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In Situ Construction and Characterization of Chlorin-Based Supramolecular Aggregates in Tumor Cells Wei-Jiao Liu,†,§ Di Zhang,§ Li-Li Li,§ Zeng-Ying Qiao,§ Ju-Chen Zhang,† Ying-Xi Zhao,§ Guo-Bin Qi,§ Dong Wan,† Jie Pan,*,† and Hao Wang*,§ †

State Key Laboratory of Hollow Fiber Membrane Materials and Processes, School of Environmental and Chemical Engineering, Tianjin Polytechnic University, 100044 Tianjin, China § CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, 100190 Beijing, China S Supporting Information *

ABSTRACT: We demonstrate in situ construction and characterization of supramolecular aggregates from chlorin p6 (Cp6) molecules in tumor cells. Fully deprotonated Cp6 molecules in neutral condition were partially protonated inside the acidic lysosomes of cells and significantly increased the hydrophobicity of them that resulted in simultaneous formation of J-type aggregates. Importantly, the formation of J-aggregates was fully characterized in artificial tissues by UV− vis, circular dichroism (CD) and transmission electron microscope (TEM) techniques. Compared to the monomers, the Jaggregates exhibited 55-fold enhanced thermal conversion efficiency (η) at the optimal excitation wavelength (690 nm). The remarkably increased heat effect contributed to the stronger photoacoustic (PA) signals, leading to at least 2 orders of magnitude increase of the tumor-to-normal tissue ratio (T/N), which was defined as the PA signal ratio between tumor site and surrounding normal tissue. We envision that this proof-of-concept study will open a new way to develop tumor environment-induced selfassembly for variable biomedical applications. KEYWORDS: supramolecular, chlorin, pH sensitive, tumor, photoacoustic imaging utilized for tumor diagnostics in vivo.34−40 Besides the studies on the instrumental setting up,37 extensive attentions have been paid for developing new probes with enhanced PA sensitivity and specificity.21,36,41 Herein, we in situ constructed and characterized the pHinduced chlorin p6 (Cp6)-based supramolecular self-aggregates as photoacoustic (PA) contrast agents for tumor imaging. The advantages of our design included that (i) deprotonated Cp6 in neutral condition as small molecules could readily diffuse and extravasate into the tumor tissue; (ii) upon internalized by tumor cells, the Cp6 was partially protonated at lower pH value of lysosomes and in situ formed J-type nanoaggregates which could prolong the residence time at tumor sites; (iii) PA signal significantly enhanced due to the increased thermal conversion efficiency upon formation of supramolecular self-aggregates; and (iv) strong PA signals leading to at least 2 orders of magnitude increase of the tumor-to-normal tissues ratio (T/N). The successful demenstration of this biological environment driven self-aggregation provides an alternative pathway to design functional biomaterials for disease diagnostic and therapeutics.

1. INTRODUCTION Numerous strategies have been devoted to construct selfassemblies from small molecules in aqueous solutions through the hydrophobic,1 hydrogen bonding,2,3 metal−ligand coordination interactions,4 etc. So far, supramolecular self-assemblies with controlled structural parameters, such as size, morphology, and surface chemistry have been achieved in water and successfully applied in drug delivery,5−10 bioimaging7,11−17 and tissue engineering.18,19 However, the complicated bioenvironments greatly affect the structure, stability and surface composition of the self-assemblies that lead to the compromised biofunctions of the designed superstructures. In this regard, an alternative strategy has been developed to preparation of selfassembled materials for biomedical applications. Basically, the scientists introduced the biocompatible building blocks to biological systems and in situ construction of self-assemblies in a certain physiological/pathological condition, for example, pH,20 enzymes,21−26 or superoxides.27 Recently, the enzymetriggered self-assemblies from small molecules in living cells had been reported.26,28 The enzymatic activity could be monitored by recording the fluorescence signals generated from selfassemblies.29,30 However, fluorescence imaging showed the intrinsic limitation in animal studies due to their poor tissue penetration ability.31−34 More recently, the photoacoustic (PA) imaging with deeper tissue penetration capability has been © 2016 American Chemical Society

Received: June 11, 2016 Accepted: August 16, 2016 Published: August 16, 2016 22875

DOI: 10.1021/acsami.6b07049 ACS Appl. Mater. Interfaces 2016, 8, 22875−22883

Research Article

ACS Applied Materials & Interfaces

mass of the solution of the Cp6 dissolved, t was the irradiation time, I was the power of the laser (280 mW/cm−2), and τs was the slope of the line of time dependent −ln (θ). Qdis was ignored because of the same value with different pH solution. 2.7. Confocal Laser Scanning Microscopy (CLSM). MCF-7 cells (1 × 105) were cultured in the confocal dish with DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% FBS (fetal bovine serum), 1% penicillin, and streptomycin at 37 °C in humidity and 5% CO2 atmosphere. The culture medium was replaced by fresh medium without FBS after 17 h incubation, and then Cp6 monomer (10 μM) and PBS (composted with 10 mM Na2HPO4, 1.8 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, and NaCl 137 mM) were added into the medium, separately. The medium was removed after coincubation of Cp6 with cells for 10, 30, and 60 min, independently, and then the cells were washed with PBS three times. Prior to CLMS experiments, cells were incubated with Cp6 monomer for 1 h, followed by incubation with lysotracker green DND-26 (100 nM) for 15 min. The treated samples were imaged by a CLSM (Zeiss LSM710) with a 60× objective lens. 2.8. Hyperspectral Imaging. MCF-7 cells (1 × 105) were plated in a microscopic cover glass with DMEM supplemented with 10% FBS, 1% penicillin, and streptomycin at 37 °C in humidity and 5% CO2 atmosphere. The culture medium was replaced by medium without FBS after cells were attached. Then Cp6 (10 μM) in PBS were added into the medium and further incubated 2 h. The cells were fixed with fresh methanol (4%) in dark for 24 h at 4 °C, followed by washing with PBS several times. The prepared cell samples were observed by utilizing Cytoviva (integrated optical microscopy and hyperspectral imaging system (CytoViva, Inc. affiliated with Auburn University). 2.9. Tumor Mimicking Patterns Preparation. The agarose pattern was prepared to mimicking the acidic condition in tumor tissues. 0.5% agarose in PBS buffer (pH = 5.4, w/w) were heated to dissolute and then cooling naturally under room temperature in a mold. After solidification, the patterns were merged into a neutral PBS buffer (pH = 7.4) solution containing Cp6 (c = 100 μM) for permeated experiments. The TEM imagines of the aggregates were obtained by ultrasection cut of the patterns. 2.10. In Vitro Experiment. MCF-7 cells were seeded in 6-well plates at a density of 2 × 106 cells per well. Then Cp6 (10 μM) was added into the medium and cultured for different times from 10 min to 4 h at 37 °C in a humidified atmosphere containing 5% CO2. Cells were digested with trypsin and collected in centrifuge tubes for PA signal detection by using MOST (MOST 128, excitation wavelength at 689 ± 1 nm). 2.11. TEM Study of Cell Section. MCF-7 cells (1 × 107) were incubated with Cp6 (100 μM) in culture medium without FBS for 1 h. Cells were harvested and washed for fixation by 3% glutaraldehyde in dark for 2 h at 4 °C. After primary fixation above, the cells were washed with PBS (5 times) and subjected to secondary fixation with 1% osmium tetroxide (100 mM-phosphate buffer) in dark at 4 °C for 1 h followed by washing with phosphate buffer (100 mM). Subsequently, cells were passed through series of 50%, 75%, 85%, 90%, and 100% ethanol v/v 15 min for each. Cells were further dehydrated with 100% ethanol (20 min × 3). Next, dry ethanol and resin (1:1) was added into the cells for 2 h after dehydration. 100% resin was further added and cultured for 1 h, followed by polymerized for 24 h at 60 °C. Finally, the resin blocks were carefully trimmed to expose the underlying agar blocks. Sections of various thicknesses were cut using Leica Ultracut UCT microtome and transferred to 300 m copper grids. Samples were stained with uranyl acetate, lead citrate and Zn(NO3)2 successively in dark at room temperature and ready for TEM studies. 2.12. Cytotoxicity of Cp6. MCF-7 cells were seeded in 96-well plates at a density of 1 × 105 cells per well. Cp6 (pH = 7.4) with different concentrations (5−500 μM) were added into the medium and cultured for 12 and 24 h for cell viability tests by kit-8 assay (CCK-8). 2.13. In Vivo Experiment. BALB/c nude female mice were purchased and all animal experiments were conducted by the regulation of department of laboratory animal science, Peking University Health Science Center. MCF-7 cells (5 × 106) in PBS (100 μL) solution were subcutaneously injected into the right flank of BALB/c nude mice. The initial body weight of mice was about 17−18 g. After 3 weeks of tumor

2. EXPERIMENTAL SECTION 2.1. UV−vis Absorption and Fluorescence of Cp6 at Different pH Conditions. The Cp6 (2.5 mM) was dissolved into McIlvaine buffer (composite with sodium dihydrogen phosphate and citric acid with total ion strength of 150 mM) as stock solution. During the experiments of UV−vis absorption, the Cp6 stocking solution was diluted into 50 μM and 5 μM by buffer solution with different pH values (4.1−7.4). Then, the UV−vis absorption was monitored in the range of 200−900 nm. The fluorescence intensities of the Cp6 at different pH (4.9−8.0) were measured using fluorescence spectrometer (F-280) with the excitation wavelength at 600 nm (isosbestic point). The concentration of Cp6 in McIlvaine buffer was 5 μM. The statistical data of FL were collected at the maximum emission point at 672 nm. 2.2. PA Imaging of Cp6 Solution with Different pH Values. The Cp6 (5 μM) in McIlvaine buffer with different pH values (in a range of 4.1−7.4) were measured in agarose tubes. The PA signals (encoded through mean pixel intensity of the same area of the images) were obtained by scanning from 660 to 720 nm by the multispectral optacoustic tomography (MOST 128). The statistical data were collected at the most distinction detection window (around 700 nm) for acidic to neutral condition. 2.3. TEM Study of Cp6-Based Self-Aggregates. The morphology and size of Cp6-based self-aggregates were examined on a Tecnai G2 20 S-TWIN TEM at an acceleration voltage of 200 kV. The TEM samples were prepared by drop-coating 2-μL of Cp6 (10 μM) solutions with pH values of 7.4 or 5.4 onto carbon-coated copper grids. The liquid was removed with a filter paper after 1 min. Then the sample was stained for 2 min Zn(NO3)2 (50 μM) after 25 s staining with uranium acetate. 2.4. Study of Cp6 Aggregation in Agarose Pattern. The artificial tissue was prepared with 0.5% agarose solution at pH = 5.4. After the pattern was cooled down to room temperature, the pattern was soaked into the solution of Cp6 (100 μM) with pH = 7.4. When Cp6 was slowly diffused into the agarose for 12 or 36 h, the agarose was taken out and warmed up to melting. Afterward, the solution was diluted to 10fold with water and detected with UV−vis absorption, CD spectra, and TEM. 2.5. Investigation of Light-Thermoconversion in a PDMS Chamber. The solutions of Cp6 (500 μL) in pH values of 7.4 or 5.4 were injected into polydimethylsiloxane (PDMS) chambers. The temperatures of respective solutions were recorded through thermal camera under the laser irradiation at wavelength of 690 nm (280 mW/ cm−2). 2.6. Calculation of the Enhanced PA Signal. To investigate the PA signal after formation of self-aggregates in acidic condition, the value of η should be calculated first and the related equations are as follows: The equation of absorption coefficient (μa, cm−1) μa = σMNA

(1)

σ = 1000 ln(10)ε/NA

(2)

where σ was absorption cross section, ε was molar absorption coefficient, and NA was Avogadro’s number. Γ was Grueneisen parameter (dimensionless)

Γ = c 2β /C p

(3)

where c was the speed of acoustic monitored in MOST, β was the volume expansion coefficient, Cp was the isobaric heat capacity (4.2 J/ mol·K).

η = β /C p = [hs(Tmax − Tevir) − Q dis]/I(1 − 10−Ab690)

(4)

hs = mC p/τs

(5)

τs = − ln(θ )/t

(6)

θ = (T − Tevir)/(Tmax − Tevir)

(7)

where Tmax was the maximum temperature increase of the solution under irradiation time (10 min), Tevir was the temperature of the environment, Ab690 was the absorption of the Cp6 at 690 nm, m was the 22876


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ACS Applied Materials & Interfaces growth, Cp6 and ICG (Indocyanine Green, 60 μM, 200 μL) were dispersed into PBS was intravenous injected into mice through tail vein. Mice were scanned with MOST (mode MOST 128, excitation wavelength at 689 ± 1 nm for Cp6, 810 ± 1 nm for ICG) at different times.

and S2). For further in vivo application, the complex condition with serum should be considered during the aggregation process. Besides the acidic condition induced aggregation, Cp6 (10 μM) in 10% fetal bovine serum (FBS) PBS exhibited some aggregates, which might be due to the hydrophilic monomer of Cp6 had insignificant affinity to hydrophobic domains of proteins (see Figure S3). In addition, the fluorescence intensities of protonated Cp6 (c = 5 μM) were gradually quenched with decrease of pH from 8.0 to 4.9 (Figure 1b) under the excitation of 600 nm. These results may be attributed that the aggregation induces an interconversion decay dominating over other decay processes from the S1 state that explains the fluorescence quenching observed.46 The nonradiative relaxation relates to the heating effect that is favorable to generate the transient thermoelastic expansion of substance and PA signals.39 According to the UV− vis absorption of protonated Cp6 in aggregated state, the maximum PA signal (detected by multispectral opt-acoustic tomography MSOT 128, iTheraMedical Germany, under the pulsed laser irradiation) were recorded at the window of 689 ± 1 nm. In this section, the solutions of Cp6 (c = 5 μM) with different pH values from 8.0 to 4.9 were injected into the agarose patterns and scanned. The average PA signal intensities were recorded through mean pixel intensity at the same area. As shown, the PA signal intensity of Cp6 was remarkably increased when pH ≤ 6. The intensities of PA signal at pH = 5.4 and 4.9 enhanced almost 700 and 1000 times compared with that of at pH = 7.4 (Figure 1b).The representative fluorescence spectra and the photographs of PA patterns of Cp6 at pH = 7.4 and 5.4 were exhibited in Figure 1c, separately. In order to further validate the dynamic equilibrium of self-aggregation by PA signal, the PA intensity was imaged at the aggregated and monomeric states, separately. The results implied that the aggregated state induced a higher PA signal than that of monomeric state at the detection window at 700 nm (see Figure S4). Meanwhile, morphology of protonated Cp6 in aggregated state was investigated by transmission electron microscopy (TEM) and DLS. The aggregates of protonated Cp6 (c = 10 μM) were formed at pH = 5.4 and their sizes represented a time-dependent behavior. As showed in Figures 1d and S5, the sizes of aggregates meatured from TEM images increased from 3 ± 2 nm at 30 min to 25 ± 2 nm at 2 h, and growed to bigger size after incubating for 8 and 24 h. This kinetic size growth tendency was also real-time recorded by DLS (Figure S6). In parallel, no detectable aggregates were observed when pH values higher than 6 over 24 h (see Figure S7). 3.2. Mechanism of PA Signal Enhanced. Why the PA signal was enhanced upon formation of Cp6-based selfaggregates? It is well-known that the PA signal generation is from the substances converting absorbed energy into heat leading to transient thermoelastic expansion and thus wideband (e.g., MHz) ultrasonic emission detected by ultrasonic transducers.47 Wang et al. described that the PA signal was proportional to the local pressure rise47

3. RESULTS AND DISCUSSION 3.1. pH-Induced Chlorin-Based Supramolecular Aggregates. Cp6, a porphyrin derivative,42 has three ionizable carboxylic acidic groups which can be protonated in acidic condition and simultaneously form self-aggregates via π−π interactions in aqueous solutions.43 According to literature,41 the pK around 7.0 was consisted with on-site protonated species and nonprotonated species, and the pK around 4.8 was mostly consisted with two sites protonated specie and all protonated specie. Thus, upon internalization of Cp6 by cells, the partially protonated Cp6 species with enhanced hydrophobic ability would aggregate and accumulate in lysosomes (pH 5.4),44 which simultaneously formed J-type self-aggregates (Scheme 1). To Scheme 1. Schematic Illustration of in Situ Construction of pH-Induced Cp6-Based Supramolecular Self-Aggregates as PA Contrast Agents for Tumor Imaging

verify self-aggregates formation of Cp6 at the acidic condition, UV−vis spectra of Cp6 (c = 50 μM) were first investigated in McIlvaine buffer (150 mM) solutions with pH ranging from 4.1 to 7.4. The ion strength of the buffer were corresponding to 1 × PBS for further application in vivo. As shown in absorption spectra of Cp6, there were exhibited a stronger Soret band near 400 nm and a weaker Q-band at 663 nm. There was no obvious red shift at 400 nm upon decrease pH. However, a broadening of Q-band, accompanied by emerging a new band around 688 nm upon pH decrease to 5.4, was obviously observed (Figure 1a). According to the previous studies,45 the bathochromic shift of Qband suggested that Cp6 were stacked in a strongly slipped arrangement (also called J-type aggregates). Of course, during the red shift and broaden Q-band, the color of Cp6 solution was changed from olive-green to dark-green when the aggregation occurred (Figure 1a, indicated by the pink arrows). The same phenomenon was observed at the low concentration of Cp6 (5 μM) at different incubation time (0.5 and 12 h, see Figures S1

q ∝ ΓημaF

where Γ was Grueneisen parameter (dimensionless), μa was the optical absorption coefficient (cm−1), η was the thermal conversion efficiency, and F was the local optical fluence (J· cm−2). Although the reconstruction of the PA signal had much more relationship with many parameters, the major parameters were the η and μa of the substrates. μa could be written as the absorption cross section which was related to the ε of absorber. 22877


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Figure 1. (a) UV−vis absorption of Cp6 (c = 50 μM) at different pH values from 4.1 to 7.4. The black arrows indicated the increase and decrease trend at corresponding wavelength (663 and 688 nm). The pink arrows indicated the color of the deprotonated Cp6 (50 μM) at pH = 7.4 and protonated Cp6 species at pH = 5.4 (50 μM). The blue arrows identified the isosbestic points at 330 and 600 nm respectively; (b) the pH-dependent fluorescence (at 672 nm) and PA signal intensities (at 670 nm) of Cp6 solutions changes from pH 4.1 to 7.4, (c) the typical fluorescence spectra and the PA signal intensities of Cp6 solutions at pH = 7.4 and 5.4 with an isosbestic point excitation at 600 nm; (d) the TEM image of protonated Cp6 self-aggregates at pH 5.4 after 2 h preparation.

Figure 2. (a) Photothermal transformation photographs were taken from thermal camera. The solutions of Cp6 (100 μM) with different pH (7.4 and 5.4) were injected into a PDMS chamber and irradiated with a 690 nm-laser; (b) the time-dependent temperature increase of the Cp6 (100 μM) solutions at different pH values (7.4 and 5.4); (c) the curve of the temperature change with irradiation and decreased after shutting off the irradiation source (pH 5.4); and (d) time constant for heat transfer from the system was determined to be τs = 263.5 s by applying the linear time data from the cooling period after 420 s.

Therefore, to analyze the PA signal, we should calculate η of Cp6. The solution of Cp6 (100 μM) with pH = 7.4 and 5.4 was injected into individual PDMS chambers48 (Figure S8).

Considered the thermal conductivity of the chamber might affect the experiemnt results, we used PDMS to build the chambers. Otherwise, during the heat and cooling process there 22878


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Figure 3. Construction and characterization of the Cp6-based supramolecular self-assemblies formed in artificial tissues: (a) Agarose with pH 5.4 was soaked into the solution of Cp6 with pH 7.4. After 12 h permeation of monomer Cp6 into agarose pattern, the pattern was melted, and the solution was measured by UV−vis and CD spectra; (b and c) the UV−vis and CD spectra of Cp6 molecules in solutions (pH 7.4) and artificial tissues, respectively; (d) in situ TEM image of Cp6 aggregates in the artificial tissue.

Cp6 solution at pH 5.4 was enhanced at least 715 times compared to that of Cp6 solution at pH 7.4, which was consistent with the experimental data (700 folds) obtained in pattern. 3.3. Demonstration of Supramolecular Self-Aggregates in Situ. To provide the direct evidence for the formation of supramolecular self-aggregates in situ, the comprehensive characterizations of superstructures in tumor-mimic phantoms were carried out. The agarose pattern (0.5% agarose in McIlvaine buffer, w/w) with pH = 5.4 was used as artificial tumor tissues (Figure 3a). The agarose pattern was merged into a neutral McIlvaine buffer (pH = 7.4) solution containing Cp6 (c = 100 μM). During the permeated process, the aggregates formation and stability were monitored. As showed in TEM images, protonated Cp6 formed uniform size of spherical aggregates (Figure S12) after permeated into acidic artificial phantoms. Meantime, the aggregates stabled distributed in the phantoms up to 36 h (Figure S13). Additionally, the Cp6 molecules diffused into the pattern and subsequently formed self-aggregates were also verified by UV−vis spectra in situ. As shown in Figure 3b, the typical Q-band at 688 nm with 25 nm bathochromic shift compared to that of the monomer was observed, implying the formation of J-type aggregates in our case. Besides bathochromic shift of the long-wavelength UV−vis absorption bands, a distinct feature for protonated Cp6 aggregate with a assembled structureal charity effect was monitored by circular dichroism (CD). This effect occurred on chiral excitonic coupling of transition dipole moments and consists of two bands with opposite signs (exciton couplet, Figure 3c) which was consistent with previous studies.50,51 Encouraged by these results, the morphology of the spherical self-aggregates with a size distribution about 20 ± 5 nm were obtained from the agarose pattern (Figure 3d) after 12 h soaking. Furthermore, comparing to the fast kinetically growth of the aggregate in solution, the aggregation in agrose patterns were limited with minior growth up to 36 h incubation at room temperature. All detailed spectra of

should have no volume change and no air bubble involved, thus the PDMS chambers were sealed similar to microfluidics fabrication. 690 nm-laser was used to illuminate the samples. First, we monitored the temperature variation with the illumination time up to 10 min by using thermal camera (America, FLIR E60 thermal sensitivity = 0.05 °C; range of temperature = −20−650 °C). The results indicated that the temperature of Cp6 solution at pH = 5.4 increased much higher than that at pH 7.4, suggesting that the aggregation induced stronger heat effect at 690 nm (Figure 2a). In parallel, the timedependent temperature variation curves of the Cp6 solutions (c = 100 μM) were profiled in Figure 2b. As could be seen from the Figure 2b, the temperature increased more than 15 °C in acidic condition (pH = 5.4) compared to that in the natural condition (pH = 7.4). Meanwhile, the concentration-dependent temperature variation of Cp6 solutions (c = 5, 10, 50, and 100 μM) were also investigated at the acidic condition (pH = 5.4) (Figure S9). The results showed that the temperature raised with the concentration increased from 5 to 100 μM. ΔT reached 7−8 °C even at the lowest concentration of Cp6 (5 μM), we believed that the obvious heat effect will directly contribute to the enhanced PA signal. To quantitatively study the heat effect, we carried out the time-dependent cooling curve after shutting off the irradiation source (Figure 2c). According to the reported equations of η49 shown in the Supporting Information eqs 1−7 and the fitting curve in Figure 2d, we could calculate the value of η (39%, c = 100 μM, at 690 nm), which was 55-fold higher than that of monomer in neutral environment (0.7% calculated from the curves showed in Figures S10 and S11). Meanwhile, the apparent molar extinction coefficient (the molar concentration was calculated based on the Cp6 units including protonated and deprotonated species) of aggregates (4800 M−1·cm−1) was 13 folds higher than molar extinction coefficient of the monomer (355 M−1·cm−1) at 690 nm. Therefore, multiplied 13 folds of ε by the 55 folds higher of η (690 nm), the theoretical PA signal of the 22879


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Figure 4. BioTEM images of Cp6-based supramolecular self-assemblies inside the lysosomes of cells, (a) without treatment, (b) treated with Cp6 (10 μM in medium). Inset: Images of local amplification. L-Lysosome, N-nucleus. (c) Confocal laser scanning microscopy (CLSM) of MCF-7 cells that were incubated with PBS and Cp6 (10 μM) with a time scale from 10 to 120 min. Lysosomes were labeled with lysotracker green DND-26 for 15 min before imaging. Scale bar: 10 μm. (d) Fluorescence intensity profile of ROIs across MCF-7 cells in merged images.

formation in the lysosomes of cells. First, the intracellular localization of Cp6 was carried out by CLSM microscopy. From the results of colocalization of Cp6 (red) and lysosomes (lysotracker green) showed in Figure 4c, we deduced that the monomer of Cp6 mainly dispersed in lysosomes of MCF-7 cells at the beginning of 1 h. The disappeared fluorescence after 2 h incubation might suggest the aggregation of protonated Cp6 (Figure 4c and 4d). More convincing evidence were provided by the hyperspectral imaging technique with the same incubation process of Cp6 to cells, which could in situ acquire the spectrum for individual pixel in the images, identifying optical property and composition information on materials.53 From the results showed in Figure 5a, the aggregates formed inside of cells was clearly observed which was consistent with the aggregated formed in buffer (pH = 5.4). And no aggregates were observed in control cells. The spectral mapping results were also confirmed that aggregates formed in MCF-7 cells treated with Cp6 (2 h, 10 μM). The maximum spectral response of protonated Cp6 aggregates in cells was 580 nm, which was consistent with that of aggregates formed in buffer (pH = 5.4) and higher than that of cell substrates (520 nm) (Figure 5b).

UV−vis and CD, as well as TEM images, for indentification of the aggregates formation during permeated process were carried out in Figures S14−S16. These favorable results indicated that protonated Cp6 could form uniform self-aggregates in situ under acidic condition and these self-aggregates had high stability under physiologically relevant conditions. 3.4. Demonstrated Self-Aggregates Inside the Acidic Lysosomes. To investigate the formation of self-aggregates inside the acidic lysosomes (pH 5.4)52 of cancer cells, we employed bioTEM to image in situ self-assembled nanoaggregates of Cp6 (10 μM in medium) incubated with MCF-7 cells (2 × 106 cells) for 2 h. As shown in Figure 4a and 4b, the average size of nanoaggregates was approximately 25 ± 5 nm, which was correlated well with the TEM results in buffer and tumor-mimic phantom, which was mainly accumulated in the lysosomes. To identified nanoaggregates observed was in source of the aggregates of prononated Cp6 species, in which coordinated with Zn 2+, the energy disperse spectroscopy (EDS) analysis were carried out to confirmed the observation above (Figure S17). Besides bio-TEM technology, CLSM and Cytoviva microcopies were also employed to check the aggregate 22880


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Figure 5. (a) Hyperspectral imaging of Cp6 aggregates formed in MCF-7 cells compared with the aggregates formed in buffer (pH = 5.4) and (b) their spectral mapping of aggregates in cells after 2 h incubation. (c) Time-dependent images of PA signals after incubation MCF-7 cells (2 × 106 cells per well of 6-well plates) with Cp6 (10 μM). (d) The quantification of the PA signals of Cp6 self-assemblies in cells after different incubation times.

Figure 6. PA signals of Cp6 (a) and ICG (b) in MCF-7 xenografted mice with different treatment times from 0 to 4 h. Red star: Tumor. Red triangle: Surrounding tissues. (c) The quantification of the PA signals of Cp6 and ICG in tumors after treatments at different times (0−4 h). (d) Tumor-tonormal tissue ratio (T/N) of Cp6 and ICG for tumor imaging (red star and red triangle identified tumor and surrounding normal tissues). The statistic signal intensity for T/N was calculated through the average signal intensity of the same area of the tumor and the surrounding tissues. The dashed lines in panels a and b indicated the skin and the outlines of the tumor.

3.5. In Vivo Self-Aggregation of Cp6 and the Specific Tumor PA Imaging. Finally, in vivo self-aggregation of protonated Cp6 and the specific tumor PA imaging were carried out. Cp6 dispersed in the PBS (200 μL, 60 μM, pH = 7.4) was injected intravenously into MCF-7 tumor-bearing mice (n = 3) (Figure S19). The PA signals were observed at 1 h postinjection (Figure 6a) and intensity kept increasing to a plateau from 2 to 4 h administration. The gradually enhancement of PA signal was ascribed to (i) the accumulation of protonated Cp6 aggregates in acidic lysosomes of tumor cells and (ii) the resulting nanosized aggregates prolonged the residence time in tumor site. The deprotonated Cp6 was quickly eliminated with low distribution in the normal tissues surrounding the tumor mass. The PA properties of Cp6-based supramolecular aggregates were compared with the indocyanine green (ICG) which was

The enhanced PA signal of self-aggregates inside lysosomes of cancer cells was further investigated under the detection window of 700 nm. The PA signal was significantly enhanced after 1 h incubation and reached the maximum at 2 h (Figure 5c). From the quantification of the PA signals with different incubation times, we found that the PA signal enhancing inside the cells was time-dependent. The signal enhanced almost 200 folds at 1 h incubation and reached to maximum at 2 h, which enhanced almost 400-fold compared with that of control (Figure 5d). For development of Cp6 as pH induced PA contrast agents in vivo, the cytoxicity of the Cp6 was also examined. MCF-7 cells were incubated with different concentrations of Cp6 (2.5−100 μM). No apparent cytotoxicity was observed after 12 and 24 h treatments (Figure S18). 22881


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approved by FDA for routine clinical use. ICG as a PA contrast agent produced a strong PA signal at 810 nm.54 ICG (200 μL, 60 μM) was injected intravenously into MCF-7 tumor-bearing mice (n = 3, Figure S19). The PA signal of ICG reached maximum at 1 h and quickly decreased after 2 h injection and even disappeared at 4 h (Figure 6b). The margin of solid tumor was not clearly visible by using ICG as the contrast agent because of the nonenvironment activatable property. The small hydrophilic ICG could be quickly removed by mice after 2-h administration. The quantitative time-dependent PA signals vs different contrast agents were profiled in Figure 6c. We defined that the PA signal ratio of tumor to surrounding tissues as tumor-tonormal tissue ratio (T/N) for determining the sensitivity and specificity of the imaging. We compared the T/N of the in vivo constructed supramolecular aggregates and ICG golden standard side-by-side. At the same concentration (200 μL, 60 μM), the T/ N of mice group treated by Cp6 was 56-times higher than that of ICG after 1 h administration (Figure 6d). Significantly, about 250-time T/N was achievable in 2−4 h detection window. The significant enhancement of the T/N was attibuted to the assembly induced retention (AIR) effect.

4. CONCLUSIONS In conclusion, we first reported in situ construction and characterization of pH induced Cp6-based supramolecular Jaggregates as PA contrast agents for tumor imaging. The resulting of self-aggregates could be induced inside the tumor cells and utilized as PA contrast agents with at least 2 orders of magnitude increase of the T/N for tumor diagnosis. This study paves the way to develop tumor environment-induced supramolecular self-assemblies for cancer diagnostics and therapeutics.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07049. Experimental details (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

W.-J.L. and D.Z. contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2013CB932701), National Natural Science Foundation of China (31671028, 21374026, 21304023, 21506161, and 51303036). Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541) and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA09030301).

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REFERENCES

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