Pharmaceutical nanotechnology
Synthesis and biological evaluation of radiolabeled photosensitizer linked bovine serum albumin nanoparticles as a tumor imaging agent

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Abstract

In this study, we reported on the synthesis and biological evaluation of radiolabeled fluorescent dye conjugated bovine serum albumin nanoparticles within the size range 190–210 nm. The bovine serum albumin nanoparticles (BSANPs) were prepared using a desolvation method, and chemical cross-linking was performed using gluteraldehyde. Furthermore, pheophorbide-a (PH-A) was loaded on the BSANPs. The results obtained from dynamic light scattering and electron microscopy have proved that nanoparticles are highly monodisperse and near-spherical shaped. The photo-physical properties of the PH-A-BSANPs were obtained using the spectrophotometric techniques. According to the results, PH-A and BSANPs show high non-covalent interaction. PH-A loaded nanoparticles were labeled with 99mTc and the radio-labeling efficiency was determined as 90 ± 1.2%. Biodistribution studies of 99mTc labeled PH-A-BSANPs and PH-A were carried out using female Albino Wistar rats, and 99mTc-PH-A-BSANPs showed a significantly higher uptake in the breast and uterus than 99mTc-PH-A. Cell culture study was carried out in MCF-7 cell line (human breast adenocarcinoma cell line). According to the cell culture studies, 99mTc-PH-A-BSANPs showed a higher uptake than 99mTc-PH-A. Moreover, PH-A-BSANPs demonstrated good photo-physical properties and BSANPs increased the uptake of PH-A on to the MCF-7 cell line. These results confirm that 99mTc labeled PH-A-BSANPs could be utilized for radioimaging.

Graphical abstract

99mTc-PH-A complex (right) conjugated bovine serum albumin nanoparticles within the size range from 190 to 210 nm for use as a cancer targeting agent. Fluorescence microscopy image of PH-A-BSANPs inside MCF-7 cells (left).

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Introduction

Recent advances in nanotechnology have stimulated novel applications in cancer therapy and diagnosis where nanoparticles are used to achieve colloidal drug delivery systems. The major goals of in colloidal drug delivery systems are controlled transport of the drug to target cells and tissues at a therapeutically optimal rate and dose regimen. Thus, they can prevent the unwanted toxic side effects of the drug and improve its therapeutic effects (Crommelin and Schreier, 1994, Zhang et al., 2004, Hans and Lowman, 2002). Nanoparticles are useful drug delivery systems for the selective transport of the drug to the target cells and tissues. Therefore, they may be combined with targeting ligand, imaging-probes and polymers via adsorption or conjugation (Wang et al., 2010, Yang et al., 2010, Lin et al., 1999, Cho et al., 2008).

Nanoparticles can be prepared using a variety of materials such as proteins, synthetic polymers and lipids. The selection of materials is dependent on size and surface characteristics, toxicity, and the biodegradability of nanoparticles (Wang et al., 2010, Mohanraj and Chen, 2006). Among polymer materials, those based on proteins are particularly preferred such as serum albumin and gelatin, which are widely used for the preparation of nanoparticles in drug delivery systems (Ofokansi et al., 2010, Labhasetwar et al., 1996). Since, they are non-toxic, biodegradable, easy to prepare, and their size distribution can be easily monitored. Moreover, proteins have some functional groups such as amino, carboxylic and hydroxyl, and these groups make easy the bonding process between drug and protein based nanoparticles through covalent linking and non-covalent adsorption (Weber et al., 2000).

Nanoparticulate colloid systems have been improved for the diagnosis of cancer. For example, strong fluorescence emitting materials, super-paramagnetic iron oxide (SPIO) and gadolinium chelates are loaded on the nanoparticles for the imaging of target tumors (Yang et al., 2010). One different approach for tumor imaging is to use radiolabeled (99mTc, 67Ga, 111In etc.) agent conjugated nanoparticles such as hematoporphyrin derivatives and porphyrin analogues bearing albumin nanoparticles. These agents demonstrate good localization in tumors, and they can easily form a complex with radionuclide for having chelating properties (Yang et al., 2010, Babbar et al., 2000, Josefsen and Boyle, 2007, Murugesan et al., 2002). Furthermore, many photosensitizer compounds exhibit hydrophobic characteristics, thus they can be combined with nanoparticles for effective distribution in the body (Chatterjee et al., 2008).

Albumin is a major protein in blood plasma, and it has a great potential as a nanocarrier in drug delivery systems. It is widely used in nanoparticulate colloid systems due to its non-toxic and biodegradable properties. Many organic and inorganic molecules can interact easily with albumin nanoparticles owing to their proper functional groups on the particle surface. In addition, albumin is a protein which is soluble, and stable in a wide range of pH and up to 60 °C. So, it is a suitable protein for preparing nanoparticles, which are widely used in colloidal drug delivery systems (Chen et al., 2009, Kratz, 2008, Sharman et al., 2004). The desolvation process is commonly used to prepare polymer based nanoparticles such as BSANPs. The addition of desolvating agents such as ethanol causes a better controlled separation and precipitation of the particles (Jun et al., 2011).

Chlorophyll derivatives are colored compounds and possess well-known naturally occurring photosensitizing properties. PH-A is synthesized from chlorophyll-a by the elimination of phytol and magnesium (Keller et al., 1996). PH-A is an efficient photosensitizer agent and extensively used in photodynamic therapy. Previous research results demonstrated that PH-A can be used for the treatment of a number of tumors and microbial infections. It has been considered as for anti-tumor and anti-inflammatory agents. Some photosensitizers (T3, 4BCPC, Photosan-3) like PH-A were labeled with 99mTc and their potential for murine tumors was evaluated, and it was found useful for monitoring cancer (Chen et al., 2009, Jori et al., 2006, Aprahamian et al., 1993). On the other hand PH-A was labeled with Tc-99m by Ocakoglu et al. and its potential was evaluated in infection imaging (Ocakoglu et al., 2011).

Recently, numerous photosensitizer linked albumin nanoparticles have been synthesized and evaluated for tumor imaging and PDT. However, in this paper PH-A was prepared from a biological material, and it was loaded on protein based nanoparticles. The generated structure is commonly natural, and it was prepared using easy synthesis methods. Due to all these important advantages of PH-A, in current study, bovine serum albumin nanoparticles (BSANPs) was prepared using a desolvation technique, and PH-A was loaded on BSANPs by non-covalent adsorption. Then, BSANPs-PH-A nanoparticles were labeled with 99mTc. Additionally, the radiopharmaceutical potential of radiolabeled photosensitizer linked bovine serum albumin nanoparticles was examined in female Albino Wistar rats and MCF-7 cells.

Section snippets

Materials

1H NMR measurements were performed on a Bruker 400 MHz spectrometer using residual solvent peaks as internal standards. Absorption spectra were recorded on a 1 cm path length quartz cell on a Shimadzu UV-2102 UV–vis spectrophotometer. Mass analysis was performed using a Waters LCT Premier (ESI).

Bovine serum albumin (BSA) was supplied by Sigma Aldrich Chemical Co. (Steinheim am Albuch, Germany). The RPMI 1640, Trypsin-EDTA, fetal bovine serum, l-glutamine, penicillin and streptomycin were from

NMR, UV–vis spectrometry and mass spectrometry

The chemical structure of PH-A was checked with 1H NMR and MS (ESI). UV (CH2C12) λmax: 666 (0.36), 610 (0.06), 538 (0.063), 508 (0.07), 412 (0.79), 1H NMR (400 MHz, CDCI3) δ ppm: 9.57 (s, 1H, CH-5), 9.45 (s, 1H, CH-10), 8.64 (s, 1H, CH-20), 7.99 (q, 1H, J = 6.4 Hz, 31), 6.25, 6.19 (q, 1H + 1H, J = 16.8 Hz, 32), 5.25, 5.14 (d, 1H + 1H, J = 20 Hz, 132-CH2), 4.51 (dq, 1H, J = 2 Hz, CH-18), 4.32 (dt, 1H, J = 8.4 Hz, CH-17), 3.67 (s, 3H, COOCH3), 3.61 (s, 3H, CH3-121), 3.55 (q, 2H, J = 8 Hz, CH2-81), 3.41 (s, 3H, 21-CH3),

Conclusions

In this study, BSANPs were prepared by a desolvation method and a photosensitizer (PH-A) was successfully loaded onto the nanoparticles’ surface. The size of the particles was measured in the range from 190 to 210 nm. The biodistribution of 99mTc-PH-A-BSANPs in healthy female rats showed high uptake in the breast and uterus. 99mTc-PH-A-BSANPs might therefore be used in scintigraphic tumor imaging for the organs. 99mTc-PH-A-BSANPs were taken up to human breast adenocarcinoma cell line (MCF-7). 99m

Acknowledgement

The authors gratefully acknowledge the financial support received from the Department of Scientific Projects at Ege University, Izmir, Turkey (Project no: 2010-NBE-003).

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