Cancer Cell Membrane-Biomimetic Nanoprobes with Two-Photon

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Cancer Cell Membrane-Biomimetic Nanoprobes with Two-Photon Excitation and Near-Infrared Emission for Intravital Tumor Fluorescence Imaging Yanlin Lv, Ming Liu, Yong Zhang, Xuefei Wang, Fan Zhang, Feng Li, Weier Bao, Jie Wang, Yuanlin Zhang, Wei Wei, Guanghui Ma, Liancheng Zhao, and Zhiyuan Tian ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07716 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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Cancer Cell Membrane-Biomimetic Nanoprobes with Two-Photon Excitation and Near-Infrared Emission for Intravital Tumor Fluorescence Imaging

Yanlin Lv1,2,§, Ming Liu3,4,§, Yong Zhang3*, Xuefei Wang1, Fan Zhang2, Feng Li2, Wei-Er Bao2, Jie Wang1, Yuanlin Zhang1, Wei Wei2*, Guanghui Ma2*, Liancheng Zhao3, Zhiyuan Tian1* 1

School of Chemistry and Chemical Engineering, University of Chinese Academy of

Sciences, Beijing 100049, P. R. China. 2

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

CAS Beijing 100190, P. R. China. 3

School of Materials Science and Engineering, Harbin Institute of Technology,

Harbin 150001, P. R. China. 4

School of Materials Science and Engineering, Wuhan Institute of Technology,

Wuhan 403052, P. R. China. §

Yanlin Lv and Ming Liu contributed to this work equally.

1

ACS Paragon Plus Environment

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ABSTRACT Biomimetic fluorescent nanoprobes capable of emitting near-infrared (NIR) fluorescence (λmax ~720 nm) upon excitation of 800-nm light were developed. The key conjugated polymer enabled two-photon absorption (TPA) and Fӧrster resonance energy transfer (FRET) processes within the nanoprobes, which imparted the nanoprobes with ideal NIR-incoming-NIR-outgoing fluorescence features. The cancer cell membrane (CM) coating endowed these nanoprobes with perfect biocompatibility and highly specific targeting ability to homologous tumors. It was believed that CM encapsulation provided additional protecting layer for the photoactive components residing in the core of nanoprobes for retaining their intrinsic fluorescing ability in physiological milieu. The long-term structural integrity, excellent photostability (fluorescence decrease < 10% upon 30-min illumination of 800-nm pulse laser), high NIR fluorescence quantum yield (~ 20%) and long probe circulation time in vivo of the target nanoprobes were also confirmed. The ability of these feature-packed nanoprobes for circumventing the challenges of absorption and light scattering caused by cellular structures and tissues was definitely confirmed via in vivo and in vitro experiments. The superior performances of these nanoprobes in terms of fluorescence signaling as well as targeting specificity were verified in intravital fluorescence imaging on tumor-bearing model mice. Specifically, these nanoprobes unequivocally enabled high-resolution visualization of the fine heterogeneous architectures of intravital tumor tissue, which proclaims the great potential of this type of probes for

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high-contrast fluorescence detection of thick biological samples in practical applications.

KEYWORDS: two-photon excitation, near-infrared emission, cancer cell membrane, homologous targeting, biological imaging

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Fluorescence imaging, which is capable of offering minimally invasive and ultrasensitive detections, has become a preferred choice in biological detection and biomedical diagnosis and provided a wealth of information regarding biological structures, functions and physiological processes.1, 2 The key enabling component for fluorescence imaging is the fluorescent probes with optimized features. However, fluorescence imaging for mapping intravital deep-seated targets generally suffers from limited penetration depth of excitation (incoming) light because most conventional probes are excited via ultraviolet or visible light, which is vulnerable to scattering and absorption caused by optically turbid tissues.1, 3 Additionally, the emission (outgoing) light with relatively short wavelength is also vulnerable to the similar attenuating influence that the optically turbid tissues exerted, which typically leads to reduced fluorescence signals and discrimination of beaconing signals from background.4-6 It is known that many biological issues reach the maximum optical transparency and greatly attenuated light scattering in the biological window ( ∼ 700−1000 nm), indicating fluorescent probes with excitation and emission lights both in NIR region are intrinsically capable of circumventing the abovementioned obstacles and therefore achieving bioimaging with satisfactory depth. Two-photon excited fluorescence (TPEF) is typically involved in the simultaneous absorption of two long-wavelength photons, usually in the NIR region, by a fluorophore and its subsequent emission of one photon with shorter wavelength.7 Based on such nonlinear optical mechanism, TPEF imaging is characterized with undoubted superiorities to traditional fluorescence imaging modes.8, 9 In addition to the imaging sharpness originating from 4


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the optical sectioning effect, TPEF imaging with NIR excitation light typically facilitates penetration across tissue or organ with minimum absorption and scattering, thus enabling larger imaging depth.10-14 Cell autofluorescence that typically masks signals from the labeled target of interest represents another ubiquitous problem in fluorescence bioimaging. Specifically, autofluorescence covers the spectrum region of most common fluorescent probes, which makes it difficult to distinguish the labeled target from background. Due to the much weaker autofluorescence of biological samples upon TPE process, TPEF-based bioimaging with NIR signals is expected to benefit from greatly alleviated interference of cell autofluorescence and thus enables high signal-to-noise (S/N) ratio in biological fluorescence detections.15 On the other hand, most NIR fluoescent probes suffer from low fluorescence quantun yield due to their narrow optical gap that typically facilitates nonradiative decay and self-aggregation features in aqueous milieu originating from their hydrophobicity,12, 16, 17

which usually significantly limits the imaging S/N ratio and therefore weakens the

ability of highlighting beaconing signals from the background. It also deserves mentioning that photostability, or photobleaching resistance, of fluorescent probes is a critical measure in fluorescence imaging, particularly for long-term tracking applications involving live cells and tissue. Thus, development of NIR fluorescent probes with high fluorescence quantun yield together with photostability is also a crucial step toward satisfactory biological fluorescence imaging. Another demand for fluorescent probes for bioimaging is their targeting ability, which is beyond doubt critical

for

cancer

diagnosis

and

imaging-assisted 5


therapy.2,

18

Various

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nanoparticle-based probes have been developed for tumor fluorescence imaging, where the probes were typically delivered to the pathological sites via EPR (enhanced permeability and retention) effect.19, 20 As a common strategy, functionalization of probes with specific ligands recognizable by the overexpressed surface receptors on cancer cells has been exploited for improving the targeting ability of probes.21 However, performance of the functionalized probes in terms of target specificity still remains a major challenge. 22 Thus, ideal fluorescence bioimaging demands photophysical features of probes with TPE-based NIR fluorescence emission for satisfactory imaging depth and minimized interference of autofluorescence, high fluorescence quantum yield and photostability for long-term imaging sensitivity, perfect biocompatibility and highly specific targeting ability for selective recognition for cancer cells. Based on conjugated copolymer with two-photon absorption (TPA) moiety and NIR fluorescing moiety covalently integrated into the polymer chains, we herein developed feature-packed fluorescent nanoprobes capable of emitting NIR fluorescence (centered ∼720 nm) upon excitation of 800-nm pulse laser and possessing perfect biocompatibility and highly specific tumor targeting ability. Specifically, the optimized molecular structure of the conjugated copolymer and the photophysical properties of the photoactive components imparted high quantum yield, photostability and NIR fluorescing ability to the polymer-based nanoparticles (hybrid NPs). In terms of biocompatibility and intrinsic targeting ability in biological detections, functional agents with coating of cell membrane (CM) provide promising alternative for the 6


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development of intelligent nanoprobes and have attracted considerable of attention.23-26 Specifically, nanoprobes with cancer CM coating typically display homotypic tumor self-recognition ability, which provides great facility for specific tumor targeting.27 Taking this, we encapsulated the abovementioned hybrid NPs with cancer CM and therefore constructed hybrid TPEF-based nanoprobes featured with superior biocompatibility and highly specific tumor targeting ability. Additionally, we proposed that CM encapsulation of probes, particularly NIR fluorescent probes, is likely to provide an additional protecting layer for the highly hydrophobic photoactive components and therefore help to retain their intrinsic fluorescing ability in physiological milieu. These salient features unequivocally proclaim such probe as an ideal agent for fluorescence imaging of thick biological samples with high contrast and spatial resolution. RESULTS AND DISCUSSION The photoactive copolymer, hereafter called PTPE-DTNT4, was synthesized by copolymerizing two types of functional monomers into molecular skeleton of the polymer chains. Specifically, a tetraphenylethene-based conjugated monomer possessing remarkably large TPA cross section (σ) and high fluorescence quantum yield

28

was used as the TPA components and energy donor (D) for enabling TPEF

feature (Scheme 1). A naphthothiadiazole-based monomer capable of emitting NIR fluorescence

29, 30

was used as the energy acceptor (A) and fluorescing unit. The

abovementioned two functional moieties were integrated into the polymer chains via palladium-catalyzed Suzuki coupling strategy, which yielded blackish green 7


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PTPE-DTNT4 solid with 4% acceptor content, number-average molecular weight of ~33869 and coefficient of dispersion (d) of 1.86. Upon TPA-based excitation of NIR photon, the energy donor monomer typically emits strong fluorescence in the range of ∼460-600 nm, which overlaps appreciably with the absorption band of the NIR fluorophore acceptor, thus enables transferring the excitation energy to NIR ﬂuorescence. Specifically, upon excitation of 800-nm photon, this copolymer generates ﬂuorescence emission centered at ∼720 nm, yielding a desired NIR-incoming-NIR-outgoing feature.

Scheme 1. Strategy for constructing CM-coated fluorescent nanoprobes with TPE and NIR fluorescence emission feature (A) and schematic illustration of fluorescence imaging of these probes in tumor-bearing mice (B).

Hybrid nanoparticles (NPs) (~110 nm), hereafter called hybrid NPs, were constructed via a coprecipitation strategy (Figure 1A).31 PTPE-DTNT4 resided in the hydrophobic core and the hydrophilic PEG segments of an amphiphilic polymer decorated on the surfaces. And the weight ratio of PTPE-DTNT4 to the amphiphilic 8


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polymer was 1:2. The cancer cell membrane was extracted via a strategy of ultrasonic cell disintegration and confirmed using dot-blot method (Figure S1). Subsequent to surface charge inversion (Figure S2), the NPs were encapsulated into cancer CM via electrostatic interactions (indicated by the inserted illustration in Figure 1B), which generated the target nanoprobes with average diameter of ~130 nm, hereafter called M-NPs. Such encapsulation brought about an increase in average hydrodynamic radius (Figure S3), again indicating the successful CM cloak on the polymeric NPs. The size of hybrid NPs and M-NPs nanoprobes nearly kept unchanged upon storing up to 30 d (Figure S4), indicating the excellent colloid stability of these nanoprobes. As shown in Figure 1C, the weak emission with maximum at ~535 nm originated from the donor moiety while the intense emission band centered at ~720 nm was assigned to the acceptor segment. The brightness of the latter was more than 10 times larger than that of the former, suggesting efficient energy transfer within the nanoprobes. Interestingly, fluorescence quantum yield (ø) of the hybrid NPs slightly increased upon CM encapsulation, from less than 18% to more than 20%. Additionally, the average fluorescence lifetime (τ) of the hybrid NPs displayed similar change, namely from 4.620 ns to 5.334 ns, upon CM encapsulation (Figure 1D). It is noted that the half-width at half-maximum (HWHM) of instrument response function (IRF) for the fluorescence kinetics measurement was ∼50 ps. Thus, CM encapsulation of polymeric NPs indeed induced increase in fluorescence lifetime for 714 ps. Such changes in both steady-state (ø) and transient-state (τ) indicating parameters were attributable to the CM coating that virtually provided additional protective layer for 9


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isolating the fluorophoric units from nonradiative decay pathways and therefore enabled the relaxation of more excited-state species to ground state via manner of photon emission.32 A

B

CM

500 nm

500 nm

D

Hybrid NPs M-NPs

C

E

Hybrid NPs M-NPs

F

Hybrid NPs

M-NPs

Figure 1. Characterization of the hybrid NPs and the target M-NPs nanoprobes. Representative TEM images of hybrid NPs (A) and M-NPs (B). (C) UV-vis absorption and fluorescence emission spectra of M-NPs. (D) Transient fluorescence (at 720nm) of hybrid NPs and M-NPs. The scattered symbols represent experimental data and the black solid lines were fits. (E) Evolution of fluorescence intensity (at 720 nm) of M-NPs under continuous 800-nm laser irradiation with time up to 30 min. (F) TPA σ of NPs and M-NPs using Rhodamine B as reference.

As compared to those nanoprobes with physically incorporated dyes, our M-NPs probes were expected to be more resistant to dye leaching and therefore possess structural integrity for relatively long time.33 It was found that the M-NPs aqueous dispersion sample displayed less than 5% decrease in fluorescence intensity after 30-d storage, suggesting its perfect structure stability (Figure S5). To evaluate the photostability of the probes, we monitored their time-elapsed fluorescence emission upon continuous illumination of an 800-nm pulse laser with the same illumination 10


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power as that used in typical biological fluorescence imaging. Upon 30-min illumination of 800-nm pulse laser, less than 10% fluorescence intensity loss of the M-NPs sample was obtained, suggesting the perfect photostability of M-NPs (Figure 1E). In contrast, ~25.3% fluorescence intensity loss of the hybrid NPs was obtained under the identical experiment condition. Similar results were obtained upon single-photon illumination of 388-nm continuous-wave laser (Figure S6). A plausible explanation was that the CM encapsulation provides protective layer for the conjugated polymer units within the nanoprobe and thus alleviates their photobleaching. Benefiting from the property of the two-photon harvesting moiety of the

PTPE-DTNT4

polymer,

the

hybrid

NPs

and

M-NPs

displayed

wavelength-dependent TPA cross section (σ) in the range of 6.06-8.0×106 GM per nanoparticle (Figure 1F),34 much higher than the counterpart values of other types of NPs reported previously.35-37 The evaluation results regarding the cytotoxicity of M-NPs nanoprobes clearly indicated their appreciable biocompatibility (Figure S7). To evaluate the recognition specificity of M-NPs nanoprobes, we prepared a series of nanoprobes by encapsulating the same batch of hybrid NPs into CM of HeLa, 4T1, J774 and red blood cells (RBC), respectively, and then incubated them with HeLa cells for evaluation. Similar to hybrid NPs, coating with 4T1, J774 or RBC membrane failed to arouse the appetite of the HeLa cells (Figure 2A). On the contrary, quite a few Hela M-NPs were observed in the Hela cells, indicating a markedly promoted uptake. Such disparity in cell uptake was also quantitatively verified by flow cytometer analysis, 11


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where the Hela M-NPs group gained a 16-fold enhancement in the intracellular fluorescence intensity as compared to the counterpart NPs uncoated or coated with membranes of heterotypic cells (Figure 2B). Additionally, we evaluated the targeting specificity of Hela M-NPs to various cell lines. Similarly, HeLa cells internalized many M-NPs while very few were found in other kinds of cells (Figure S8). Such a good uptake could also be well maintained after long-time exposure to 800-nm pulse laser (Figure S9). Together, these results clearly demonstrated the high targeting specificity that the HeLa CM enabled and therefore the superiority of homologous CM over the heterotypic ones.

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A

i

ii

iii

iv

v

vi

B Control Hybrid NPs RBC M-NPs J774 M-NPs 4T1 M-NPs HeLa M-NPs

NPs Nucleus Membrane

C

0.5 h

6h

12 h

24 h

48 h 300

Hybrid NPs

725

FA-NPs

1150

1575

M-NPs

2000

D

Left

Right

Left/Right Hybrid NPs - NPs ** FA M-NPs

**

Figure 2. Evaluation of specific targeting performance of the M-NPs probes. (A) CLSM images of HeLa cells (i) and HeLa cells after incubation with hybrid NPs (ii), 4T1 M-NPs (iii), J774 M-NPs (iv), RBC M-NPs (v) and HeLa M- NPs (vi), respectively. Scale bar was 5 µm. The concentration of NPs in all cases was 20 µg/mL. (B) Flow cytometry analysis of HeLa cells after incubation with various NPs. (C) In vivo real-time fluorescence images of Balb/c nude mice simultaneously bearing HeLa cervical (left) and MCF-7 breast (right) tumor xenograft after intravenous injection of hybrid NPs (EPR effect), FA-NPs (EPR effect + FA-mediated target), and HeLa M-NPs (EPR effect + homologous target), respectively. The injection volume of stock NPs dispersion (400 µg/mL) in all cases was 100 µL. (D) Quantitative time-dependent distributions of model nanoprobes in left & right tumor sites and their intensity ratio. Results were expressed as means ± s.d. *p < 0.05, **p < 0.01, analyzed by one-way ANOVA. 13


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The in vivo targeting ability of M-NPs probes was also evaluated by gauging their time-elapsed biodistribution in model mice simultaneously bearing two different types of tumor xenografts at parallel sites (HeLa tumor at left and MCF-7 tumor at right). For comparison, the counterpart dynamic data of hybrid NPs and hybrid NPs functionalized with folic acid (FA-NPs) were also acquired. As illustrated in Figure 2C, fluorescence signals of probes at the HeLa tumor site could be clearly observed 30 min and peaked 6 h after intravenous injection in three cases. Although sharing the similar kinetics, the counterpart beaconing signals in three cases were found increased in the order of hybrid NPs, FA-NPs, and M-NPs at the same time intervals. Such an efficient target capability of M-NPs can be attributed to the superior long-circulation effect (Figure S10) and endocytosis (Figure S11). In addition, M-NPs exhibited superior recognition specificity to the homologous tumor, which was sourced from the same cell line as the membrane for encapsulation of NPs. No detectable fluorescence signal of M-NPs was observed at the MCF-7 site during the observation period due to non-homologous nature. Specifically, the indicator value (IHeLa/IMCF-7) for recognition specificity of nanoprobe in M-NPs group could reach up to 10 (Figure 2D), which was markedly superior to those in the cases of hybrid NPs and even FA-NPs. Although HeLa model was used herein, it could be envisioned that the homologous targeting ability of cancer CM coating was pervasive to other types of tumors.27 Next, 2D cell culture systems were applied to evaluate the ability of our probes for circumventing the challenges of absorption and light scattering caused by cellular structures and tissues. As shown in Figure 3A, a 300-µm-thick mock tissue brought 14


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out

only

∼10%

attenuation

of

signal

intensity

in

the

TPE-based

NIR-incoming-NIR-outgoing fluorescence imaging mode (Ex = 800 nm, Em = 600-780 nm). In sharp contrast, mock tissue with the same thickness in the case of traditional single-photon excitation (SPE) (Ex = 405 nm, Em = 600-780 nm) resulted in marked decrease in fluorescence intensity with loss percentage up to ∼58% (Figure 3B). In the case at the other end, where SPE (Ex = 405 nm) was used and visible fluorescence signal (Em = 460-600 nm) was collected, a 300-µm-thick mock tissue dramatically induced attenuation of more than 81% beaconing signal (Figure S12). Unequivocally,

for

mapping

intravital

deep

target,

our

probes

with

NIR-incoming-NIR-outgoing feature have overwhelming superiority in terms of circumventing the attenuating effects of absorption and light scattering caused by optically turbid tissues. 3D multicellular tumor spheroid (MCTS) model with histological characteristics similar to the solid tumor nidus were also used for evaluating the performance of M-NPs nanoprobes. In the case of TPE-based imaging mode, the obtained fluorescence image displayed appreciable brightness uniformity throughout the whole spheroid (Figure 3C) due to the superior permeability (Figure S13). In sharp contrast, the varying thickness of cellular tissue along the light path in the SPE-based image exerted marked influence on the fluorescence brightness: only the periphery of spheroids displayed relatively strong fluorescence while the inner area around the core was nearly as dim as the background (Figure 3D). It is noted that nanoprobes residing in the core of a compact spheroid with diameter of ∼500 µm fluoresced upon 15


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excitation of incoming photons passing through cellular structures with thickness of ∼250 µm. In contrast, nanoprobes residing in the periphery of spheroids were nearly directly exposed to the incoming photons. Thus, such fluorescence imaging results of MCTS models clearly confirmed the ability of M-NPs nanoprobes for circumventing the daunting impediments of light absorption and scattering caused by cellular structures, unequivocally indicating their superiority for imaging deep-seated target in biological applications. Additionally, the excellent intratumoral penetration of our probes also paved the way for the exquisite observation of histological architecture. A

0 µm

300 µm

**

B

0 µm

300 µm

C

D

Figure 3. Comparison of penetration performance of TPE- and SPE-based fluorescence imaging in 2D cells and 3D multicellular spheroid models. Typical TPEF (A) and SPEF (B) images acquired with laser crossing 0-µm-thick (left panel) and 300-µm-thick mock tissue (middle panel), respectively, and the comparison of corresponding fluorescence intensity (right panel). Scale bar was 5 µm. Schematic illustrations of TPEF (C) and SPEF (D) CLSM 3D multicellular spheroid 16


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imaging, the corresponding transverse section fluorescence images of multicellular spheroids and fluorescence line profile passing through the multicellular spheroids, respectively. Scale bar was 100 µm. The concentration of NPs in all cases was 20 µg/mL. Results were expressed as means ± s.d. (n = 3) *p < 0.05, **p < 0.01, analyzed by one-way ANOVA.

A B F

F

B

J

Infiltration

Circulation

J

Permeation

C

G

K

D

H

L

E

I

M

Figure 4. In vivo tumor fluorescence imaging using our M-NPs probes. (A) Photograph of tumor-bearing mice and schematic illustration of the target lesion with specific regions containing vessels, peripheral vessels and adjacent perivascular tumor tissues magnified in (B), (F) and (J), respectively. 3D reconstructed images of the target lesion showing spatial distributions of nanoprobes in vessels (C), peripheral vessels (G) and the adjacent perivascular tumor tissues (K). (D, H, L) Magnified regions of interest selected from C, G, and K, respectively, and the corresponding profile of the amount of nanoprobes over the distance away from the vessel. (E, I, M) IHC-Fr of tumor section from the sites shown in C, 17


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G, and K, respectively. The vessels and nuclei were respectively stained with FITC (green) and Hoechst (blue) for visualization and the red spots arose from the nanoprobes. The injection volume of stock NPs dispersion (400 µg/mL) in all cases was 100 µL. Scale bar was 25 µm.

The aforementioned results prompted us to verify the salient features of M-NPs in tumor-specific in vivo fluorescence imaging. Specifically, an 800-nm pulse laser was used as the excitation source and NIR fluorescence signal from the hybridd Hela tumor with internalized M-NPs was collected (Figure 4A). For clear demonstration, specific regions containing vessels, peripheral vessels and adjacent perivascular tumor tissues at the target lesion, respectively, were presented with schematic illustration (Figure 4B, F, and J). As shown in Figure 4C, the TPEF imaging mode enabled 3D reconstruction of a representative tumor site containing a selected blood vessel, which outlined the initial stage of EPR effect characterized with a dominant accumulation of M-NPs probes in the vessel and extravasation of a few probes with distance up to 10 µm (Figure 4D). Similar result was obtained via the immunohistochemical (IHC) characterization of the same site (Figure 4E), where almost all fluorescence signals of M-NPs resided in the blood vessel. Over time, more and more nanoprobes extravasated through the vessel into adjacent perivascular tumor tissue and cells, presenting beaconing signals over the perivascular area far away from the blood vessel (Figure 4G). A 3D volumetric analysis of a selected area demonstrated a relatively wide spatial distribution of the probes with some probes infiltrating as far as 100 µm away from the vessel (Figure 4H), in sharp contrast to the counterpart results illustrated in Figure 4D. Correspondingly, quite a few M-NPs were found scattered 18


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the perivascular tumor tissues in the IHC slice (Figure 4I). The further analysis of another selected area mainly containing perivascular tumor tissue revealed a more extensive intratumoral distribution of M-NPs probes (Figure 4K-M). Specifically, considerable amount of probes migrated to more distant area and were well coupled with or in close proximity to the tumor cells, unequivocally confirming the satisfactory penetration and recognition capacities of M-NPs probes. It is particularly noteworthy that the abovementioned 3D reconstructed fluorescence images definitely displayed great image sharpness and clear edges, which unambiguously revealed the dynamic distribution of probes in regions of interest with high contrast and therefore enabled high-resolution visualization of the fine heterogeneous architecture of tumor tissue. Such performance benefiting from our NIR-incoming-NIR-outgoing M-NPs probes with superior targeting specificity is expected to pave the way for accurate diagnosis of clinical stage and treatment of solid tumors by providing the microenvironment information regarding the interfacial features at the perimeter of tumor tissue as well as tumor growth and metastasis. CONCLUSIONS In conclusion, we developed conjugated copolymer based cancer cell membrane-biomimetic fluorescent nanoprobes characterized with two-photon excitation and NIR fluorescence emission. The optimized molecular structure of the photoactive copolymer not only imparted the NIR-incoming-NIR-outgoing feature but also contributed to structural stability and photostability of the nanoprobes. On the other hand, cancer CM coating endowed the nanoprobes with perfect biocompatibility 19


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and highly targeting specificity to homologous tumor. Such type of feature-packed nanoprobes demonstrated superior performance in both in vitro and intravital biological fluorescence imaging in terms of targeting specificity as well as circumventing the impediment of penetration depth of the excitation light and the attenuation of the beaconing fluorescence signals caused by the cellular structures and tissues. Specifically, in vivo imaging experiments on tumor-bearing model mice clearly verified the ability of the probe for generating fluorescence image of intravital tumor fine structure with high contrast and sharpness, which proclaiming such type of probe as an ideal agent for biological fluorescence imaging of thick samples in practical applications. MATERIALS AND METHODS Chemicals in synthesis were purchased from J&K Chemical Co. or Energy Chemical Co. and were used directly without further purification. P-L-Lysine was purchased

from

Sigma-Aldrich.

1,2-distearoyl-sn-glycero-3-phosphoethanola-

mine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) was purchased from Avanti® Polar Lipids, Inc. Alexa Fluor® 488 phalloidin and Hoechst 33342 were purchased from Life technologies. Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco. Other chemicals used here were purchased from Alfa Aesar Co. or Xilong Scientific Co., Ltd. The tetraphenylethene-based monomer and naphthothiadiazole-based monomer were

firstly

synthesized

following

the

previous

literatures

with

minor

modifications,28-30 and the target fluorescent conjugated polymer PTPE-DTNT4 was 20


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obtained via Suzuki coupling strategy based on the abovementioned monomers. The detailed synthesis and characterization results of the polymer will be reported elsewhere. HeLa cells (2×108) were suspended in 2 mL of Hepes B buffer (2.38 g·L-1Hepes, 0.476 g·L-1 MgCl2, 0.292 g·L-1 EDTA, 0.154 g·L-1 DTT, 0.746 g·L-1KCl, pH=7.6) adding 1% Halt TM Protease Inhibitor Cocktail. Cells were enucleated in IKA® T18 basic homogenizer. The resulting supernatants were collected and laid on a discontinuous sucrose density gradient composed of 55% (w/v), 40% (w/v) and 30% (w/v) sucrose in Hepes B buffer. After ultracentrifugation at 28000 rpm for 2 h, three banded samples were clearly detectable (Figure S1). These three bands were collected and diluted with Hepes C buffer (11.914 g·L-1Hepes, 5.844 g·L-1NaCl, 13.492 g·L-1KCl,

pH=7.6)

for

successive

protein

characterizations.

Through

ultracentrifugation at 28000 rpm for 30 min, the membrane fractions were collected and stored in Hepes C buffer at -20 °C. Other cell membranes were obtained by the same method. The distribution of proteins associated to plasma membranes along the gradient were analyzed by dot-blot. 5 µL of each band was spotted on a protonated polyvinylidene fluoride (PVDF) membrane, the membrane was blocked with 1% goat serum, followed by ordinal incubation with anti-mouse CD44 antibody (1:200 dilution) and HRP (Horse radish Peroxidase) conjugated mouse anti-human IgG secondary antibody (1:2000 dilution) (Santa Cruz Biotechnology). The CD44 protein on membrane was observed with DAB stain (Figure S1). 21


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TPE-NIR nanoprobes were prepared by co-precipitation method. 2 mg DSPE-mPEG2000 and 1 mg TPE-NIR polymer PTPE-DTNT4 were dissolved in THF, and rapidly injected into 10 mL water under brief sonication. The THF was removed via N2 bubbling and the aqueous suspension sample containing polymeric nanoparticle was eventually obtained. Addition of 1 mg P-L-lysine to 10 mL the resulting aqueous suspension induced the surface charge inversion of the polymeric NPs (hybrid NPs(+)). Cancer cell membrane was quantified by BCA. 100 µL aqueous suspension sample containing polymeric nanoparticle after surface charge inversion treatment and 120 µL 1 mg·mL-1 cell membrane dispersion was fully mixed in 2 mL Hepes C buffer solution at 4 ℃ for at least 6 h. The CM-coated NPs were rinsed using water three times to remove redundant cell membrane, giving the final nanoprobes, M-NPs, suspended in PBS for characterization and animal experiments. The size and zeta potential of the hybrid NPs and M-NPs were measured using a Malvern Zetasizer. For hybrid NPs and M-NPs probes dispersed in PBS buffer, more than 3 parallel samples were measured. Specifically, the polymeric NPs with positively charged surface were encapsulated into tumor cell membrane via electrostatic interaction. In contrast to the polymeric NPs prior to cell membrane encapsulation treatment, these target nanoprobes displayed zeta potential of -10.1mV (Figure S2) and hydrodynamic radius of ~190 nm (Figure S3B), indicative of the cloak of cell membrane around the previously hybrid polymeric NPs. The

22


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fluorescence stability of hybrid NPs and M-NPs was evaluated via F7000 fluorescence spectrometers (HITACHI, Japan) with excitation wavelength of 388 nm. The as-prepared hybrid polymeric NPs and M-NPs were kept at ambient temperature in dark for 30 d and their hydrodynamic radii and fluorescence emission spectra of were acquired each day, respectively. As shown in Figure S4, there is no obvious change in the size of the polymeric NPs and the target M-NPs nanoprobes in 30 days, indicating the excellent colloid stability of the aqueous dispersion sample containing M-NPs nanoprobes. For the fluorescing ability, it was found that the M-NPs sample displayed less than 5% decrease in fluorescence intensity after 30-day storage, suggesting the perfect structure stability of such nanoprobe sample (Figure S5). Additionally, the photostability of the aqueous dispersion sample containing M-NPs nanoprobes was evaluated by monitoring its fluorescence emission feature upon continuous irradiation of 388-nm laser with time up to 30 min. It can be seen from Figure S6, the sample displayed appreciable photostability upon continuous irradiation of high-energy light. The two-photon absorption cross section (σ) of NPs and M-NPs was determined via the two-photon induced fluorescence method. A tunable femtosecond laser (120 fs, 1 kHz) was used as the excitation source. Rhodamine B (Rh B) in methanol was used as the reference.34 And the TPA cross-section (σ) of NPs unit was calculated from the following equations:

σ =σs ×

F φ cs ns × × × Fs φs c n 23


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where F denotes the two-photon fluorescence intensity; ø denotes the fluorescence quantum yield; c denotes the molar concentration, n denotes the refractive index of the solvent. The subscripts s represents Rh B in methanol. The steady-state absorption spectra were measured on a UV-3600 Plus UV-VIS-NIR spectrophotometer (SHIMADZU, Japan). The stationary fluorescence spectra were measured on F7000 fluorescence spectrometers (HITACHI, Japan), and the absolute fluorescence quantum yields were measured on steady-state and time-resolved fluorescence spectrophotometers (FLS 980, Edinburgh, UK) equipped with an integrating sphere. The fluorescence lifetime results of hybrid polymeric NPs and HeLa M-NPs samples were determined using time-resolved fluorescence spectrophotometers (FLS 980, Edinburgh, UK). Human cervical cancer HeLa cell line was used for cancer cell membrane extraction, cytotoxicity, cellular specific target, and tumor model. Human breast cell MCF-7, mammary breast cancer 4T1 cell line, mouse monocyte macrophage J774A.1 cell line and erythrocyte served as negative control. The cancer cells were routinely cultured in DMEM or 1640 media (supplemented with 10% fetal bovine serum and 1% penicillin streptomycin antibiotics), incubated at 37 ℃

in a humidified

atmosphere with 5% CO2, and split 1: 3 every other day. Red blood cells (RBCs) were obtained from mice. HeLa cells were seeded in 96-well plates at a density of 5 × 103 cells per well and incubated for 24 h. Afterwards, the HeLa cells were treated with TPE-NIR NPs

24


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and HeLa M-NPs at nanoprobe concentration from 0 to 20 µg/mL for 24 h. CCK-8 assay was applied for evaluating the cell viability. Fluorescence microscopy images were obtained by the confocal laser scanning microscopy (CLSM, Ultra VIEW VoX, PerkinElmer, USA). HeLa cells were seeded in Petri dish and incubated with NPs, HeLa M-NPs, J774 M-NPs, 4T1 M-NPs and RBC M-NPs for 2 h, respectively. Cellular F actin (cell membrane) was labeled by Alexa Fluor® 488 phalloidin, and cell nucleus was stained by Hoechst 33342. Flow cytometry analysis was performed using BD Aria III flow cytometer and analyzed using FlowJo 7.6. Single-photon and two-photon fluorescence imaging with different penetration depth was investigated using confocal laser scanning microscopy (CLSM, Ultra VIEW VoX, PerkinElmer, USA) with the excitation wavelength of 405 nm and multi-photon laser confocal scanning fluorescence microscope (A1R MP, Nikon, Japan) with the excitation wavelength of 800 nm , respectively. 1% intralipid was used as the simulated tissue phantom for its turbidity and scattering properties similar to those of the real tissue.31 HeLa cells were incubated with HeLa M-NPs for 24 h. HeLa cells were inoculated in supplemented DMEM with 250 cells per drop and cultivated for 5 days. The formative HeLa spheroids were incubated with HeLa M-NPs for 48 h. Two-dimensional single-photon fluorescence imaging was investigated using spinning disc laser confocal microscope (Opera, PerkinElmer, USA), and two-photon fluorescence imaging was performed using a multi-photon laser confocal scanning fluorescence microscope (FV1200MPE-M, Olympus, Japan). 25


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The tumor-bearing Balb/c nude mouse was anesthetized and fixed on the objective stage. And three-dimensional multi-photon fluorescence imaging of tumor in vivo was captured by a multi-photon laser confocal scanning fluorescence microscope (FV1200MPE-M, Olympus, Japan) with the excitation wavelength at 800nm, and analyzed by Imaris 8.1.2. Animals were purchased, maintained, and handled with protocols approved by the guide of care and use of laboratory animals. Female Balb/c nude mice (4–6 week old, 18–22 g) were used for all the animal experiments. Mice were purchased from the Beijing laboratory animal center and were housed in the specific-pathogen-free animal facility for at least 1 week before the experiments. ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (grant no. 21373218, 21573234, 21622608 and 91233107), National Key 26


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R&D

Program

of

China

(2017YFA0207900),

and

Beijing

Talents

Fund

(2015000021223ZK20). Y. Zhang thank the support from the Fundamental Research Funds for the Central Universities (Harbin Institute of Technology) and the Open Fund of the State Key Laboratory of Luminescent Materials and Devices (South China University of Technology, skllmd-2015-12). REFERENCES (1) Li, D. Y.; Zhao, X. Y.; Qin, W.; Zhang, H. Q.; Fei, Y.; Liu, L. W.; Yong, K. T.; Chen, G. D.; Tang, B.; Qian, J., Toxicity Assessment and Long-Term Three-Photon Fluorescence Imaging of Bright Aggregation-Induced Emission Nanodots in Zebrafish. Nano Res. 2016, 9, 1921-1933. (2) Vinegoni, C.; Swisher, C. L.; Feruglio, P. F.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R., Real-Time High Dynamic Range Laser Scanning Microscopy. Nat. Commun. 2016, 7: 11077. (3) Zandt, B. J.; Liu, J. H.; Veruki, M. L.; Hartveit, E., AII Amacrine Cells: Quantitative Reconstruction and Morphometric Analysis of Electrophysiologically Identified Cells in Live Rat Retinal Slices Imaged with Multi-Photon Excitation Microscopy. Brain Struct. Funct. 2017, 222, 151-182. (4) Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud'homme, R. K., Review of Long-Wavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores, and Multifunctional Nano Carriers. Chem. Mater. 2012, 24, 812-827. (5) Anees, P.; Joseph, J.; Sreejith, S.; Menon, N. V.; Kang, Y. J.; Yu, S. W. K.; Ajayaghosh, A.; Zhao, Y. L., Real Time Monitoring of Aminothiol Level in Blood Using a Near-Infrared 27


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TOC graphic

Fluorescence Cancer cell 800-nm imaging membrane excitation

Injection

33


720-nm emission

Cancer Cell Membrane-Biomimetic Nanoprobes with Two-Photon

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