Chlorophyll-Based Near-Infrared Fluorescent Nanocomposites: Preparation and Optical Properties.

Near-infrared (NIR) fluorescence has attracted much attention in biomedical fields because it offers deep tissue penetration and high spatial resolution. Herein, a method is developed for the preparation of NIR fluorescent nanocomposites (NCs) by encapsulating natural chlorophyll (Chl) into the micelles of octylamine-modified poly(acrylic acid) (OPA). Both femtosecond transient absorption spectra and isothermal titration calorimetry thermogram reveal that the micelles of OPA provide a hydrophobic environment for the improved fluorescence efficiency. Hence the resulted Chl NCs possess unique properties such as ultrasmall size, outstanding photostability, good biocompatibility, and superbright NIR fluorescence emission. In vivo imaging of sentinel lymph node is achieved in nude mice, demonstrating the potential of Chl NCs in biomedical applications. This work provides a new strategy for the preparation of highly biocompatible NIR fluorescence labeling nanocomposites.

Introduction

Recently, the development of life science and biomedicine has stimulated the rapid improvement of in vivo fluorescence-based imaging techniques. The fabrication of fluorescent-emitting materials has developed rapidly. So far, the most widely used fluorescent-emitting materials are organic dyes, fluorescent proteins, and Cd-containing quantum dots (QDs). These materials generally emit visible fluorescence and have a deficiency for in vivo imaging due to poor tissue penetration depths. In contrast, near-infrared (NIR) fluorescence can be less interfered by autofluorescence, scattering, and absorption of biological substances, providing deep-penetrating radiation to probe biological events in living organisms.[1−3]

So far, two kinds of NIR fluorescent emitting materials are well known for in vivo applications. One is organic dyes including indocyanine green,[4] IR-780,[5] IR-1061,[6] etc. However, they also have some inevitable disadvantages such as low fluorescence intensity, insufficient photostability, and poor water solubility, which hinder their bioapplications. The other is nanomaterials including CdTe/CdSe,[7] Ag2S,[8] graphene oxide,[9] Si nanoparticles,[10] gold nanoparticles,[11] semiconducting polymer dots,[12] etc. Unfortunately, the toxicity and relatively large size of the present NIR nanomaterials are concerned for in vivo applications. Therefore, NIR fluorescent emitting materials with high fluorescence intensity, small size, less cytotoxicity, and good water dispersity are urgently required to be developed for in vivo applications.

Employing biocompatible fluorescent molecules extracted from vegetables to prepare desirable fluorescent nanoprobes is an attractive strategy for in vivo imaging.[13,14] Chlorophyll (Chl) with NIR fluorescent emission at 675 nm, an environmentally friendly fluorescent dye that is widely distributed in higher plants, is generally used as a food additive.[13,14] Therefore, Chl can be an excellent NIR fluorescent biolabeling candidate for in vivo imaging. Nonetheless, natural Chl with poor water solubility and minimum fluorescence emission cannot be directly applied for in vivo imaging. The effective improvement of water solubility and fluorescence intensity of natural Chl is the prerequisite for its bioapplications. In a pioneering work, Chu et al. reported that employing liposome or pluronic F68 to prepare Chl nanocomposites can effectively improve the water solubility and fluorescence intensity of natural Chl.[13,14] However, the resulted nanocomposites lack appropriate functional sites on the surface, which may result in negative effects for further bioapplications.

Herein, natural Chl extracted from spinach leaves was encapsulated with octylamine-modified poly(acrylic acid) (OPA).[15] Thus, the resulted Chl-based nanocomposites (NCs) with small size, bright NIR fluorescence emission, good water solubility, and excellent biocompatibility are achieved. The as-prepared Chl NCs are successfully applied in sentinel lymph node (SLN) imaging, demonstrating its prospect for in vivo applications.

Experimental Section

Materials and Instruments

Spinach was purchased from a local supermarket (Wuhan, China). Bladder cancer cell lines EJ (EJ cells) were purchased from China Center for Type Culture Collection (Wuhan, China). All the media for cell culture were purchased from Gibco Corp (CA). Ultrapure water (18 MΩ·cm) was prepared with a Millipore Milli-Q system (Billerica). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (CA). All the other chemical reagents, including chloroform, octylamine, 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC), ethanol, poly(acrylic acid) (molecular weight = 1800), dimethylsulfoxide (DMSO), and N,N-dimethylformamide (DMF), were purchased from China National Pharmaceutical Group Corporation.

Extraction and Characterization of Chl

Chl was extracted from spinach leaves. Briefly, 10 g of smashed spinach leaves were mixed with 200 mL of ethanol and shaken at 120 rpm for 2 h. Subsequently, the Chl–ethanol solution was filtered and centrifuged at 10 000 rpm for 3 min.

Since one Chl molecule contains one magnesium atom, the concentration of Chl can be calculated by means of its magnesium contents according to the atomic emission spectrometry by the following equationwhere X and Y represent the contents of Chl and magnesium, respectively.

Preparation and Characterization of Chl NCs

OPA (50 mg) dissolved in ethanol was mixed with the Chl–ethanol solution. Subsequently, a membrane layer was obtained under vacuum and dissolved in 4 mL of a borate saline buffer (5 × 10–2 M, pH 9) to obtain Chl NCs. The resulted Chl NCs were centrifuged at 10 000 rpm for 3 min and stored at 4 °C. The concentration of Chl NCs was represented by the contents of Chl in them, which can be calculated by means of its magnesium contents by atomic emission spectrometry.

UV–vis spectra were recorded with a UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan), and photoluminescence spectra were recorded with a Fluorolog-3 fluorescence spectrophotometer (Shimadzu, Tokyo, Japan). The absolute photoluminescence quantum yield was measured using an absolute PLQY measurement system C9902-02 (Hamamatsu photon, Hamamatsu, Japan) with all samples excited at 450 nm. The hydrodynamic diameters of Chl NCs were measured by a Malvern Zeta-sizer Nano-ZS instrument (Malvern, UK). Transmission electron microscope images were obtained in a JEM2010FEF (JEOL, Tokyo, Japan) microscope operated at an accelerating voltage of 150 kV. Chl NCs were diluted to an appropriate concentration and stained with 2% of phosphotungstic acid for TEM measurements.

Fluorescence Imaging In Vivo

Two hundred microliters of Chl NCs aqueous solution (1.2 mg mL–1 of Chl) was intradermally injected into the paws of nude mice. The fluorescence images of SLN were acquired by the CRi Maestro in vivo fluorescence imaging system (Woburn, MA). The fluorescence images were analyzed using Maestro 3.0 software. The excitation wavelength, emission wavelength, and exposure time were 635 nm, 675 nm, and 500 ms, respectively. The nude mice were killed at 1 day after the injection of Chl NCs, and their organs were resected to obtain the biodistribution images of Chl NCs in vivo.

Results and Discussion

Preparation and Characterization of Chl NCs

Chl was extracted from spinach leaves according to a previous report.[13,14] OPA, a significant amphiphilic polymer that is widely utilized to make hydrophobic nanoparticles water soluble,[15] was synthesized and used to encapsulate Chl based on hydrophobic interactions (Figures a and S1 and Table S1). As shown in Figure b, Chl NCs display good water solubility and bright fluorescence emission after OPA encapsulation. Compared with Chl dispersed in the buffer, the photoluminescence quantum yield (PLQY) of Chl NCs is obviously improved from ca. 0 to ca. 10.8%. This is in agreement with Chu’s results that the fluorescent intensity of liposome-coated chlorophyll nanocomposites is higher than that of Chl dispersed in water.[13]

Figure 1

(a) Schematic diagram for the preparation of Chl NCs. (b) Bright field and the corresponding fluorescence images of Chl (1: Chl in borate saline buffer; 2: Chl in ethanol; 3: Chl NCs in borate saline buffer). (c) Absorption spectra and (d) normalized fluorescence spectra of Chl under different environments (1: Chl in borate saline buffer; 2: Chl in ethanol; 3: Chl NCs in borate saline buffer). (e) Photostability of Chl NCs (black), Chl in ethanol (green), and indocyanine green (red) under continuous irradiation with a 50 W mercury lamp. (f) TEM image, (g) size distribution histogram, and (h) hydrodynamic diameter distribution of Chl NCs.

Moreover, both the absorption and the photoluminescence (PL) spectra of Chl exhibit profiles identical before and after OPA encapsulation (Figure c,d), suggesting that the molecular structure of Chl is maintained during the water-solubilization processes. In addition, compared with the indocyanine green and Chl dispersed in ethanol, Chl NCs show good photostability (Figure e), implying that they can be applied in long-time imaging of a biological event.

The transmission electron microscopy (TEM) image shows that Chl NCs are spherical, with no obvious irregular aggregates observed (Figure f). The corresponding statistic diameters are 20 ± 5 nm (Figure g). Moreover, the average hydrodynamic diameter of Chl NCs is 9.3 nm (Figure h), indicating that Chl NCs are beneficial to metabolism in vivo.

The Origins of Chl NC Fluorescence

To understand the origins of Chl NC fluorescence, femtosecond transient absorption (TA) spectroscopy, a powerful tool that is widely used to investigate the excited-state decay process of fluorophores,[16] was utilized to collect the TA spectra of different Chl samples. As shown in Figure a, no obvious stimulated emission (SE) is observed, indicating that Chl dispersed in a buffer has the minimum fluorescence. These results are in accordance with the results that the PLQY of Chl dispersed in a buffer is almost 0 (Figure d, sample 1).

Figure 2

TA spectra of Chl in a borate saline buffer (a), Chl in ethanol (c), and Chl NCs in a borate saline buffer (e). The corresponding schematic illustrations of the possible excited-state decay process (b: Chl in a borate saline buffer; d: Chl in ethanol; f: Chl NCs in a borate saline buffer). In (2b), (2d), and (2f), the B band, Qx band, and Qy band are referred to previous reports.[19]

As shown in Figure c,e, the region from 640 to 690 nm (negative signals) can be attributed to stimulated emission (SE),[17] which is in agreement with the results of steady fluorescence (Figure d, samples 2 and 3). The region from 470 to 640 nm (positive signals) can be attributed to excited-state absorption (ESA),[17] which coincided with the results of Figure c. The region with a wavelength shorter than 470 nm is corresponding to the ground-state bleaching (GSB).[18,19]

As shown in Table S2 and Figure S2, the global analysis for the TA data presented contains three components of different time scales. The first component is a very short component of only the femtosecond scale, which can be attributed to the fast internal conversion process due to the existence of an isosbestic point at 490 and 640 nm (Figure S3).[18,19] Another component is a lifetime component of the picosecond scale, which can be attributed to the solvent relaxation and in accordance with previous reports.[18,19] Thus the remaining component of the nanosecond scale can be safely assigned to the radiation recombination (fluorescence); this is in agreement with the results in Figure d (samples 2 and 3). The results of TA spectra indicate that Chl NCs in a borate saline buffer and Chl in ethanol have similar excited-state dynamic processes.

Possible excited-state decay processes of Chl in different environments are illustrated in Figure and Table S2. For Chl dispersed in a borate saline buffer (Figure b), the energies of excited-state Chl are quickly dissipated by water; thus no obvious fluorescence can be observed. On the contrary, dispersion in ethanol (Figure d) or encapsulation into OPA micelles (Figure f) is beneficial to enhance their fluorescence, suggesting that a hydrophobic environment is provided.

Formation Procedure of Chl NCs

To investigate the formation procedure of Chl NCs, isothermal titration calorimetry (ITC), a powerful method that is widely used for the investigation of the energies of molecular interactions by recording a thermogram during a titration,[20,21] was utilized to understand the formation procedure of Chl NCs. First, the critical micelle concentration (CMC) of OPA was measured by an interface tensiometer. As shown in Figure a, surface tension remains constant when the concentration of OPA is higher, 9 μM, which can be considered to reach the CMC of OPA.

Figure 3

(a) Surface tension of OPA as a function of concentration. (b) ITC curve of heat change as a function of OPA concentration.

Subsequently, OPA dispersed in a borate saline buffer was dropped into Chl suspension in the cell of isothermal titration microcalorimeter. With the addition of OPA, the heat presents an obvious decrease and reaches a stationary phase at an OPA concentration of ∼31 μM (Figure b). It can be inferred that when OPA concentration exceeds the CMC (∼31 μM, point A), Chl molecules begin to be transferred into OPA micelles. This is in agreement with the results of the interface tensiometer that the CMC of OPA is ∼9 μM (Figure a).

As shown in Figure b, when OPA concentration is higher than 185 μM, the heat decreases prominently (point B); it can be inferred that Chl is completely transferred into OPA micelles. Finally, with the further addition of OPA, a relatively small change in heat that relates to dilution can be observed. The corresponding fitting results show that the enthalpy change (ΔH) and the entropy contribution (ΔS) are 29.67 kJ·mol–1 and 212.2 J·mol–1·K–1, respectively. These results suggest that Chl molecules are spontaneously encapsulated into OPA micelles, providing an enhanced fluorescence of Chl under hydrophobic environments.

Cytotoxicity and In Vivo Toxicity of Chl NCs

To assess the biocompatibility of Chl NCs in vitro, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), a standard cell viability assay reagent,[22,23] was utilized to examine the cytotoxicity of Chl NCs on EJ cells. Briefly, EJ cells were incubated with Chl NCs at different concentrations. As shown in Figure a, the viability of EJ cells is maintained above 90% after being incubated with Chl NCs for 24 and 48 h, indicating the good biocompatibility of highly bright Chl NCs at these dosages.

Figure 4

(a) MTT assay on EJ cells exposed to Chl NCs at different concentrations for 24 and 48 h. Evolution of mouse body weight after the injection of Chl NCs (b) and PBS only (c). (d–f) In vivo fluorescence images of nude mice after the injection of Chl NCs at 1 h, 1 day, and 3 day postinjection, respectively. (g) Ex vivo fluorescence image of excised organs after injecting Chl-OPA at 1 day postinjection. (h) In vivo SLN mapping with the injection of Chl NCs at 30 min postinjection. The excitation wavelength was 635 nm, and the emission wavelength was 675 nm. All images were acquired under the same condition.

The biosafety of Chl NCs in vivo was further evaluated; Chl NCs were then intravenously injected into healthy nude mice to evaluate toxicity, clearance, and biodistribution.[10] As shown in Figure b,c, no significant changes in weight can be observed for more than 4 weeks postinjection, with no changes in drinking, eating, activity, or exploratory behavior were observed. This strongly suggests the low toxicity of the as-prepared Chl NCs at this dosage. As shown in Figure d–f, the fluorescence almost completely disappears after 3 days, implying that Chl NCs can be easily cleaned by nude mice. All results show that Chl NCs can be used for the fabrication of biocompatibile fluorescence nanoprobes. The biodistribution of Chl NCs at day 1 after the injection of Chl NCs is shown in Figure g, indicating that Chl NCs are mainly uptaken by the liver, spleen, and kidneys.

In Vivo Imaging with Chl NCs

One promising tumor-related application of NIR fluorescence is SLN imaging. SLN is defined as the first lymph node to receive lymphatic drainage from primary cancer.[24] In general, cancer may have spread if cancer cells are found in the SLN.[25] Therefore, the imaging of the SLN is an important technique for surgeons to decide whether a patient needs surgery.[8] Herein, the prospect of the as-prepared Chl NCs in vivo was also demonstrated. As shown in Figure h, the axillary region of nude mice emits bright fluorescence with the injection of Chl NCs into nude mice paw at 30 min postinjection. Combined with the result that chlorophyll dispersed in water does not emit fluorescence (Figure b, sample 1), it can be concluded that there is no leaching problem for the embedded chlorophyll in our experiments. To verify the accurate location of SLNs by Chl NCs, the SLNs were resected and imaged; the results of Figure S4a showed that a bright fluorescence emission was observed in the excised SLN with the injection of Chl NCs. In contrast, no fluorescence can be observed in the controls (Figure S4b), indicating that the SLNs are accurately located by Chl NCs. Thus Chl NCs are demonstrated to have the potential for in vivo imaging.

Compared with convention fluorescent labeling materials, four main advantages of Chl NCs should be highlighted. First, Chl is generally used as a food additive; thus, nontoxic Chl-based NCs can be applied for in vivo imaging. Second, Chl NCs emit at 675 nm, allowing for in vivo imaging of deep tissues.[26] Third, the preparation process of Chl NCs is simple, avoiding complex synthesis procedures and decreasing time and labor consumption. Last but not the least, Chl NCs can be easily visualized with that of the present commercial imaging devices, benefiting surgery operations such as tumor resection.

Conclusions

In summary, based on natural Chl extracted from spinach leaves and biocompatible OPA, a strategy for the preparation of NIR fluorescence NCs is reported. The as-prepared NCs possess unique properties including bright NIR fluorescence, small diameter, excellent photostability, and good biocompatibility. Both TA spectra and ITC thermogram results suggest that Chl molecules are spontaneously transferred into OPA micelles, providing a hydrophobic environment for the improvement of Chl fluorescence efficiency. The successful application of Chl NCs in SLN imaging demonstrates its prospects in the field of in vivo imaging.