Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer
© Cohen and Margel; licensee BioMed Central Ltd. 2012
Received: 7 May 2012
Accepted: 23 July 2012
Published: 14 August 2012
The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors.
The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method.
Results and discussion
Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis.
These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.
Early detection of adenomatous colonic polyps is a major concern in the prevention of colon cancer [1, 2]. Colorectal cancer can largely be prevented by the early detection and removal of adenomatous polyps. A variety of colorectal cancer screening modalities are available, including stool-based tests and radiological and endoscopic examinations of the colon [3, 4]. These methods are considered to be either lacking in sensitivity or invasive, and colon cancer continues to be a major cause of death in the western world. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors.
The key to effective specific imaging in deep tissues is the use of NIR light . Absorption, light scattering and autofluorescence are limitations to fluorescence imaging that are significantly reduced in the NIR region (approximately 700 to 1000 nm). Biological chromophores, particularly hemoglobin, strongly absorb visible light, thereby limiting the depth of penetration at shorter wavelengths to a few millimeters. Other biological compounds such as water and lipids strongly absorb light in the infrared region. In addition, the decreased light scattering and autofluorescence in the NIR region provide the non-fluorescent background necessary for optimal detection of a fluorophore within the surgical field [6, 7]. Nanoparticle-based NIR probes have been shown to have significant advantages over free organic NIR dyes such as enhanced photostability and biocompatibility, improved fluorescent signal (a large number of dye molecules per nanoparticle) and easy conjugation of biomolecules to the nanoparticle surface functional groups .
There is growing interest in the fabrication of albumin nanoparticles due to their biocompatibility, biodegradability and non-antigenicity [9, 10]. Albumin is one of the most commonly used and characterized proteins in the pharmaceutical field . Soluble albumin and albumin nanoparticles are currently used as a delivery vehicle in chemotherapy as they are known to accumulate and to be catabolized by cancerous tumors [12–14]. NIR dyes such as Indocyanine Green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin . Previous studies also showed that the derivatization of cyanine dyes with carboxylic group/s increases their binding affinity to albumin . Encapsulation of dye molecules within the albumin matrix plays a role in shielding the dye against reactive oxygen species thereby reducing the risk of photobleaching.
In this work, we have exploited the high affinity of cyanine dyes to albumin for preparation of fluorescent NIR albumin nanoparticles. The dye chosen for use throughout this work is a previously synthesized carboxylic acid derivative of the commercially available NIR dye IR-783 (CANIR, Figure 1). Leakage of the entrapped NIR dye into PBS containing 4% albumin and into human bowel juice was not detected. NIR fluorescent HSA nanoparticles were bioactivated by covalently conjugating targeting agents such as PNA, anti-CEA antibodies (anti-CEA) and anti-TAG-72 antibodies (anti-TAG-72) to the nanoparticle surface. These bioactive NIR fluorescent HSA nanoparticles were found to specifically detect colon cancer tumors, as demonstrated in vivo in a mouse model.
Results and discussion
Leakage of the encapsulated CANIR dye into PBS dispersion in absence or presence of 4% soluble HSA was not detected following ultrafiltration, as described in the experimental section. There was also no leakage observed when these fluorescent nanoparticles were dispersed in human bowel juice instead of PBS. These results indicate that the CANIR is strongly associated with the albumin nanoparticles, as already known from the literature , and the free dye does not leach into the continuous phase (PBS or bowl juice).
Optimization of the CANIR concentration entrapped within the HSA nanoparticles
In order to optimize the fluorescence intensity of the NIR fluorescent nanoparticles, different concentrations of the CANIR dye were added to the 4% aqueous HSA solution, followed by the formation of the fluorescent nanoparticles via the precipitation process described in the experimental part. The concentration of the entrapped CANIR dye that provided the maximum fluorescence intensity of the NIR fluorescent HSA nanoparticles was 3.0 μg per 0.5 mg particles in 1 mL PBS. At higher dye concentrations, quenching of fluorescence was observed, due to the short distance between the dye molecules entrapped within the nanoparticles, leading to non-emissive energy transfer between them.
Photobleaching of the NIR fluorescent HSA nanoparticles
In vivo optical imaging of human colon tumors in a mouse model
Similar mouse trials with 4 h recovery time were performed with targeting agents other than PNA, e.g., monoclonal antibodies against CEA and against TAG-72. For this purpose we conjugated these antibodies to the NIR fluorescent albumin nanoparticles and tested their efficiency for tumor detection in LS174t and HT29 colorectal cancer cell lines. CEA, a highly glycosylated glycoprotein, and TAG-72, a human mucin (MUC1)-like glycoprotein complex, are both highly expressed in most human carcinomas, and therefore used as biomarkers in several modalities of human carcinoma [21–23]. Tumors originating from HT29 cell line serve as a negative control since this cell-line expresses the CEA and TAG-72 antigens to a much lower extent, at least 103 times, lower than the LS174t cell-line [24, 25].
We should also note that the mouse colon treated with the anti-TAG-72-conjugated fluorescent HSA nanoparticles shown in Figure 5A3 has two clear fluorescent signals (both with a SBR of approximately 50). The tumor in the distal part of the colon is visible to the eye, whereas the more proximal fluorescent signal was only revealed as pathological subsequent to histological analysis. This result may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles, over regular colonoscopy.
The NIR fluorescent HSA nanoparticles discussed in this work have the potential to assist in early diagnosis of colonic neoplasms. The PNA, anti-CEA and anti-TAG-72-conjugated nanoparticles specifically label in vivo the mice colon tumor tissues. While detection of cancer by these bioactivated nanoparticles has been demonstrated, the conjugation of a few targeting agents is likely to significantly enhance the fluorescent signal and the specificity, and therefore enhance their efficiency as diagnostic biomaterials.
In future work, the particles could be used for in vivo detection using either whole body fluorescence imaging or a newly developed fluorescent endoscopy camera. We plan to extend this study to include additional tumor-targeting ligands including antibodies, proteins and small peptides such as epidermal growth factor (EGF) and TRAIL [26, 27]. In addition, cancer drugs such as paclitaxel and doxorubicin may also be encapsulated within these NIR fluorescent HSA nanoparticles, so that these nanoparticles may be used for both diagnosis and therapy.
Materials and methods
The following analytical-grade chemicals were purchased from commercial sources and were used without further purification: 2-[2-[2-Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2 H-indol-2-ylidene]ethylidene]-1 cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3 H-indolium inner salt sodium salt (IR-783), HSA, 4-mercaptobenzoic acid, N,N-dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), sepharose 4B, peanut agglutinin (PNA) and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA), hematoxylin and eosin from Sigma (Rehovot, Israel); N-hydroxysulfosuccinimide (Sulfo-NHS) from Thermo Scientific, U.S.A; 2-morpholino ethanesulfonic acid (MES, pH 6) from Fisher Scientific, U.S.A; Tissue-Tek® O.C.T. from Sakura, Japan. Phosphate Buffered Saline (PBS), Minimum Essential Medium (MEM) eagle, McCoy's 5A medium and Dulbecco's modification of eagle's medium (DMEM), glutamine, penicillin and streptomycin from Bet Haemek, Israel; LS174t and HT29 tumor cell lines from American Type Culture Collection (ATCC); monoclonal anti-CEA (T86-66) and anti-TAG-72 (CC-49) antibodies were purified from the hybridoma supernatant at Alomone Labs, Israel; human bowel juice were obtained from Prof. A. Nissan, Surgery Department, Rabin Medical Center, Beilinson Hospital, Israel. Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).
Synthesis of the NIR fluorescent CANIR dye
Synthesis of the NIR fluorescent HSA nanoparticles
The NIR fluorescent HSA nanoparticles were prepared by a desolvation technique via a precipitation process [29, 30]. Briefly, ethanol (12.5 mL) was added to an aqueous solution (6.25 mL) of HSA (250 mg) and CANIR (1.25 mg). The obtained solution was then stirred at room temperature for 15 min at 800 rpm. Then, the temperature was raised to 70°C and the mixture was stirred for additional 4 h, followed by 78°C for 12 h. The obtained NIR fluorescent particles dispersed in water were then washed from impurities by sepharose 4B gel filtration.
Non-fluorescent HSA nanoparticles were prepared similarly to those of the NIR fluorescent HSA nanoparticles, in the absence of the CANIR dye.
Determination of the encapsulated CANIR concentration in the NIR fluorescent HSA nanoparticles
The concentration of the encapsulated CANIR was determined for 0.5 mg/mL nanoparticles, via a calibration curve obtained by measuring the integral of absorbance peaks of standard solutions of free CANIR in PBS (at wavelengths 630 – 900 nm).
Optimization of the CANIR concentration encapsulated within the HSA nanoparticles
Different concentrations of the CANIR dye were added to the 4% aqueous HSA solution (weight % ratio of the CANIR dye and the NIR fluorescent HSA nanoparticles were 0.1, 0.25, 0.5, 0.7 and 1), followed by the formation of the fluorescent nanoparticles. The NIR fluorescent HSA nanoparticles dispersed in PBS were diluted with PBS to 0.5 mg/mL and their fluorescence intensities at 823 nm were then measured.
Leakage extent of the CANIR encapsulated in the fluorescent HSA nanoparticles into PBS and human bowel juice
4% HSA solution in PBS containing the NIR fluorescent HSA nanoparticle (1 mg/mL) was shaken at 37°C for 4 h and then filtered via a 300-kDa filtration tube (VS0241 VIVA SPIN) at 4000 rpm. The fluorescence intensity of the supernatant was then measured at 750 nm. A similar procedure was performed with human bowel juice as a continuous phase substituting the 4% HSA solution in PBS.
Conjugation of the tumor-targeting ligands (PNA, anti-CEA and anti-TAG-72) to the NIR fluorescent HSA nanoparticles
PNA was covalently conjugated to the nanoparticle surface carboxylate groups via the cabodiimide activation method . Briefly, EDC (1 mg) and Sulfo-NHS (1 mg) were each dissolved in 0.1 M MES (pH 6.0, 1 mL) containing 0.5 M NaCl. The EDC solution (1 mg/mL, 10 μL) was added to an aqueous solution of PNA (0.25 mg, 62.5 μL), followed by the addition of the sulfo-NHS solution (1 mg/mL, 25 μL). The mixture was then shaken at room temperature for 15 min, followed by the addition of the NIR fluorescent HSA nanoparticles (2.5 mg, 1 mL PBS). The mixture was then shaken for an additional 90 min. The obtained PNA-conjugated fluorescent nanoparticles were then washed from excess reagents by sepharose 4B gel filtration. FITC-PNA, anti-CEA and anti-TAG-72 were conjugated to the NIR fluorescent HSA nanoparticles via a similar procedure substituting the PNA by the other bioactive reagents. The concentration of bound PNA (2.4 μg/mg nanoparticles) was determined with FITC-PNA via a calibration curve of FITC. The concentration of bound anti-CEA and anti-TAG-72 (2.2 ± 0.2 μg/mg nanoparticles) were determined by ELISA.
Characterization of the NIR fluorescent HSA nanoparticles
Particle size and size distribution were determined by DLS with photon cross-correlation spectroscopy (Nanophox particle analyzer, Sympatec GmbH, Germany) and by Scanning Electron Microscopy (SEM) (JEOL, JSM-840 Model, Japan). For the SEM study, the diameter of more than 100 particles was measured with the image analysis software AnalySIS Auto (Soft Imaging SystemGmbH, Germany).
Fluorescence measurements of the FITC-PNA were performed using a multiplate reader TECAN SpectraFluor Plus (Neotec Scientific Instruments) with excitation wavelength of 485 ± 10 nm and emission wavelength of 535 ± 10 nm, integration time was 40 μs and gain was set to 70.
Fluorescence spectra of the non-conjugated and bioactive conjugated NIR fluorescent HSA nanoparticles were recorded using a spectrofluorometer (Cary eclipse, Agilent Technologies Inc.). Excitation and emission slits were fixed at 5 nm, and λex was set at 750 nm. For photobleaching experiments, the samples were diluted to 0.05 M and λex was set at 800 nm and λem at 830 nm. Each of the samples was illuminated continuously, and fluorescence intensity was measured over a period of 20 min. The intensity values were normalized.
Human colorectal adenocarcinoma cell lines were used for each of the experiments. The LS174t cell line was maintained in Minimum Essential Medium (MEM) eagle supplemented with heat-inactivated FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and l-glutamine (2 mM). The HT29 cell line was maintained in McCoy’s 5A medium supplemented with FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and l-glutamine (2 mM).
Detecting of human colon tumor with the non-conjugated and bioactive- conjugated NIR fluorescent HSA nanoparticles in a mouse model
Experiments were performed according to protocols of the Israeli National Council for Animal Experiments by Harlan Biotech, Israel. Cancerous cells (30 uL containing 2x106 LS174t or HT29 cells) were injected into the mouse intestinal wall. 2 weeks later the nude mice (50 mice) were anaesthetized and treated with non-conjugated and bioactive-conjugated NIR fluorescent HSA nanoparticles (0.1%, 200 μL), via the anus, using guidance of a mini-colonoscope. 20 min later each colon was washed with PBS (5 x 1 mL) and mice were allowed to recover for 4 h. The mice were sacrificed and the colons were removed. Each colon was spread on a solid surface, and imaging was performed by using the Odyssey® Infrared Imaging System, (Li-Cor Biosciences, Lincoln, NE, USA) with excitation wavelength 780 nm and emission wavelength 800 nm.
Following the in vivo mice experiments the tissues were frozen in liquid nitrogen in Tissue-Tek® O.C.T. compound and cryosectioned at 5 microns in the cryostat. The sections were picked up on a glass slide and stained with hematoxylin and eosin (H&E) for histopathological examination. The sections were also spread on a stab, stained with H&E and visualized by SEM.
This study was supported by the Israeli Ministry of Commerce & Industry (BMP Consortium of Biomedical Photonics). The authors thank Harlan Biotech Israel for their assistance in performing the in vivo mice experiments. Thanks also Prof. Ilana Ariel from Hadassah Medical Center for her assistance in performing the histological analysis.
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