Intracellular near-Infrared Microlaser Probes Based on Organic

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Biological and Medical Applications of Materials and Interfaces

Intracellular Near-Infrared Microlaser Probes Based on Organic Microsphere-SiO Core-Shell Structures for Cell Tagging and Tracking 2

Zheng Lv, Zhongwei Man, Zhenzhen Xu, Changfu Feng, Yong Yang, Qing Liao, Xu Wang, Lemin Zheng, and Hongbing Fu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09380 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Applied Materials & Interfaces

Intracellular Near-Infrared Microlaser Probes Based on Organic Microsphere-SiO2 Core-Shell Structures for Cell Tagging and Tracking Zheng Lv[a], Zhongwei Man[a], Zhenzhen Xu*[a], Changfu Feng[b], Yong Yang[a], Qing Liao[a], Xu Wang[c], Lemin Zheng[c],and Hongbing Fu*[a,b] [a]

Beijing Key Laboratory for Optical Materials and Photonic Devices, Department of Chemistry,

Capital Normal University, Beijing 100048, P. R. China [b]

Tianjin Key Laboratory of Molecular Optoelectronic Sciences Department of Chemistry,

School of Sciences, Tianjin University Tianjin Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, People's Republic of China [c]

School of Basic Medical Sciences, and Key Laboratory of Molecular Cardiovascular Sciences

of Ministry of Education, the Institute of Cardiovascular Sciences and Institute of Systems Biomedicine, Peking University Health Science Center, Beijing 100191, P. R. China KEYWORDS: NIR emission, intracellular microlaser probe, SiO2 shell, crystalline organic microspheres, tagging and tracking

ABSTRACT：Conventional near-infrared (NIR) luminescent probes, such as DsRed and Cy5, utilize spontaneous emission (SE) signals, which are broad (FWHM > 50 nm) and often have

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low quantum yield. Herein, we developed smart NIR intracellular whispering-gallery mode (WGM) microlaser probes made by organic microspheres of (E)-3-(4-(di-ptolylamino)phenyl)-1(1-hydroxynaphthalen-2-yl)prop-2-en-1-one (DPHP) coated with a silica shell. The overall small diameter (D: adjustable between 2−10 µm) and the biocompatible silica shell ensure our coreshell microspheres (CSmSPs) to be engulfed in cells as a microlaser operating around 720 nm with a low threshold of 0.78 µJ/cm2. Considering that WGM mode spacing depending strongly on its size, it will be possible to distinguish millions of individual macrophages through welldefined WGM lasing peaks (FWHM ≤ 2 nm) of CSmSPs of different sizes. Furthermore, we monitored the transformation of normal macrophages to foamy ones by encoding them with our NIR CSmSPs microlaser probes, which deliver constant WGM lasing signals with a spectral fluctuation < 0.02 nm and excellent stability.

INTRODUCTION Individual cells are the building blocks of complex organism. Tagging and real-time tracking of different types and large amounts of cells are very important for understanding the biological process as well as the functional capacities and responses of each type of cell in complex tissues.1 For example, as the major cause of cardiovascular disease, the atherosclerosis progression is accompanied with the transformation of normal macrophages to foamy ones.2-4 Many different tagging techniques, such as the spectral encoding method, have been intensively investigated for identifying and discriminating different cells. Consequently, various luminescent probes based on organic dyes and fluorescent proteins,5-7 fluorescent silicon nanoparticles,8-9and quantum dots,10-12 have been developed (Scheme 1). Among them, NIR luminescent probes have aroused much research interests due to various advantages, such as deep tissue penetration length, little interference from auto-fluorescence and minimal photo-damage to living


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organisms.13-14 Nile red, Cy5 and red fluorescent protein DsRed are representative and commercially available examples. However, their broad spontaneous emission (SE) spectra (typically with a full width at half maximum (FWHM) > 50 nm, Scheme 1, dash lines) limit the number of discernable probes for multiplexed detection, which is a bottleneck for their application in the cell tagging. WGM microresonators trap light as a result of total internal reflection within the small cavity, and are widely exploited not only for lasers and nonlinear devices but also for sensing and imaging in biophotonics. 15-17 Recently, two independent research teams led by Seok Hyun Yun and Malte Gather, respectively, employed a similar strategy by using intracellular WGM microresonator made of polystyrene microspheres (PS) (Scheme 1, green solid line) or natural lipid droplets (Scheme 1, orange solid line), with organic dyes as gain materials, demonstrating stand-alone intracellular lasers.18-20As shown in Scheme 1, the spectra of the WGM lasing (solid lines) are much narrower than those corresponding SE spectra. This, combined with the sizedependent microcavity modes, enable tagging numerous cells easily. Furthermore, much more cells will be tagged by multiplexing with different dyes. Nevertheless, the low loading density of organic dyes (to avoid the aggregation induced fluorescence quenching effect) demands i) a high quality (Q) factor of the WGM microresonator and ii) a high pumping intensity to realize microlaser.21 The former requires a large optical microresonator size at least >10 µm, making it difficult to be engulfed by cells; and the latter means relatively high lasing threshold (over 103 µJ/cm2) that might damage the cell.22-23 Furthermore, the report on NIR intracellular microlaser is rare due to the scarcity of NIR gain materials.24-25 Meanwhile, the non-linear optical WGM resonators driven by NIR lasers have been reported recently. 26-29


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In the past few years, we reported that self-assembled organic microcrystals provide welldefined crystal facets to form built-in cavities for laser applications, such as nanowire FabryPerot (FP) and microdisk WGM microlasers.30-31 Herein; we developed smart NIR intracellular WGM microlaser probes based on self-assembled microspheres of DPHP coated with a silica shell. The overall small diameter (D: adjustable between 2-10 µm) and the biocompatible silica shell minimize the perturbations of our core-shell microspheres (CSmSPs) to the cell.20 The intracellular lasers based on CSmSPs operate at 720 nm with a low threshold of 0.78 µJ/cm2 (Scheme 1, red solid line). In principle, it is possible to distinguish millions of individual macrophages through well-defined WGM lasing peaks of CSmSPs of different sizes. As a proofof-concept, we monitored the transformation of normal macrophages to foamy ones by encoding them with our NIR CSmSP microlaser probes, which deliver constant WGM lasing signals with a spectral fluctuation < 0.02 nm and excellent stability. We synthesized DPHP CSmSPs using a simple reprecipitation method and the modified stober method.32-33 First, the stock tetrahydrofuran (THF) solution of DPHP was injected to 2.0 mL of cetyltrimethyl ammonium bromide (CTAB) in H2O (concentration C ~1.0× 10−3 M) solution. After aging for 30 minutes at 50 ℃ in the water bath, a colloidal dispersion of DPHP microspheres was obtained. By controlling the concentration of the DPHP solution, organic microspheres with different sizes can be obtained (Figure S1). 50 µL of tetraethyl orthosilicate (TEOS) in ethanol (0.30g/L) was added dropwise into the above-mentioned suspension with rapid stirring. After adding 100 µL of ammonia water (2.80%), 15 µL of (3-aminopropyl) triethoxysilane (APTES) in ethanol (0.30 g/L) were slowly injected. The mixture was stirred for 75 minutes. After centrifugation and washed by deionized water, the CTAB molecules was removed. The collected red powder formed by DPHP CSmSPs can be re-dispersed into


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phosphate buffered saline (PBS) solution or deionized water. Moreover, compared with the uncoated DPHP microspheres suspended in phosphate buffered saline (PBS), CSmSPs with the same concentration can still keep stable after standing for 3 days (Figure S2), exhibiting high colloidal stability. Under white light (Figure 1a, right), the DPHP CSmSPs suspension exhibits an intense orange color, indicating the high throughput of the preparation method. Measurements of the surface potential of these CSmSPs show that they are positively charged, and the zeta-potential retained a value of about 23.9 mV as shown in Figure S3. Shapes and sizes of CSmSPs were measured by confocal microscopy and scanning electron microscopy (SEM). When this solution (C ∼20×10−3 M), the DPHP CSmSPs are well-defined spheres morphology with the diameter around 3−5 µm (Figure 1b). SEM images of DPHP CSmSPs also present the uniform spherical structures with smooth surfaces (Figure 1c), similar to the uncoated DPHP microspheres (Figure S4). The energy-dispersive X-ray spectroscopy (EDS) experiment exhibit the ratio of Si/O about 1/2 for DPHP CSmSPs, in agreement with CSmSPs stoichiometry (Figure S5). Moreover, the EDS line analysis in transmission electron microscopy (TEM) of the cross section demonstrates that CSmSPs with a microsphere core (3 µm) and the SiO2 shell (0.5 µm) structure has been successfully fabricated (Figure 1d). The X-ray diffraction (XRD) measurement reveals no significant difference between the DPHP CSmSPs and the uncoated DPHP microspheres, indicating that DPHP CSmSPs are crystalline in nature (Figure S6). The biocompatibility was tested by utilizing the Cell counting Kit-8 colorimetric assay based on RAW 264.7 cells. After 24 h incubation with CSmSPs, even at an ultrahigh concentration of 100 µg•mL-1, the viability of RAW 264.7 cells remained over 85% (Figure S7), indicating the better biocompatibility than the uncoated DPHP microspheres (Figure S8).


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For intracellular operation, the 10 µL of CSmSPs in PBS solution (60 µg·mL-1) were incubated with and internalized into macrophages in confocal dish. Because of the small size, and the positively charged surface which can favor the interaction with electronegative cell membranes, 34

nearly 50% of the DPHP CSmSPs were internalized by macrophages within 24 h (Figure S9).

Furthermore, each cell can engulf multiple probes as high as 14. We next tested the ability of the DPHP CSmSPs as intracellular microlaser probe using a homemade microscope setup (Scheme S1). Figure 2a shows the photoluminescence (PL) spectra of the isolated cell containing one probe with diameter of 4 µm placed on the confocal dish under different pump densities (P). Below the threshold, the PL spectrum exhibits a broad spontaneous emission with a series of sharp peaks.22,

25, 35

With an increasing pump density above the

threshold from P=1.20 to 2.19 µJ•cm-2, strong laser emission develops as a set of sharp peaks. The laser peaks are around 720 nm and extend to 790 nm in the NIR region, which would take an advantage of low auto-fluorescence compared to the previously developed microlaser system operated at visible wavelength (Figure S10). We defined a parameter, namely, probe efficiency χ, as the ratio between emission intensity per nanometer and the optical pump density. And the χ around 720 nm increase from 80 below the threshold to 3826 above the threshold. By contrast, the χ around 650 nm at the spontaneous emission remains a low value of around 80 (Figure S11). When close to the threshold, the FWHM of lasing peak is 1.67 nm with a Q factor about 430 (Figure S12). These enable the lasing spectra to be distinguished easily from the broad autofluorescence form tissues compared with those molecular probes. As shown in Figure 2b, the plot of the integrated intensities of emission peak around 720nm as a function of pump density, showing clearly a threshold of Pth = 0.78 µJ•cm-2, which is nearly equal to the CSmSPs probe on glass substrate (Figure S13) and two orders of magnitude lower


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than the intracellular PS lasers probe.16,17,22 The relatively low threshold energy of CSmSPs integrated into the cells means this microlaser probe can operate without compromising their biological function. It should be pointed out that the spacing between the two laser peaks stayed the same throughout the experiment. Even at the very high pump density (P > 20 Pth) with continuous irradiation 30 s (~millions of shorts), the CSmSPs laser probe can still operate without obvious spacing fluctuation (

Intracellular near-Infrared Microlaser Probes Based on Organic

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