and macrophages can interact with luminol to generate blue light with a peak at 425 nm. The blue light can activate the NBs through the BRET and the following FRET between DiI and DiD will red-shift the light into red light with a peak at 670 nm. (B) Luminol can freely penetrate into inflamed tissues, e.g. tumors, to interact with MPO and activate the DiI-DiD NBs through the BRET-FRET process for bioluminescence imaging. The echogenicity of DiI-DiD NBs also makes the tissues detected by contrast-enhanced ultrasound imaging.


19

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

Page 20 of 24

Figure 2. Fabrication and characterization of DiI-DiD NBs. (A) Normalized emission spectra of luminol and excitation and emission spectra of DiI and DiD. (B) Fluorescence spectra of LNPs doped with varying amounts of DiI with an excitation of 550 nm. (C) Fluorescence spectra of LNPs doped with different DiI/DiD ratios with an excitation of 550 nm. (D) Fluorescence spectra of LNPs under different total concentration of DiI and DiD with an excitation of 550 nm. (E) Fluorescence spectra and photos (insert) of DiI-DiD LNPs and DiI-DiD NBs. (F) Size distribution of DiI-DiD LNP and NBs measured by nanoparticle tracking analysis. (G) Fluorescence images of DiI-DiD NBs under the


20

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

ACS Nano

confocal microscope. Quantitative results of MPO assay obtained with an IVIS spectra instrument when different optical filters (H) and tissue-like phantom at different depth (I) were applied. *P

Bioluminescence Imaging of Inflammation in Vivo Based on

Recommend Documents