Materials Science and Engineering C 33 (2013) 923–931
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Synthesis and characterization of near IR fluorescent albumin nanoparticles for optical detection of colon cancer Sarit Cohen a, Michal Pellach a, Yossi Kam b, Igor Grinberg a, Enav Corem-Salkmon a, Abraham Rubinstein b, 1, Shlomo Margel a,⁎ a b
Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel
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Article history: Received 13 July 2012 Received in revised form 16 October 2012 Accepted 13 November 2012 Available online 21 November 2012 Keywords: HSA nanoparticles Fluorescent nanoparticles NIR fluorescence NIR fluorescent albumin nanoparticles Optical imaging
a b s t r a c t Near IR (NIR) fluorescent human serum albumin (HSA) nanoparticles hold great promise as contrast agents for tumor diagnosis. HSA nanoparticles are considered to be biocompatible, non-toxic and non-immunogenic. In addition, NIR fluorescence properties of these nanoparticles are important for in vivo tumor diagnostics, with low autofluorescence and relatively deep penetration of NIR irradiation due to low absorption of biomatrices. The present study describes the synthesis of new NIR fluorescent HSA nanoparticles, by entrapment of a NIR fluorescent dye within the HSA nanoparticles, which also significantly increases the photostability of the dye. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent albumin nanoparticles, increasing the potential fluorescent signal in tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent HSA nanoparticles was demonstrated in a chicken embryo model and a rat model. In future work we also plan to encapsulate cancer drugs such as doxorubicin within the NIR fluorescent HSA nanoparticles for both colon cancer imaging and therapy. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It has become common knowledge that the early detection of colon cancer is the key to its survival. Early detection of adenomatous colonic polyps is also of major concern in the prevention of colon cancer [1–4]. Current techniques used for colon cancer screening include double-contrast barium enema, fecal occult blood tests and colonoscopy. 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 [5]. There is therefore a need for development of more effective methods for early detection of colonic tumors. Optical imaging techniques provide functional and anatomical characterization of biological tissues, revealing important information on significant physiological parameters [6]. While optical imaging based on white light allows observation of only superficial structures, fluorescence imaging allows for observation beyond the surface. Another advantage of fluorescence imaging compared to white-light technology is the high signal-to-noise ratio that can be
⁎ Corresponding author. Tel.: +972 3 5318861; fax: +972 3 6355208. E-mail address: [email protected] (S. Margel). 1 Affiliated with the Harvey M. Krueger Family Center for Nanoscience and Nanotechnology and the David R. Bloom Center of Pharmacy of the Hebrew University of Jerusalem. 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.022
achieved. However, significant autofluorescence of bodily tissues remains a limiting factor, with fluorescence imaging in the visible region of the electromagnetic spectrum. Materials with fluorescence in the near-infrared (NIR) region (700–1000 nm) intended for use as imaging agents are of great interest, as they result in a lower background signal and deeper penetration into biomatrices [7,8]. 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 functional groups on the nanoparticle surface [9]. Nanoparticles based on silica, calcium phosphate and lipoprotein containing NIR dyes have already been developed [10,11]. Tumors can be actively targeted by nanoparticles conjugated to molecular probes that recognize tumor-specific biomarkers. Known targeting agents include antibodies, lectins, small peptides and small targeting molecules, all with upregulated receptors on the tumor cell membrane. With targeting agents conjugated to the nanoparticles, the nanoparticles bind to specific cell-surface receptors, and are often uptaken into the cell via receptor-mediated endocytosis. The intracellular concentration of nanoparticles is consequently enhanced in cancer cells compared to normal cells [12]. There is growing interest in the fabrication of albumin nanoparticles due to their biocompatibility, biodegradability and non-antigenicity.
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Albumin is one of the most commonly used and characterized proteins in the pharmaceutical field. HSA is the most abundant plasma protein with a molecular weight of 66.5 kDa and a blood half-life of 19 days [13]. Among many other physiological functions, albumin serves as a carrier for a variety of substances such as Ca2+, bilirubin, fatty acids and drugs [14]. Albumin is also known to accumulate and be catabolized by cancerous tumors [15–19]. The accumulation of HSA in tumor tissues is probably due to the enhanced microvascular permeability that allows large molecules to penetrate through to the tumor tissue, together with an impaired or absent lymphatic system (enhanced permeability and retention, or EPR) [20]. Furthermore, Stehle et al. have suggested that albumin plays an important role as a nutrient for proliferating tumors [21]. There is also evidence of active targeting of albumin, that binds to a cell surface 60-kDa glycoprotein (gp60) receptor [22]. Soluble albumin as well as albumin nanoparticles are therefore currently used as delivery vehicles in chemotherapy. NIR dyes such as indocyanine green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin [23]. Molecular modeling illustrates that albumin has two binding sites with different polarities to which hydrophobic or amphiphilic ligands bind, and there is often selectivity towards one of the two binding sites. A strong physical between the NIR cyanine dyes and albumin at these binding sites is formed, also affecting the photophysical properties of the dyes [24–26]. In this work, we have exploited the high affinity of NIR cyanine dyes to albumin for the preparation of NIR albumin nanoparticles, as well as the high affinity of albumin to tumor tissue, for their use as tumor-specific contrast agents. For increasing the specificity of the nanoparticles towards colon cancer, the NIR fluorescent HSA nanoparticles were covalently conjugated to colon cancer targeting agents such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibody (anti-CEA) [27,28]. In order to test the performance of the fluorescent nanoparticles as imaging agents for solid tumors, initial experiments were performed on tumors grown on the chorioallantoic membrane (CAM) of embryonic chicken eggs. Further assessment of the nanoparticles was performed in a rat model. The nanoparticles were found to specifically detect colon cancerous tumors, as demonstrated in both tumor implants in the chicken embryo model and in a rat model.
2.2. Synthesis of the NIR fluorescent dye A carboxylic acid derivative of the NIR dye IR-783 (CANIR) was prepared by the treatment of IR-783 (1) with 4-mercaptobenzoic acid in DMF (see Fig. 1), a method similar to that reported in the literature [29,30]. The structure was confirmed by MS, 1H and 13C NMR (see supplementary data). 2.3. Synthesis of NIR fluorescent HSA nanoparticles The NIR fluorescent HSA nanoparticles were prepared by a desolvation technique [31,32]. 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 isolated from impurities by sepharose gel filtration. The fluorescence intensity of the HSA nanoparticles was varied by changing the CANIR concentration within the HSA particles. The effectiveness of dye encapsulation was confirmed by testing for dye leaching in absence of or presence of 4% HSA, shaking RT for 4 h, and then ultrafiltration. The absorbance and fluorescence spectra of the filtrate were then measured. Non-fluorescent HSA nanoparticles were prepared similarly to NIR fluorescent HSA nanoparticles, in the absence of the CANIR dye. 2.4. Nanoparticle characterization
2. Materials and methods
Hydrodynamic particle size and size distribution were determined by dynamic light scattering (DLS) with photon cross-correlation spectroscopy (Nanophox particle analyzer, Sympatec GmbH, Germany). The mean dry diameter was determined by Scanning Electron Microscopy (SEM) (JEOL, JSM-840 Model, Japan), measuring 100 particles, using an image analysis software (AnalySIS Auto, Soft Imaging SystemGmbH, Germany). Absorbance spectra were obtained using a Cary 100 UV–Visible spectrophotometer (Agilent Technologies Inc.). Excitation and emission spectra were measured by a Cary eclipse spectrofluorometer (Agilent Technologies, Inc.). ζ-potential measurements were performed by gradual titration of the pH (11–2.5) with 1 M HCl (Zetasizer zeta potential analyzer 3000 Has Model, Malvern Instruments, England).
2.1. Materials
2.5. Determination of encapsulated CANIR
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)2H-indol-2-ylidene]ethylidene]-1 cyclohexen-1-yl]ethenyl]-3,3dimethyl-1-(4-sulfobutyl)-3H-indolium inner salt sodium salt (IR-783), HSA, 4-mercaptobenzoic acid, N,N-dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), sepharose 4B, Matrigel, peanut agglutinin (PNA), dimethylhydrazine (DMH) and fluorescein isothiocyanate–conjugated peanut agglutinin (FITC–PNA) 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; Phosphate Buffered Saline (PBS), Minimum Essential Medium (MEM) eagle, McCoy's 5A medium and Dulbecco's modification of eagle's medium (DMEM), fetal bovine serum (FBS), glutamine, penicillin and streptomycin from Biological Industries, Bet Haemek, Israel; LS174t, HT29 and SW480 cell lines from American Type Culture Collection (ATCC); monoclonal anti-CEA antibodies (T86-66) were purified from the hybridoma supernatant at Alomone Labs, Israel. Male Sabra rats (200–250 g) were obtained from Harlan Laboratories, Israel. Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).
A calibration curve of free CANIR was obtained by measuring the integrals of absorbances of standard solutions (0.5–10 μg/mL) in PBS, at wavelengths 630–900 nm. The concentration of encapsulated CANIR was determined by preparing a suspension of NIR HSA 0.5 mg/mL nanoparticles in which the integral of the absorbance spectrum at 630–900 nm was determined. An estimation of encapsulated CANIR per 0.5 mg of nanoparticles was determined according to the calibration curve. 2.6. Optimization of quantity of encapsulated CANIR Different samples of NIR fluorescent HSA nanoparticles were prepared as described in Section 2.3, with six different concentrations of the CANIR dye (0.02–1.0% w/w relative to HSA). Each nanoparticle dispersion was diluted to 0.5 mg/mL in PBS, illuminated at 750 nm, and their relative fluorescence intensities at 823 nm were measured and compared. 2.7. Photostability of NIR fluorescent HSA nanoparticles An aqueous solution of CANIR (0.05 M) in PBS was prepared, and the fluorescence intensity with λex set at 800 nm and λem set at
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Fig. 1. Synthetic scheme of the CANIR dye.
830 nm was measured. A dispersion of NIR fluorescent HSA nanoparticles also in PBS was prepared, and diluted to give a similar fluorescence intensity to the dye at the same wavelengths. The excitation slit was opened to 20 nm and the emission slit was opened to 5 nm. Each of the samples was illuminated continuously with a xenon lamp, and the fluorescence intensity was measured over a period of 20 min by a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc.). Intensity values were normalized for comparison. 2.8. Conjugation of the tumor-targeting ligands to the nanoparticles PNA was covalently conjugated to the NIR fluorescent HSA nanoparticles by 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 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 isolated from excess reagents by sepharose gel filtration. FITC–PNA or anti-CEA were conjugated to the NIR fluorescent HSA nanoparticles via a similar procedure. The concentration of bound PNA was determined with FITC–PNA via a calibration curve of FITC–PNA fluorescence using a multiplate reader (TECAN SpectraFluor Plus, Neotec Scientific Instruments). The concentration of bound anti-CEA was determined using a mouse IgG ELISA kit (Biotest, Israel). 2.9. Cell lines Human colorectal adenocarcinoma cell lines used include LS174t, HT29 and SW480. 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). 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). SW480 cell line was maintained in Dulbecco's MEM supplemented with FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). 2.10. Chicken chorioallantoic membrane (CAM) grafting procedures Tumor cells were grafted on CAM according to the literature [33]. Briefly, fertile chicken eggs obtained from a commercial supplier were incubated at 37 °C at 60–70% humidity in a forced-draft incubator. At 3 days of incubation, an artificial air sac was created and allowed the CAM to drop. A window was opened in the shell and the CAM was exposed on day 8 of incubation. Tumor cells were collected by trypsinization, washed with culture medium and pelleted by gentle centrifugation. After removing the medium, 5 × 10 6 cells were resuspended in 30 μL ice-cold Matrigel and inoculated on the CAM at the site of the blood vessels. Eggs were then sealed and placed back into the incubator. On day 6 post-grafting (day 14 of
incubation), the tumor size ranged from 3 to 5 mm in diameter with visible neoangiogenesis.
2.11. Detection of colonic tumors with NIR fluorescent HSA nanoparticles 2.11.1. CAM tumor labeling Chicken embryos with 6-day-old tumors (LS174t, HT29 and SW480 cancerous tumor cell lines), implanted on the CAM, were treated with the non-conjugated and PNA or anti-CEA-conjugated NIR fluorescent HSA nanoparticles (40 μL, 0.7 mg/mL). 20 min later, the tumors and non-pathological CAM were removed, washed with PBS, and spread on a mat black background for imaging, using a Maestro™ in vivo imaging system. Fluorescence intensity measurements were calculated as an average intensity over the tumor surface area, using ImageJ software.
2.11.2. Rat tumor labeling The rat studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH, revised 1985), at the Hebrew University Animal Facility. Male Sabra rats were kept under constant environmental conditions, (22 °C, 12 h light/dark cycles) and fed with standard laboratory chow and tap water. Induction of colon neoplasia was performed by a weekly subcutaneous injection of 100 μL of the carcinogen dimethylhydrazine (DMH) at a dose of 4 mg/100 g rat body weight, for 5 weeks. The rats were kept for an additional 10 weeks under the same conditions [34–36], and 24 h prior to nanoparticle administration, the rats were transferred to metabolic cages (with free access to water only). After anesthesia (intraperitoneal injection of a mixture of 100 mg/kg body weight of ketamine (Ketaset, 0.1 g/mL, Fort Dodge, USA) and xylazine HCl (100 mg/mL)) polyps were identified by a mini-colonoscope for small laboratory animals. The colons of 3 anesthetized rats with DMH-induced polyps were washed 3 times with PBS to remove feces residues. PNA–HSA-nanoparticles in PBS (500 μL, 700 μg/mL) were instilled into each rat colon. After 20 min the colons were rinsed 4 times with PBS, the rats were then euthanized (by chest wall puncturing) and their colons were removed, cut open, and spread on a polyethylene transparent films with the mucosal aspects upwards, for immediate imaging. Specimens from polyps and adjacent tissues of each colon were stained and examined pathologically for colon cancer severity.
2.11.3. Fluorescence imaging Fluorescence imaging of the chicken embryo tumors was performed with a Maestro II in vivo fluorescence imaging system (Cambridge Research and Instrumentation, Inc., Woburn, MA) at λex: 770–700 nm and λem > 790 nm. The liquid crystal tunable filter was programmed to acquire image cubes from λ = 790 nm to 860 nm with an increment of 10 nm per image. Fluorescence imaging of rat colons was performed using an Odyssey® Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) with λex = 780 nm, λem = 800 nm, intensity set to 1.
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3. Results and discussion 3.1. Synthesis of CANIR and NIR fluorescent HSA nanoparticles The dye chosen for use throughout this work is a carboxylic acid derivative of the commercially available IR-783 (CANIR), as described in Section 2.2. CANIR is a dark green powder obtained in high yield (90%) and the structure was confirmed by MS, 1H and 13C NMR (see supplementary data). The NIR fluorescent HSA nanoparticles were prepared by a desolvation technique, in the presence of CANIR. The NIR fluorescent HSA nanoparticles are designed for oral administration, in a capsule for release in the colon. NIR dyes such as indocyanine green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin. This strong physical binding of NIR polymethine dyes to albumin was exploited for entrapment of the dye within HSA nanoparticles. To achieve maximal fluorescence of nanoparticle-labeled tumor cells, the dye should be strongly associated with the nanoparticles. Following nanoparticle synthesis, no leaching of the encapsulated CANIR dye into the aqueous phase was observed over time, either in the absence or presence of 4% soluble HSA, determined by isolation of the aqueous phase using ultrafiltration. This indicated that all the CANIR was associated with the albumin nanoparticles, confirming complete entrapment of CANIR, as well as its strong physical interaction to HSA within the nanoparticles.
Fig. 3. Absorbance and emission spectra of free CANIR dye (solid lines) and CANIR–HSA nanoparticles (dashed lines). The maximum absorbance of free CANIR and NIR fluorescent HSA nanoparticles occurs at approximately 790 nm and 810 nm, respectively. The fluorescence emission maxima occur at approximately 818 nm and 823 nm, respectively.
physical binding to the HSA, which places the dye in a more hydrophobic environment, affects the dipole moment of the dye. 3.4. Optimization of nanoparticle fluorescence
3.2. Size and size distribution The dry diameter of the NIR fluorescent HSA nanoparticles was measured to be 100 ± 15 nm, as illustrated by the typical SEM photomicrograph shown in Fig. 2A. The hydrodynamic diameter of these nanoparticles dispersed in water was 140 ±15 nm, as illustrated by the typical DLS measurement shown in Fig. 2B. The differences in the particle size between the two methods are due to the fact that the SEM measurements determine the dry diameter, whereas light scattering measurements determine the hydrodynamic diameter, which takes into account absorbed water, water layers adsorbed on the particle's surface, and Brownian motion.
In order to optimize the fluorescence intensity of the NIR fluorescent nanoparticles, different concentrations of CANIR dye were added to the 4% aqueous HSA solution, prior to formation of the fluorescent nanoparticles, via the precipitation process described in the experimental section. 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 (Table 1). The concentration of the entrapped CANIR dye that was found to give the maximum fluorescence intensity of the NIR fluorescent HSA nanoparticles was 3.0 μg per 0.5 mg particles in mL PBS. At higher dye concentrations, quenching of the fluorescence was observed, as closeness of the dye molecules entrapped within the nanoparticles results in non-emissive energy transfer between dye molecules.
3.3. Optical spectra 3.5. Photobleaching measurements Absorbance and emission spectra of both free dye in solution and the nanoparticles were recorded (Fig. 3). A 20 nm red-shift of the absorbance spectrum of the NIR fluorescent HSA nanoparticles, compared to free CANIR dye in solution, was observed. This indicates that the
Photobleaching is the irreversible light-induced destruction of the fluorophore, affected by factors such as oxygen, oxidizing or reducing agents, temperature, exposure time and illumination levels.
Fig. 2. SEM image (A) and hydrodynamic size histogram (B) of the NIR fluorescent HSA nanoparticles.
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Table 1 Effect of CANIR concentration on the fluorescence intensity of the NIR fluorescent HSA nanoparticles. [CANIR]/[HSA] (weight % ratio)
[CANIR]/[HSA nanoparticles] (μg)/(0.5 mg/mL)
Relative fluorescence intensity of the HSA nanoparticles at 823 nm
0.25 0.5 0.7 1.0
0.6 3.0 5.6 7.2
32 137 136 122
[CANIR]/[HSA] refers to the initial weight % ratio of the CANIR dye and the NIR fluorescent HSA nanoparticles. The NIR fluorescent HSA nanoparticles were prepared according to the experimental section. The quantity of CANIR encapsulated was determined for 0.5 mg/mL nanoparticles according to a calibration curve, as described in the experimental part.
As previously shown, encapsulation of dye into nanoparticles can stabilize the dye against photobleaching [9,37]. The photostability of the free CANIR dye was examined and compared to the CANIR encapsulated within the HSA nanoparticles. Samples of the free CANIR dye and the CANIR entrapped HSA nanoparticles were illuminated at 800 nm and their fluorescence intensities were measured. We have demonstrated that, during illumination, the fluorescence intensity of the CANIR containing HSA nanoparticles remains almost unaltered while that of the free CANIR decreases significantly (t=20 min, 32%, pb 0.001), as shown in Fig. 4. Encapsulation of the CANIR within the HSA nanoparticles probably protects the dye against reactive oxygen species, thereby stabilizing the dye against photobleaching.
3.6. ζ-potential The ζ-potential of the nanoparticles may affect their stability, that is, a negative nanoparticle surface charge will create repulsion between nanoparticles and prevent aggregation. The surface charge also affects the ability of the nanoparticles to penetrate through biological barriers such as mucus to colonic tumors [38]. ζ-potential measurements for both the non-fluorescent and the NIR fluorescent HSA nanoparticles dispersed in aqueous continuous phase were performed (Fig. 5). The titration curves show a similar general behavior for both non-fluorescent and fluorescent HSA nanoparticles, and the encapsulated dye does not significantly affect the surface charge. The isoelectric point of the non-fluorescent and fluorescent HSA nanoparticles occurs at pH 5.3. Small differences between the two curves may be attributed to small amounts of dye at the particle surface as well as the effect of the dye on the orientation of the HSA molecules.
Fig. 5. ζ-potential of the NIR fluorescent HSA nanoparticles (A) and HSA nanoparticles (B).
3.7. Confirmation of ligand conjugation Logically, the quantity of ligand conjugated to the nanoparticles should be, on the one hand, sufficient for cell recognition regardless of the orientation at which the particle reaches the cell, but on the other hand limited, to prevent steric hindrance of the ligand–receptor interaction. Fluorescently labeled PNA was used for determining the approximate amount of the bioactive reagent conjugated to the nanoparticles, via a calibration curve of FITC–PNA fluorescence. The concentration of bound anti-CEA was determined using a mouse IgG ELISA kit. The quantities calculated of PNA and anti-CEA conjugated to the nanoparticles were 2.4 and 2.1 μg, per mg of nanoparticles, respectively. The effectiveness of the quantity conjugated was demonstrated with effective ligand–receptor interaction, and thus an increase in fluorescence of the tumors, with upregulated corresponding receptors. An additional experiment with a double amount of conjugated ligand led to a decrease in the fluorescent signal obtained, indicating possible steric hindrance. 3.8. Tumor growth on chicken embryo CAM A chicken embryo CAM model has been used in this work for testing the specific tumor detection by both the non-conjugated and the bioactive (e.g., PNA and anti-CEA)-conjugated NIR fluorescent HSA nanoparticles. Compared to commonly used animal models, this model is less expensive and allows for imaging of numerous tumors in a shorter time period [39]. All cancer cell lines evaluated in this study were able to form solid tumors, 3 to 5 mm in diameter depending on the cell line. Fig. 6 shows typical SW480 cell line derived tumors delimited by plastic rings on a chicken CAM. 3.9. Optical detection of human colon tumors on a CAM model
Fig. 4. Photostability of the CANIR–HSA nanoparticles (A) and free CANIR (B) as a function of time, under constant illumination at 800 nm for 20 min.
3.9.1. Optical detection of LS174t and HT29 tumors To demonstrate the feasibility of using the NIR fluorescent HSA nanoparticles for colon tumor detection, anti-CEA was covalently conjugated to the nanoparticles. CEA, a highly glycosylated glycoprotein, is highly expressed in most human carcinomas and therefore used as a marker in several modalities of human carcinoma management. According to the literature this antigen is upregulated on the mucosal side of various colorectal cancer cell lines such as LS174t colorectal cancer cell line, compared to the HT29 cell line in which this antigen expressed to a much lower extent (at least 10 3 times) [40,41]. As shown in Fig. 7, the LS174t tumors treated with anti-CEAconjugated nanoparticles gained greater fluorescence compared to those treated with the non-conjugated nanoparticles, presumably due to effective receptor–ligand interaction. However, the HT29 tumors treated with anti-CEA-conjugated nanoparticles gained less
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differences in fluorescence intensity between the different tumor types were not statistically significant.
Fig. 6. Light photograph of SW480 cell line derived tumors delimited by plastic rings on chicken CAM in situ in the egg. SW480 cells formed tumors (asterisks) 8 days after transplantation that apparently attracted host blood vessels (arrow).
fluorescence (about half) compared to those treated with the nonconjugated nanoparticles, showing significance of the HSA nanoparticle surface. No non-specific labeling of non-pathological CAM was observed, indicating specificity of the nanoparticles towards tumor tissue. In addition, no autofluorescence was observed in untreated tumors, showing that all fluorescent signals were due to the specific labeling by the fluorescent nanoparticles. The relative fluorescence intensities of the tumors treated by the non-conjugated and the anti-CEA-conjugated nanoparticles are summarized in Fig. 8. The fluorescence intensity of the LS174t cell line treated with the anti-CEA-conjugated fluorescent albumin nanoparticles is 6 times higher than that of the HT29 treated cell line (Figs. 7 and 8). This can be attributed to the higher expression of CEA receptors on the LS174t cell line compared to the HT29 cell line [40]. The non-conjugated albumin fluorescent nanoparticles also labeled the tumors, however, the
3.9.2. Optical detection of LS174t, HT29 and SW480 colorectal tumors PNA binds to the terminal sugar β-D-galactosyl-(1–3)-N-acetyl-Dgalactosamine of the Thomsen–Friedenreich (TF) antigen [42]. According to the literature, this antigen is upregulated on the mucosal side of various colorectal cancer cell lines such as LS174t and HT29, compared to the SW480 cell line in which this antigen expressed to a much lower extent (at least 103 times) [42]. The fluorescence intensities of the LS174t and the HT29 tumors treated by the PNA-conjugated fluorescent HSA nanoparticles were found to be 7 times higher than that of the SW480, due to upregulated PNA receptors present in LS174t and HT29 tumors compared to SW480. The LS174t and HT29 tumors were detected by the PNA-conjugated NIR fluorescent HSA nanoparticles with fluorescence intensity double that of the non-conjugated nanoparticles. On the other hand, the fluorescence intensity of the SW480 tumors treated with the non-conjugated nanoparticles was approximately double those treated by the PNA-conjugated nanoparticles. These results indicate that the SW480 tumors respond to albumin more than to the conjugated PNA. The specific detection of the cancerous tumors by the non-conjugated fluorescent albumin nanoparticles does not significantly differ between cell lines, and is attributed to the presence of albumin receptors on the tumor cells as well as albumin playing an important role as a nutrient for proliferating tumors [21]. 3.9.3. Optical detection of human colon tumors on a rat model After treatment with PNA-conjugated fluorescent HSA nanoparticles, the rat colons were washed thoroughly, the rats were euthanized and colons were removed. Relative spectral analysis was performed on pre-defined mean mucosal areas of 25 mm 2 along the rat colons. Fluorescent images of the colon specimens taken from the rats (Fig. 9) demonstrate the selective labeling of polyps and dysplasia by the PNA-conjugated fluorescent HSA nanoparticles in the rat colonic mucosa.
Fig. 7. Merged fluorescent and greyscale images from a typical experiment of tumor cell lines (LS174t and HT29) implanted on chicken embryo CAM treated with non-conjugated (A) and anti-CEA-conjugated (B) NIR fluorescent HSA nanoparticles. Images of non-pathological CAM treated with the anti-CEA-conjugated NIR fluorescent HSA nanoparticles are shown in (C). Images of the untreated tumor cell lines (LS174t and HT29) are shown in (D). This experiment was repeated an additional 3 times with similar results.
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The fluorescence intensity of the polyps was calculated to be 15.5 times higher than that of the surrounding non-pathological tissue (Fig. 9). 3.10. Advantages of fluorescent HSA nanoparticles Overall, there are two mechanisms by which the tumors can be specifically labeled by the fluorescent HSA nanoparticles: by “active” receptor–ligand interaction, or by “passive” exploitation of the HSA nanoparticles as nutrients for tumor growth, and thus effective fluorescent labeling. Without a conjugated targeting agent, the “passive” mechanism may dominate, although it has been suggested that receptors for albumin are upregulated on some cancerous tumors. When using biomolecule-conjugated nanoparticles, we have demonstrated that with upregulated corresponding receptors on tumor tissue, an enhancement of the fluorescent signal is obtained. 4. Summary and conclusions
Fig. 8. Relative fluorescence intensity of the LS174t, HT29 and SW480 tumors labeled with non-conjugated (HSA), PNA-conjugated (PNA–HSA), and anti-CEA-conjugated (anti-CEA-HSA) nanoparticles. Non-conjugated (HSA) nanoparticles labeled all three tumor types with only slight differences between them (a). The highest fluorescence was obtained for tumors treated with biomolecule-conjugated nanoparticles in which there is upregulation of the corresponding receptors (b). The lowest fluorescence was obtained for tumors treated with biomolecule-conjugated nanoparticles with a comparative downregulation of corresponding receptors (c). Data is presented as the mean ± SE. Values not sharing a common letter (a–c) differ significantly from each other (p b 0.05). The representative calculations are an average of 3 experiments.
NIR fluorescent HSA nanoparticles have been prepared by a desolvation method by which the CANIR dye was encapsulated within HSA nanoparticles of 100 ±15 nm diameter. These nanoparticles may be very useful for in vivo tumor diagnosis due to the lower autofluorescence background signal and deeper penetration into biomatrices of the NIR florescence range. We have demonstrated in the present study that the encapsulation process significantly stabilizes the NIR dye against photobleaching. We have also established in chicken embryo and rat models, that the PNA and anti-CEA-conjugated fluorescent albumin nanoparticles maintain their activity and specifically detect the
Fig. 9. White light and logarithmically scaled fluorescent images of the tumor-bearing colons of rats treated with DMH (A, B, C) and two normal colons (D, E), after treatment with PNA-conjugated HSA nanoparticles. Arrows indicate polyps or regions where biopsies were taken for pathological testing, confirming the presence of pathological tissue. The table below the image is the average fluorescence intensity (arbitrary units) of selected sites along the rat colons. Pathological = tumor or polyp area. Normal = surrounding non-pathological tissue. n represents the number of sites measured. The ratio of pathological to normal tissue is 15:5.
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cancerous tissue, leaving surrounding non-pathological tissue unlabeled. The non-conjugated NIR albumin nanoparticles also detect the colonic tumor cells, probably via a combination of “active” and “passive” targeting. The results of this study indicate that for efficient fluorescent detection of colonic tumors it is likely that the use of more than one targeting agent is required. In future work, we plan to extend this study to include other tumor-targeting ligands including antibodies, proteins and small peptides such as epidermal growth factor (EGF), tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72 antibodies) and TRAIL [43,44]. In addition, cancer drugs such as paclitaxel and doxorubicin may also be encapsulated within these NIR fluorescent HSA nanoparticles, so that the particles may be used for both diagnosis and therapy. Acknowledgments This study was supported by the Israeli Ministry of Commerce and Industry (BMP Consortium of Biomedical Photonics). The authors thank Dr. Benny Perlstein for his assistance in this study. Thanks also to Dr. Ronen Yehuda for his assistance with the fluorescent images. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.msec.2012.11.022. References [1] R. Labianca, G.D. Beretta, S. Mosconi, L. Milesi, M.A. Pessi, Ann. Oncol. 16 (2005) 127–132. [2] R.W. Burt, J.S. Barthel, K.B. Dunn, D.S. David, E. Drelichman, J.M. Ford, F.M. Giardiello, S.B. Gruber, A.L. Halverson, S.R. Hamilton, J. Natl. Compr. Canc. Netw. 8 (2010) 8–61. [3] R.S. Nelson, A.G. Thorson, Curr. Oncol. Rep. 11 (2009) 482–489. [4] J.S. Mandel, T.R. Church, J.H. Bond, F. Ederer, M.S. Geisser, S.J. Mongin, D.C. Snover, L.M. Schuman, N. Engl. J. Med. 343 (2000) 1603–1607. [5] V.R. Kondepati, M. Keese, R. Mueller, B.C. Manegold, J. Backhaus, Vib. Spectrosc. 44 (2007) 236–242. [6] H. Zeng, A. Weiss, R. Cline, C.E. MacAulay, Bioimaging 6 (1998) 151–165. [7] S. Jiang, M.K. Gnanasammandhan, Y. Zhang, J. R. Soc. Interface 7 (2009) 3–18. [8] S. Santra, D. Dutta, G.A. Walter, B.M. Moudgil, Technol. Cancer Res. Treat. 4 (2005) 593–602. [9] P. Sharma, S. Brown, G. Walter, S. Santra, B. Moudgil, Adv. Colloid Interface Sci. 123–126 (2006) 471–485. [10] E.İ. Altınoğlu, J.H. Adair, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 461–477. [11] X. He, K. Wang, Z. Cheng, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 349–366. [12] O.C. Farokhzad, R. Langer, ACS Nano 3 (2009) 16–20. [13] T. Peters Jr., All About Albumin: Biochemistry, Genetics, and Medical Applications, Academic Press, 1995. [14] M. Gekle, Annu. Rev. Physiol. (2005) 573–594. [15] D. Cirstea, T. Hideshima, S. Rodig, L. Santo, S. Pozzi, S. Vallet, H. Ikeda, G. Perrone, G. Gorgun, K. Patel, Mol. Cancer Ther. 9 (2010) 963–975. [16] M. Harries, P. Ellis, P. Harper, J. Clin. Oncol. 23 (2005) 7768–7771. [17] F. Petrelli, K. Borgonovo, S. Barni, Expert Opin. Pharmacother. 11 (2010) 1413–1432. [18] S. Honary, M. Jahanshahi, P. Golbayani, P. Ebrahimi, K. Ghajar, J. Nanosci. Nanotechnol. 10 (2010) 7752–7757. [19] K. Wosikowski, E. Biedermann, B. Rattel, N. Breiter, P. Jank, R.L. ser, G. Jansen, G.J. Peters, Clin. Cancer Res. 9 (2003) 1917–1926. [20] F. Kratz, J. Control. Release 132 (2008) 171–183. [21] G. Stehle, H. Sinn, A. Wunder, H.H. Schrenk, J.C.M. Stewart, G. Hartung, W. MaierBorst, D.L. Heene, Crit. Rev. Oncol. Hematol. 26 (1997) 77–100. [22] M.J. Hawkins, P. Soon-Shiong, N. Desai, Adv. Drug Deliv. Rev. 60 (2008) 876–885. [23] R.J. Williams, M. Lipowska, G. Patonay, L. Strekowski, Anal. Chem. 65 (1993) 601–605. [24] M.Y. Berezin, H. Lee, W. Akers, G. Nikiforovich, S. Achilefu, Photochem. Photobiol. 83 (2007) 1371–1378. [25] J.F. Zhou, M.P. Chin, S.A. Schafer, in: Laser Surgery: Advanced characterization, therapeutics and systems IV, 2128, CA SPIE, Bellingham, 1994, pp. 495–505. [26] K. Awasthi, G. Nishimura, Photochem. Photobiol. Sci. 10 (2011) 461–463. [27] D.P. Tang, R. Yuan, Y.Q. Chai, J. Phys. Chem. B 110 (2006) 11640–11646. [28] S. Sakuma, T. Yano, Y. Masaoka, M. Kataoka, K.i. Hiwatari, H. Tachikawa, Y. Shoji, R. Kimura, H. Ma, Z. Yang, L. Tang, R.M. Hoffman, S. Yamashita, Eur. J. Pharm. Biopharm. 74 (2010) 451–460. [29] L. Strekowski, C.J. Mason, H. Lee, R. Gupta, J. Sowell, G. Patonay, J. Heterocycl. Chem. 40 (2003) 913–916. [30] W. Wang, S. Ke, S. Kwon, S. Yallampalli, A.G. Cameron, K.E. Adams, M.E. Mawad, E.M. Sevick-Muraca, Bioconjug. Chem. 18 (2007) 397–402.
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Sarit Cohen is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. in Chemistry (2006) and M.Sc. in Organic Chemistry (2008) (both magna cum laude) from Bar-Ilan University. Working under the supervision of Prof. Shlomo Margel, Sarit's field of interest has shifted to medical applications of albumin nanoparticles, and she is currently researching the usage of fluorescent albumin nanoparticles for early detection of colon cancer.
Michal Pellach is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. (Hons) in Pharmacology, with a chemistry major, from University of NSW, Australia (2005). After working as an organic chemist at Biolab. Ltd., Israel (2006-2007), Michal began researching polymeric micro- and nanoparticles as diagnostic biomaterials at Bar-Ilan university, and received her M.Sc. (magna cum laude) from Bar-Ilan university (2009). Her Ph.D. research involves the synthesis of fluorescent polymeric nanoparticles for early colon cancer diagnosis.
Yossi Kam, is a Ph.D. student at the School of Pharmacy Institute for Drug Research of the Hebrew University of Jerusalem, Israel. He completed his M.Sc. studies in Biomedical Engineering at the Ben Gurion University, Israel. His Ph.D. studies are under the supervision of Prof. Abraham Rubinstein, Prof. Aviram Nissan and Dr. Eylon Yavin. Mr. Kam's research project is aimed at identifying mutant and unique citosolic mRNA in colon cancer cells by molecular beacon and the use of targetable polymeric vehicles for early detection of colon cancer.
Igor Grinberg is currently pursuing postdoctoral research under the supervision of Prof. Shlomo Margel, at the Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. He received his B.Sc. (2002), M.Sc. (2007), and his Ph.D. (2011) in Biology and Life Sciences, at Bar-Ilan University. Igor's work involves the development of the chicken embryo model as well as in vivo (small animal) models for investigating various types of nanoparticles for various medical applications.
S. Cohen et al. / Materials Science and Engineering C 33 (2013) 923–931 Enav Corem-Salkmon is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. (summa cum laude) in Biotechnology with from Bar-Ilan University (2005) and M.Sc. (magna cum laude) in Biotechnology (2007) from Bar-Ilan University. Working under the supervision of Prof. Shlomo Margel, Enav's Ph.D. research involves biomedical applications of iron oxide nanoparticles, including convention enhanced delivery of anti-cancer drugs as well as usage of fluorescent iron oxide nanoparticles for early detection of colon cancer.
Abraham Rubinstein is a Professor of Pharmaceutical Sciences at The Hebrew University of Jerusalem. After his graduation (Ph.D. in physical pharmacy and pharmacokinetics) he spent two years as a postdoctoral fellow in the University of Wisconsin-Madison (GI physiology, motility and oral drug delivery) and joined the School of Pharmacy in Jerusalem as a faculty member. He is the author of more than 100 research articles, reviews and book chapters. Three of the technologies he developed were purchased by start-up companies. His research interests are focused on site-specific therapy and real-time diagnostics of inflammation and malignant processes in the GI tract.
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Shlomo Margel received his Ph.D. from the Weizmann Institute of Science, Israel (1976). He completed his postdoctoral research at the California Institute of Technology, and is a full professor at the Institute of Nanotechnology & Advanced Materials of Bar-Ilan University, Israel. He has published 185 papers in international journals and 29 patents. His major interests include polymers and biopolymers, surface chemistry, immobilization techniques, colloidal chemistry, thin coatings, biological and medical applications of polymers and micro- and nanoparticles (cancer diagnostics and therapy, neurodegenerative disorders, drug delivery, etc.).
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis and characterization of near IR fluorescent albumin nanoparticles for optical detection of colon cancer Sarit Cohen a, Michal Pellach a, Yossi Kam b, Igor Grinberg a, Enav Corem-Salkmon a, Abraham Rubinstein b, 1, Shlomo Margel a,⁎ a b
Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel
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Article history: Received 13 July 2012 Received in revised form 16 October 2012 Accepted 13 November 2012 Available online 21 November 2012 Keywords: HSA nanoparticles Fluorescent nanoparticles NIR fluorescence NIR fluorescent albumin nanoparticles Optical imaging
a b s t r a c t Near IR (NIR) fluorescent human serum albumin (HSA) nanoparticles hold great promise as contrast agents for tumor diagnosis. HSA nanoparticles are considered to be biocompatible, non-toxic and non-immunogenic. In addition, NIR fluorescence properties of these nanoparticles are important for in vivo tumor diagnostics, with low autofluorescence and relatively deep penetration of NIR irradiation due to low absorption of biomatrices. The present study describes the synthesis of new NIR fluorescent HSA nanoparticles, by entrapment of a NIR fluorescent dye within the HSA nanoparticles, which also significantly increases the photostability of the dye. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent albumin nanoparticles, increasing the potential fluorescent signal in tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent HSA nanoparticles was demonstrated in a chicken embryo model and a rat model. In future work we also plan to encapsulate cancer drugs such as doxorubicin within the NIR fluorescent HSA nanoparticles for both colon cancer imaging and therapy. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It has become common knowledge that the early detection of colon cancer is the key to its survival. Early detection of adenomatous colonic polyps is also of major concern in the prevention of colon cancer [1–4]. Current techniques used for colon cancer screening include double-contrast barium enema, fecal occult blood tests and colonoscopy. 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 [5]. There is therefore a need for development of more effective methods for early detection of colonic tumors. Optical imaging techniques provide functional and anatomical characterization of biological tissues, revealing important information on significant physiological parameters [6]. While optical imaging based on white light allows observation of only superficial structures, fluorescence imaging allows for observation beyond the surface. Another advantage of fluorescence imaging compared to white-light technology is the high signal-to-noise ratio that can be
⁎ Corresponding author. Tel.: +972 3 5318861; fax: +972 3 6355208. E-mail address: [email protected] (S. Margel). 1 Affiliated with the Harvey M. Krueger Family Center for Nanoscience and Nanotechnology and the David R. Bloom Center of Pharmacy of the Hebrew University of Jerusalem. 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.11.022
achieved. However, significant autofluorescence of bodily tissues remains a limiting factor, with fluorescence imaging in the visible region of the electromagnetic spectrum. Materials with fluorescence in the near-infrared (NIR) region (700–1000 nm) intended for use as imaging agents are of great interest, as they result in a lower background signal and deeper penetration into biomatrices [7,8]. 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 functional groups on the nanoparticle surface [9]. Nanoparticles based on silica, calcium phosphate and lipoprotein containing NIR dyes have already been developed [10,11]. Tumors can be actively targeted by nanoparticles conjugated to molecular probes that recognize tumor-specific biomarkers. Known targeting agents include antibodies, lectins, small peptides and small targeting molecules, all with upregulated receptors on the tumor cell membrane. With targeting agents conjugated to the nanoparticles, the nanoparticles bind to specific cell-surface receptors, and are often uptaken into the cell via receptor-mediated endocytosis. The intracellular concentration of nanoparticles is consequently enhanced in cancer cells compared to normal cells [12]. There is growing interest in the fabrication of albumin nanoparticles due to their biocompatibility, biodegradability and non-antigenicity.
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Albumin is one of the most commonly used and characterized proteins in the pharmaceutical field. HSA is the most abundant plasma protein with a molecular weight of 66.5 kDa and a blood half-life of 19 days [13]. Among many other physiological functions, albumin serves as a carrier for a variety of substances such as Ca2+, bilirubin, fatty acids and drugs [14]. Albumin is also known to accumulate and be catabolized by cancerous tumors [15–19]. The accumulation of HSA in tumor tissues is probably due to the enhanced microvascular permeability that allows large molecules to penetrate through to the tumor tissue, together with an impaired or absent lymphatic system (enhanced permeability and retention, or EPR) [20]. Furthermore, Stehle et al. have suggested that albumin plays an important role as a nutrient for proliferating tumors [21]. There is also evidence of active targeting of albumin, that binds to a cell surface 60-kDa glycoprotein (gp60) receptor [22]. Soluble albumin as well as albumin nanoparticles are therefore currently used as delivery vehicles in chemotherapy. NIR dyes such as indocyanine green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin [23]. Molecular modeling illustrates that albumin has two binding sites with different polarities to which hydrophobic or amphiphilic ligands bind, and there is often selectivity towards one of the two binding sites. A strong physical between the NIR cyanine dyes and albumin at these binding sites is formed, also affecting the photophysical properties of the dyes [24–26]. In this work, we have exploited the high affinity of NIR cyanine dyes to albumin for the preparation of NIR albumin nanoparticles, as well as the high affinity of albumin to tumor tissue, for their use as tumor-specific contrast agents. For increasing the specificity of the nanoparticles towards colon cancer, the NIR fluorescent HSA nanoparticles were covalently conjugated to colon cancer targeting agents such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibody (anti-CEA) [27,28]. In order to test the performance of the fluorescent nanoparticles as imaging agents for solid tumors, initial experiments were performed on tumors grown on the chorioallantoic membrane (CAM) of embryonic chicken eggs. Further assessment of the nanoparticles was performed in a rat model. The nanoparticles were found to specifically detect colon cancerous tumors, as demonstrated in both tumor implants in the chicken embryo model and in a rat model.
2.2. Synthesis of the NIR fluorescent dye A carboxylic acid derivative of the NIR dye IR-783 (CANIR) was prepared by the treatment of IR-783 (1) with 4-mercaptobenzoic acid in DMF (see Fig. 1), a method similar to that reported in the literature [29,30]. The structure was confirmed by MS, 1H and 13C NMR (see supplementary data). 2.3. Synthesis of NIR fluorescent HSA nanoparticles The NIR fluorescent HSA nanoparticles were prepared by a desolvation technique [31,32]. 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 isolated from impurities by sepharose gel filtration. The fluorescence intensity of the HSA nanoparticles was varied by changing the CANIR concentration within the HSA particles. The effectiveness of dye encapsulation was confirmed by testing for dye leaching in absence of or presence of 4% HSA, shaking RT for 4 h, and then ultrafiltration. The absorbance and fluorescence spectra of the filtrate were then measured. Non-fluorescent HSA nanoparticles were prepared similarly to NIR fluorescent HSA nanoparticles, in the absence of the CANIR dye. 2.4. Nanoparticle characterization
2. Materials and methods
Hydrodynamic particle size and size distribution were determined by dynamic light scattering (DLS) with photon cross-correlation spectroscopy (Nanophox particle analyzer, Sympatec GmbH, Germany). The mean dry diameter was determined by Scanning Electron Microscopy (SEM) (JEOL, JSM-840 Model, Japan), measuring 100 particles, using an image analysis software (AnalySIS Auto, Soft Imaging SystemGmbH, Germany). Absorbance spectra were obtained using a Cary 100 UV–Visible spectrophotometer (Agilent Technologies Inc.). Excitation and emission spectra were measured by a Cary eclipse spectrofluorometer (Agilent Technologies, Inc.). ζ-potential measurements were performed by gradual titration of the pH (11–2.5) with 1 M HCl (Zetasizer zeta potential analyzer 3000 Has Model, Malvern Instruments, England).
2.1. Materials
2.5. Determination of encapsulated CANIR
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)2H-indol-2-ylidene]ethylidene]-1 cyclohexen-1-yl]ethenyl]-3,3dimethyl-1-(4-sulfobutyl)-3H-indolium inner salt sodium salt (IR-783), HSA, 4-mercaptobenzoic acid, N,N-dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), sepharose 4B, Matrigel, peanut agglutinin (PNA), dimethylhydrazine (DMH) and fluorescein isothiocyanate–conjugated peanut agglutinin (FITC–PNA) 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; Phosphate Buffered Saline (PBS), Minimum Essential Medium (MEM) eagle, McCoy's 5A medium and Dulbecco's modification of eagle's medium (DMEM), fetal bovine serum (FBS), glutamine, penicillin and streptomycin from Biological Industries, Bet Haemek, Israel; LS174t, HT29 and SW480 cell lines from American Type Culture Collection (ATCC); monoclonal anti-CEA antibodies (T86-66) were purified from the hybridoma supernatant at Alomone Labs, Israel. Male Sabra rats (200–250 g) were obtained from Harlan Laboratories, Israel. Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).
A calibration curve of free CANIR was obtained by measuring the integrals of absorbances of standard solutions (0.5–10 μg/mL) in PBS, at wavelengths 630–900 nm. The concentration of encapsulated CANIR was determined by preparing a suspension of NIR HSA 0.5 mg/mL nanoparticles in which the integral of the absorbance spectrum at 630–900 nm was determined. An estimation of encapsulated CANIR per 0.5 mg of nanoparticles was determined according to the calibration curve. 2.6. Optimization of quantity of encapsulated CANIR Different samples of NIR fluorescent HSA nanoparticles were prepared as described in Section 2.3, with six different concentrations of the CANIR dye (0.02–1.0% w/w relative to HSA). Each nanoparticle dispersion was diluted to 0.5 mg/mL in PBS, illuminated at 750 nm, and their relative fluorescence intensities at 823 nm were measured and compared. 2.7. Photostability of NIR fluorescent HSA nanoparticles An aqueous solution of CANIR (0.05 M) in PBS was prepared, and the fluorescence intensity with λex set at 800 nm and λem set at
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Fig. 1. Synthetic scheme of the CANIR dye.
830 nm was measured. A dispersion of NIR fluorescent HSA nanoparticles also in PBS was prepared, and diluted to give a similar fluorescence intensity to the dye at the same wavelengths. The excitation slit was opened to 20 nm and the emission slit was opened to 5 nm. Each of the samples was illuminated continuously with a xenon lamp, and the fluorescence intensity was measured over a period of 20 min by a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc.). Intensity values were normalized for comparison. 2.8. Conjugation of the tumor-targeting ligands to the nanoparticles PNA was covalently conjugated to the NIR fluorescent HSA nanoparticles by 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 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 isolated from excess reagents by sepharose gel filtration. FITC–PNA or anti-CEA were conjugated to the NIR fluorescent HSA nanoparticles via a similar procedure. The concentration of bound PNA was determined with FITC–PNA via a calibration curve of FITC–PNA fluorescence using a multiplate reader (TECAN SpectraFluor Plus, Neotec Scientific Instruments). The concentration of bound anti-CEA was determined using a mouse IgG ELISA kit (Biotest, Israel). 2.9. Cell lines Human colorectal adenocarcinoma cell lines used include LS174t, HT29 and SW480. 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). 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). SW480 cell line was maintained in Dulbecco's MEM supplemented with FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). 2.10. Chicken chorioallantoic membrane (CAM) grafting procedures Tumor cells were grafted on CAM according to the literature [33]. Briefly, fertile chicken eggs obtained from a commercial supplier were incubated at 37 °C at 60–70% humidity in a forced-draft incubator. At 3 days of incubation, an artificial air sac was created and allowed the CAM to drop. A window was opened in the shell and the CAM was exposed on day 8 of incubation. Tumor cells were collected by trypsinization, washed with culture medium and pelleted by gentle centrifugation. After removing the medium, 5 × 10 6 cells were resuspended in 30 μL ice-cold Matrigel and inoculated on the CAM at the site of the blood vessels. Eggs were then sealed and placed back into the incubator. On day 6 post-grafting (day 14 of
incubation), the tumor size ranged from 3 to 5 mm in diameter with visible neoangiogenesis.
2.11. Detection of colonic tumors with NIR fluorescent HSA nanoparticles 2.11.1. CAM tumor labeling Chicken embryos with 6-day-old tumors (LS174t, HT29 and SW480 cancerous tumor cell lines), implanted on the CAM, were treated with the non-conjugated and PNA or anti-CEA-conjugated NIR fluorescent HSA nanoparticles (40 μL, 0.7 mg/mL). 20 min later, the tumors and non-pathological CAM were removed, washed with PBS, and spread on a mat black background for imaging, using a Maestro™ in vivo imaging system. Fluorescence intensity measurements were calculated as an average intensity over the tumor surface area, using ImageJ software.
2.11.2. Rat tumor labeling The rat studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH, revised 1985), at the Hebrew University Animal Facility. Male Sabra rats were kept under constant environmental conditions, (22 °C, 12 h light/dark cycles) and fed with standard laboratory chow and tap water. Induction of colon neoplasia was performed by a weekly subcutaneous injection of 100 μL of the carcinogen dimethylhydrazine (DMH) at a dose of 4 mg/100 g rat body weight, for 5 weeks. The rats were kept for an additional 10 weeks under the same conditions [34–36], and 24 h prior to nanoparticle administration, the rats were transferred to metabolic cages (with free access to water only). After anesthesia (intraperitoneal injection of a mixture of 100 mg/kg body weight of ketamine (Ketaset, 0.1 g/mL, Fort Dodge, USA) and xylazine HCl (100 mg/mL)) polyps were identified by a mini-colonoscope for small laboratory animals. The colons of 3 anesthetized rats with DMH-induced polyps were washed 3 times with PBS to remove feces residues. PNA–HSA-nanoparticles in PBS (500 μL, 700 μg/mL) were instilled into each rat colon. After 20 min the colons were rinsed 4 times with PBS, the rats were then euthanized (by chest wall puncturing) and their colons were removed, cut open, and spread on a polyethylene transparent films with the mucosal aspects upwards, for immediate imaging. Specimens from polyps and adjacent tissues of each colon were stained and examined pathologically for colon cancer severity.
2.11.3. Fluorescence imaging Fluorescence imaging of the chicken embryo tumors was performed with a Maestro II in vivo fluorescence imaging system (Cambridge Research and Instrumentation, Inc., Woburn, MA) at λex: 770–700 nm and λem > 790 nm. The liquid crystal tunable filter was programmed to acquire image cubes from λ = 790 nm to 860 nm with an increment of 10 nm per image. Fluorescence imaging of rat colons was performed using an Odyssey® Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) with λex = 780 nm, λem = 800 nm, intensity set to 1.
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3. Results and discussion 3.1. Synthesis of CANIR and NIR fluorescent HSA nanoparticles The dye chosen for use throughout this work is a carboxylic acid derivative of the commercially available IR-783 (CANIR), as described in Section 2.2. CANIR is a dark green powder obtained in high yield (90%) and the structure was confirmed by MS, 1H and 13C NMR (see supplementary data). The NIR fluorescent HSA nanoparticles were prepared by a desolvation technique, in the presence of CANIR. The NIR fluorescent HSA nanoparticles are designed for oral administration, in a capsule for release in the colon. NIR dyes such as indocyanine green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin. This strong physical binding of NIR polymethine dyes to albumin was exploited for entrapment of the dye within HSA nanoparticles. To achieve maximal fluorescence of nanoparticle-labeled tumor cells, the dye should be strongly associated with the nanoparticles. Following nanoparticle synthesis, no leaching of the encapsulated CANIR dye into the aqueous phase was observed over time, either in the absence or presence of 4% soluble HSA, determined by isolation of the aqueous phase using ultrafiltration. This indicated that all the CANIR was associated with the albumin nanoparticles, confirming complete entrapment of CANIR, as well as its strong physical interaction to HSA within the nanoparticles.
Fig. 3. Absorbance and emission spectra of free CANIR dye (solid lines) and CANIR–HSA nanoparticles (dashed lines). The maximum absorbance of free CANIR and NIR fluorescent HSA nanoparticles occurs at approximately 790 nm and 810 nm, respectively. The fluorescence emission maxima occur at approximately 818 nm and 823 nm, respectively.
physical binding to the HSA, which places the dye in a more hydrophobic environment, affects the dipole moment of the dye. 3.4. Optimization of nanoparticle fluorescence
3.2. Size and size distribution The dry diameter of the NIR fluorescent HSA nanoparticles was measured to be 100 ± 15 nm, as illustrated by the typical SEM photomicrograph shown in Fig. 2A. The hydrodynamic diameter of these nanoparticles dispersed in water was 140 ±15 nm, as illustrated by the typical DLS measurement shown in Fig. 2B. The differences in the particle size between the two methods are due to the fact that the SEM measurements determine the dry diameter, whereas light scattering measurements determine the hydrodynamic diameter, which takes into account absorbed water, water layers adsorbed on the particle's surface, and Brownian motion.
In order to optimize the fluorescence intensity of the NIR fluorescent nanoparticles, different concentrations of CANIR dye were added to the 4% aqueous HSA solution, prior to formation of the fluorescent nanoparticles, via the precipitation process described in the experimental section. 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 (Table 1). The concentration of the entrapped CANIR dye that was found to give the maximum fluorescence intensity of the NIR fluorescent HSA nanoparticles was 3.0 μg per 0.5 mg particles in mL PBS. At higher dye concentrations, quenching of the fluorescence was observed, as closeness of the dye molecules entrapped within the nanoparticles results in non-emissive energy transfer between dye molecules.
3.3. Optical spectra 3.5. Photobleaching measurements Absorbance and emission spectra of both free dye in solution and the nanoparticles were recorded (Fig. 3). A 20 nm red-shift of the absorbance spectrum of the NIR fluorescent HSA nanoparticles, compared to free CANIR dye in solution, was observed. This indicates that the
Photobleaching is the irreversible light-induced destruction of the fluorophore, affected by factors such as oxygen, oxidizing or reducing agents, temperature, exposure time and illumination levels.
Fig. 2. SEM image (A) and hydrodynamic size histogram (B) of the NIR fluorescent HSA nanoparticles.
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Table 1 Effect of CANIR concentration on the fluorescence intensity of the NIR fluorescent HSA nanoparticles. [CANIR]/[HSA] (weight % ratio)
[CANIR]/[HSA nanoparticles] (μg)/(0.5 mg/mL)
Relative fluorescence intensity of the HSA nanoparticles at 823 nm
0.25 0.5 0.7 1.0
0.6 3.0 5.6 7.2
32 137 136 122
[CANIR]/[HSA] refers to the initial weight % ratio of the CANIR dye and the NIR fluorescent HSA nanoparticles. The NIR fluorescent HSA nanoparticles were prepared according to the experimental section. The quantity of CANIR encapsulated was determined for 0.5 mg/mL nanoparticles according to a calibration curve, as described in the experimental part.
As previously shown, encapsulation of dye into nanoparticles can stabilize the dye against photobleaching [9,37]. The photostability of the free CANIR dye was examined and compared to the CANIR encapsulated within the HSA nanoparticles. Samples of the free CANIR dye and the CANIR entrapped HSA nanoparticles were illuminated at 800 nm and their fluorescence intensities were measured. We have demonstrated that, during illumination, the fluorescence intensity of the CANIR containing HSA nanoparticles remains almost unaltered while that of the free CANIR decreases significantly (t=20 min, 32%, pb 0.001), as shown in Fig. 4. Encapsulation of the CANIR within the HSA nanoparticles probably protects the dye against reactive oxygen species, thereby stabilizing the dye against photobleaching.
3.6. ζ-potential The ζ-potential of the nanoparticles may affect their stability, that is, a negative nanoparticle surface charge will create repulsion between nanoparticles and prevent aggregation. The surface charge also affects the ability of the nanoparticles to penetrate through biological barriers such as mucus to colonic tumors [38]. ζ-potential measurements for both the non-fluorescent and the NIR fluorescent HSA nanoparticles dispersed in aqueous continuous phase were performed (Fig. 5). The titration curves show a similar general behavior for both non-fluorescent and fluorescent HSA nanoparticles, and the encapsulated dye does not significantly affect the surface charge. The isoelectric point of the non-fluorescent and fluorescent HSA nanoparticles occurs at pH 5.3. Small differences between the two curves may be attributed to small amounts of dye at the particle surface as well as the effect of the dye on the orientation of the HSA molecules.
Fig. 5. ζ-potential of the NIR fluorescent HSA nanoparticles (A) and HSA nanoparticles (B).
3.7. Confirmation of ligand conjugation Logically, the quantity of ligand conjugated to the nanoparticles should be, on the one hand, sufficient for cell recognition regardless of the orientation at which the particle reaches the cell, but on the other hand limited, to prevent steric hindrance of the ligand–receptor interaction. Fluorescently labeled PNA was used for determining the approximate amount of the bioactive reagent conjugated to the nanoparticles, via a calibration curve of FITC–PNA fluorescence. The concentration of bound anti-CEA was determined using a mouse IgG ELISA kit. The quantities calculated of PNA and anti-CEA conjugated to the nanoparticles were 2.4 and 2.1 μg, per mg of nanoparticles, respectively. The effectiveness of the quantity conjugated was demonstrated with effective ligand–receptor interaction, and thus an increase in fluorescence of the tumors, with upregulated corresponding receptors. An additional experiment with a double amount of conjugated ligand led to a decrease in the fluorescent signal obtained, indicating possible steric hindrance. 3.8. Tumor growth on chicken embryo CAM A chicken embryo CAM model has been used in this work for testing the specific tumor detection by both the non-conjugated and the bioactive (e.g., PNA and anti-CEA)-conjugated NIR fluorescent HSA nanoparticles. Compared to commonly used animal models, this model is less expensive and allows for imaging of numerous tumors in a shorter time period [39]. All cancer cell lines evaluated in this study were able to form solid tumors, 3 to 5 mm in diameter depending on the cell line. Fig. 6 shows typical SW480 cell line derived tumors delimited by plastic rings on a chicken CAM. 3.9. Optical detection of human colon tumors on a CAM model
Fig. 4. Photostability of the CANIR–HSA nanoparticles (A) and free CANIR (B) as a function of time, under constant illumination at 800 nm for 20 min.
3.9.1. Optical detection of LS174t and HT29 tumors To demonstrate the feasibility of using the NIR fluorescent HSA nanoparticles for colon tumor detection, anti-CEA was covalently conjugated to the nanoparticles. CEA, a highly glycosylated glycoprotein, is highly expressed in most human carcinomas and therefore used as a marker in several modalities of human carcinoma management. According to the literature this antigen is upregulated on the mucosal side of various colorectal cancer cell lines such as LS174t colorectal cancer cell line, compared to the HT29 cell line in which this antigen expressed to a much lower extent (at least 10 3 times) [40,41]. As shown in Fig. 7, the LS174t tumors treated with anti-CEAconjugated nanoparticles gained greater fluorescence compared to those treated with the non-conjugated nanoparticles, presumably due to effective receptor–ligand interaction. However, the HT29 tumors treated with anti-CEA-conjugated nanoparticles gained less
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differences in fluorescence intensity between the different tumor types were not statistically significant.
Fig. 6. Light photograph of SW480 cell line derived tumors delimited by plastic rings on chicken CAM in situ in the egg. SW480 cells formed tumors (asterisks) 8 days after transplantation that apparently attracted host blood vessels (arrow).
fluorescence (about half) compared to those treated with the nonconjugated nanoparticles, showing significance of the HSA nanoparticle surface. No non-specific labeling of non-pathological CAM was observed, indicating specificity of the nanoparticles towards tumor tissue. In addition, no autofluorescence was observed in untreated tumors, showing that all fluorescent signals were due to the specific labeling by the fluorescent nanoparticles. The relative fluorescence intensities of the tumors treated by the non-conjugated and the anti-CEA-conjugated nanoparticles are summarized in Fig. 8. The fluorescence intensity of the LS174t cell line treated with the anti-CEA-conjugated fluorescent albumin nanoparticles is 6 times higher than that of the HT29 treated cell line (Figs. 7 and 8). This can be attributed to the higher expression of CEA receptors on the LS174t cell line compared to the HT29 cell line [40]. The non-conjugated albumin fluorescent nanoparticles also labeled the tumors, however, the
3.9.2. Optical detection of LS174t, HT29 and SW480 colorectal tumors PNA binds to the terminal sugar β-D-galactosyl-(1–3)-N-acetyl-Dgalactosamine of the Thomsen–Friedenreich (TF) antigen [42]. According to the literature, this antigen is upregulated on the mucosal side of various colorectal cancer cell lines such as LS174t and HT29, compared to the SW480 cell line in which this antigen expressed to a much lower extent (at least 103 times) [42]. The fluorescence intensities of the LS174t and the HT29 tumors treated by the PNA-conjugated fluorescent HSA nanoparticles were found to be 7 times higher than that of the SW480, due to upregulated PNA receptors present in LS174t and HT29 tumors compared to SW480. The LS174t and HT29 tumors were detected by the PNA-conjugated NIR fluorescent HSA nanoparticles with fluorescence intensity double that of the non-conjugated nanoparticles. On the other hand, the fluorescence intensity of the SW480 tumors treated with the non-conjugated nanoparticles was approximately double those treated by the PNA-conjugated nanoparticles. These results indicate that the SW480 tumors respond to albumin more than to the conjugated PNA. The specific detection of the cancerous tumors by the non-conjugated fluorescent albumin nanoparticles does not significantly differ between cell lines, and is attributed to the presence of albumin receptors on the tumor cells as well as albumin playing an important role as a nutrient for proliferating tumors [21]. 3.9.3. Optical detection of human colon tumors on a rat model After treatment with PNA-conjugated fluorescent HSA nanoparticles, the rat colons were washed thoroughly, the rats were euthanized and colons were removed. Relative spectral analysis was performed on pre-defined mean mucosal areas of 25 mm 2 along the rat colons. Fluorescent images of the colon specimens taken from the rats (Fig. 9) demonstrate the selective labeling of polyps and dysplasia by the PNA-conjugated fluorescent HSA nanoparticles in the rat colonic mucosa.
Fig. 7. Merged fluorescent and greyscale images from a typical experiment of tumor cell lines (LS174t and HT29) implanted on chicken embryo CAM treated with non-conjugated (A) and anti-CEA-conjugated (B) NIR fluorescent HSA nanoparticles. Images of non-pathological CAM treated with the anti-CEA-conjugated NIR fluorescent HSA nanoparticles are shown in (C). Images of the untreated tumor cell lines (LS174t and HT29) are shown in (D). This experiment was repeated an additional 3 times with similar results.
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The fluorescence intensity of the polyps was calculated to be 15.5 times higher than that of the surrounding non-pathological tissue (Fig. 9). 3.10. Advantages of fluorescent HSA nanoparticles Overall, there are two mechanisms by which the tumors can be specifically labeled by the fluorescent HSA nanoparticles: by “active” receptor–ligand interaction, or by “passive” exploitation of the HSA nanoparticles as nutrients for tumor growth, and thus effective fluorescent labeling. Without a conjugated targeting agent, the “passive” mechanism may dominate, although it has been suggested that receptors for albumin are upregulated on some cancerous tumors. When using biomolecule-conjugated nanoparticles, we have demonstrated that with upregulated corresponding receptors on tumor tissue, an enhancement of the fluorescent signal is obtained. 4. Summary and conclusions
Fig. 8. Relative fluorescence intensity of the LS174t, HT29 and SW480 tumors labeled with non-conjugated (HSA), PNA-conjugated (PNA–HSA), and anti-CEA-conjugated (anti-CEA-HSA) nanoparticles. Non-conjugated (HSA) nanoparticles labeled all three tumor types with only slight differences between them (a). The highest fluorescence was obtained for tumors treated with biomolecule-conjugated nanoparticles in which there is upregulation of the corresponding receptors (b). The lowest fluorescence was obtained for tumors treated with biomolecule-conjugated nanoparticles with a comparative downregulation of corresponding receptors (c). Data is presented as the mean ± SE. Values not sharing a common letter (a–c) differ significantly from each other (p b 0.05). The representative calculations are an average of 3 experiments.
NIR fluorescent HSA nanoparticles have been prepared by a desolvation method by which the CANIR dye was encapsulated within HSA nanoparticles of 100 ±15 nm diameter. These nanoparticles may be very useful for in vivo tumor diagnosis due to the lower autofluorescence background signal and deeper penetration into biomatrices of the NIR florescence range. We have demonstrated in the present study that the encapsulation process significantly stabilizes the NIR dye against photobleaching. We have also established in chicken embryo and rat models, that the PNA and anti-CEA-conjugated fluorescent albumin nanoparticles maintain their activity and specifically detect the
Fig. 9. White light and logarithmically scaled fluorescent images of the tumor-bearing colons of rats treated with DMH (A, B, C) and two normal colons (D, E), after treatment with PNA-conjugated HSA nanoparticles. Arrows indicate polyps or regions where biopsies were taken for pathological testing, confirming the presence of pathological tissue. The table below the image is the average fluorescence intensity (arbitrary units) of selected sites along the rat colons. Pathological = tumor or polyp area. Normal = surrounding non-pathological tissue. n represents the number of sites measured. The ratio of pathological to normal tissue is 15:5.
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cancerous tissue, leaving surrounding non-pathological tissue unlabeled. The non-conjugated NIR albumin nanoparticles also detect the colonic tumor cells, probably via a combination of “active” and “passive” targeting. The results of this study indicate that for efficient fluorescent detection of colonic tumors it is likely that the use of more than one targeting agent is required. In future work, we plan to extend this study to include other tumor-targeting ligands including antibodies, proteins and small peptides such as epidermal growth factor (EGF), tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72 antibodies) and TRAIL [43,44]. In addition, cancer drugs such as paclitaxel and doxorubicin may also be encapsulated within these NIR fluorescent HSA nanoparticles, so that the particles may be used for both diagnosis and therapy. Acknowledgments This study was supported by the Israeli Ministry of Commerce and Industry (BMP Consortium of Biomedical Photonics). The authors thank Dr. Benny Perlstein for his assistance in this study. Thanks also to Dr. Ronen Yehuda for his assistance with the fluorescent images. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.msec.2012.11.022. References [1] R. Labianca, G.D. Beretta, S. Mosconi, L. Milesi, M.A. Pessi, Ann. Oncol. 16 (2005) 127–132. [2] R.W. Burt, J.S. Barthel, K.B. Dunn, D.S. David, E. Drelichman, J.M. Ford, F.M. Giardiello, S.B. Gruber, A.L. Halverson, S.R. Hamilton, J. Natl. Compr. Canc. Netw. 8 (2010) 8–61. [3] R.S. Nelson, A.G. Thorson, Curr. Oncol. Rep. 11 (2009) 482–489. [4] J.S. Mandel, T.R. Church, J.H. Bond, F. Ederer, M.S. Geisser, S.J. Mongin, D.C. Snover, L.M. Schuman, N. Engl. J. Med. 343 (2000) 1603–1607. [5] V.R. Kondepati, M. Keese, R. Mueller, B.C. Manegold, J. Backhaus, Vib. Spectrosc. 44 (2007) 236–242. [6] H. Zeng, A. Weiss, R. Cline, C.E. MacAulay, Bioimaging 6 (1998) 151–165. [7] S. Jiang, M.K. Gnanasammandhan, Y. Zhang, J. R. Soc. Interface 7 (2009) 3–18. [8] S. Santra, D. Dutta, G.A. Walter, B.M. Moudgil, Technol. Cancer Res. Treat. 4 (2005) 593–602. [9] P. Sharma, S. Brown, G. Walter, S. Santra, B. Moudgil, Adv. Colloid Interface Sci. 123–126 (2006) 471–485. [10] E.İ. Altınoğlu, J.H. Adair, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 461–477. [11] X. He, K. Wang, Z. Cheng, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (2010) 349–366. [12] O.C. Farokhzad, R. Langer, ACS Nano 3 (2009) 16–20. [13] T. Peters Jr., All About Albumin: Biochemistry, Genetics, and Medical Applications, Academic Press, 1995. [14] M. Gekle, Annu. Rev. Physiol. (2005) 573–594. [15] D. Cirstea, T. Hideshima, S. Rodig, L. Santo, S. Pozzi, S. Vallet, H. Ikeda, G. Perrone, G. Gorgun, K. Patel, Mol. Cancer Ther. 9 (2010) 963–975. [16] M. Harries, P. Ellis, P. Harper, J. Clin. Oncol. 23 (2005) 7768–7771. [17] F. Petrelli, K. Borgonovo, S. Barni, Expert Opin. Pharmacother. 11 (2010) 1413–1432. [18] S. Honary, M. Jahanshahi, P. Golbayani, P. Ebrahimi, K. Ghajar, J. Nanosci. Nanotechnol. 10 (2010) 7752–7757. [19] K. Wosikowski, E. Biedermann, B. Rattel, N. Breiter, P. Jank, R.L. ser, G. Jansen, G.J. Peters, Clin. Cancer Res. 9 (2003) 1917–1926. [20] F. Kratz, J. Control. Release 132 (2008) 171–183. [21] G. Stehle, H. Sinn, A. Wunder, H.H. Schrenk, J.C.M. Stewart, G. Hartung, W. MaierBorst, D.L. Heene, Crit. Rev. Oncol. Hematol. 26 (1997) 77–100. [22] M.J. Hawkins, P. Soon-Shiong, N. Desai, Adv. Drug Deliv. Rev. 60 (2008) 876–885. [23] R.J. Williams, M. Lipowska, G. Patonay, L. Strekowski, Anal. Chem. 65 (1993) 601–605. [24] M.Y. Berezin, H. Lee, W. Akers, G. Nikiforovich, S. Achilefu, Photochem. Photobiol. 83 (2007) 1371–1378. [25] J.F. Zhou, M.P. Chin, S.A. Schafer, in: Laser Surgery: Advanced characterization, therapeutics and systems IV, 2128, CA SPIE, Bellingham, 1994, pp. 495–505. [26] K. Awasthi, G. Nishimura, Photochem. Photobiol. Sci. 10 (2011) 461–463. [27] D.P. Tang, R. Yuan, Y.Q. Chai, J. Phys. Chem. B 110 (2006) 11640–11646. [28] S. Sakuma, T. Yano, Y. Masaoka, M. Kataoka, K.i. Hiwatari, H. Tachikawa, Y. Shoji, R. Kimura, H. Ma, Z. Yang, L. Tang, R.M. Hoffman, S. Yamashita, Eur. J. Pharm. Biopharm. 74 (2010) 451–460. [29] L. Strekowski, C.J. Mason, H. Lee, R. Gupta, J. Sowell, G. Patonay, J. Heterocycl. Chem. 40 (2003) 913–916. [30] W. Wang, S. Ke, S. Kwon, S. Yallampalli, A.G. Cameron, K.E. Adams, M.E. Mawad, E.M. Sevick-Muraca, Bioconjug. Chem. 18 (2007) 397–402.
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Sarit Cohen is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. in Chemistry (2006) and M.Sc. in Organic Chemistry (2008) (both magna cum laude) from Bar-Ilan University. Working under the supervision of Prof. Shlomo Margel, Sarit's field of interest has shifted to medical applications of albumin nanoparticles, and she is currently researching the usage of fluorescent albumin nanoparticles for early detection of colon cancer.
Michal Pellach is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. (Hons) in Pharmacology, with a chemistry major, from University of NSW, Australia (2005). After working as an organic chemist at Biolab. Ltd., Israel (2006-2007), Michal began researching polymeric micro- and nanoparticles as diagnostic biomaterials at Bar-Ilan university, and received her M.Sc. (magna cum laude) from Bar-Ilan university (2009). Her Ph.D. research involves the synthesis of fluorescent polymeric nanoparticles for early colon cancer diagnosis.
Yossi Kam, is a Ph.D. student at the School of Pharmacy Institute for Drug Research of the Hebrew University of Jerusalem, Israel. He completed his M.Sc. studies in Biomedical Engineering at the Ben Gurion University, Israel. His Ph.D. studies are under the supervision of Prof. Abraham Rubinstein, Prof. Aviram Nissan and Dr. Eylon Yavin. Mr. Kam's research project is aimed at identifying mutant and unique citosolic mRNA in colon cancer cells by molecular beacon and the use of targetable polymeric vehicles for early detection of colon cancer.
Igor Grinberg is currently pursuing postdoctoral research under the supervision of Prof. Shlomo Margel, at the Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. He received his B.Sc. (2002), M.Sc. (2007), and his Ph.D. (2011) in Biology and Life Sciences, at Bar-Ilan University. Igor's work involves the development of the chicken embryo model as well as in vivo (small animal) models for investigating various types of nanoparticles for various medical applications.
S. Cohen et al. / Materials Science and Engineering C 33 (2013) 923–931 Enav Corem-Salkmon is a Ph.D. student at Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel. She received her B.Sc. (summa cum laude) in Biotechnology with from Bar-Ilan University (2005) and M.Sc. (magna cum laude) in Biotechnology (2007) from Bar-Ilan University. Working under the supervision of Prof. Shlomo Margel, Enav's Ph.D. research involves biomedical applications of iron oxide nanoparticles, including convention enhanced delivery of anti-cancer drugs as well as usage of fluorescent iron oxide nanoparticles for early detection of colon cancer.
Abraham Rubinstein is a Professor of Pharmaceutical Sciences at The Hebrew University of Jerusalem. After his graduation (Ph.D. in physical pharmacy and pharmacokinetics) he spent two years as a postdoctoral fellow in the University of Wisconsin-Madison (GI physiology, motility and oral drug delivery) and joined the School of Pharmacy in Jerusalem as a faculty member. He is the author of more than 100 research articles, reviews and book chapters. Three of the technologies he developed were purchased by start-up companies. His research interests are focused on site-specific therapy and real-time diagnostics of inflammation and malignant processes in the GI tract.
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Shlomo Margel received his Ph.D. from the Weizmann Institute of Science, Israel (1976). He completed his postdoctoral research at the California Institute of Technology, and is a full professor at the Institute of Nanotechnology & Advanced Materials of Bar-Ilan University, Israel. He has published 185 papers in international journals and 29 patents. His major interests include polymers and biopolymers, surface chemistry, immobilization techniques, colloidal chemistry, thin coatings, biological and medical applications of polymers and micro- and nanoparticles (cancer diagnostics and therapy, neurodegenerative disorders, drug delivery, etc.).
