J Nanopart Res (2011) 13:4703–4715 DOI 10.1007/s11051-011-0436-6
RESEARCH PAPER
Investigation of in vitro efficiency of magnetic nanoparticle-conjugated 125I-uracil glucuronides in adenocarcinoma cells ¨ nak • E. Ilker Medine • Perihan U ¨ • Serhan Sakarya Feriha Ozkaya
Received: 26 March 2010 / Accepted: 21 May 2011 / Published online: 9 June 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Modification of the magnetic properties of a drug can be used to direct the drug to the desired site, enhancing its therapeutic effectiveness and reducing side effects. In this study, surface-modified magnetic nanoparticles were immobilized with uracil glucuronide derivatives and then labeled with I-125. The morphology, structure, and composition of the magnetic particles were examined by TEM, SEM, VSM, and XRD. The particles sizes were about 50 nm. The labeling yield was 93.8% for uracil-O-glucuronideimmobilized magnetic particles and 95.0% for uracilN-glucuronide-immobilized magnetic particles. The cell incorporation rates of N- and O-glucuronides were higher than those of uracil. The incorporation rates of uracil-, O-glucuronide-, and N-glucuronide-conjugated magnetic particles were all high. The cell
Electronic supplementary material The online version of this article (doi:10.1007/s11051-011-0436-6) contains supplementary material, which is available to authorized users. ¨ zkaya ¨ nak F. O E. I. Medine (&) P. U Institute of Nuclear Sciences, Department of Nuclear Applications Bornova, Ege University, 35100 Izmir, Turkey e-mail:
[email protected] S. Sakarya Science and Technology Research and Development Center (ADUBILTEM), Adnan Menderes University, 09100 Aydın, Turkey
incorporation rates of ligand-conjugated magnetic particles increased under a magnetic field. Keywords Magnetic nanoparticles Uracil Glucuronide Iodine Cell culture Drug delivery Nanomedicine
Introduction Cancer is one of the most threatening human diseases, and currently, chemotherapy is the most common method of cancer treatment. Although chemotherapeutic drugs are very effective, their potency leads to many undesirable and even toxic side effects. The major disadvantage of most chemotherapeutic agents is that they are relatively nonspecific. These therapeutic drugs are administered intravenously, resulting in general systemic distribution that leads to deleterious side effects as the drug attacks normal, healthy cells in addition to the target tumor cells (Chunfua et al. 2004). Replacing the systemic delivery of chemotherapeutic drugs with regional cancer treatment approaches is one way of overcoming side effects. Therapeutic efficiency may also be improved since higher drug concentrations may be achieved at the tumor sites when using regional therapy. Magnetically controlled targeted chemotherapy has been proposed as a more generally applicable drug-targeted delivery method for cancer therapy (Hafeli et al. 2001), allowing controlled drug
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delivery and more effective therapy while eliminating both under and overdosing. Additional advantages include the maintenance of drug levels within a desired range, the need for fewer administrations and increased patient compliance (Asmatulua et al. 2005). Magnetic drug targeting allows the concentration of drugs at a defined target site that is generally and, importantly, sited away from the reticular endothelial system (RES), with the aid of a magnetic field. Typically, the planned drug and a suitable magnetically active component are formulated into a pharmacologically stable formulation. In general, this compound is injected through the artery supplying the tumor tissue in the presence of an external magnetic field with sufficient field strength and gradient to retain the carrier at the target site (Lubbe et al. 2001; Rudge et al. 2000). Magnetic carriers are given their magnetic responsiveness to a magnetic field by incorporating materials such as magnetite, iron, nickel, cobalt, neodymium–iron–boron or samarium–cobalt (Hafeli 2004). Iron oxide nanoparticles that can act as an effective magnetic drug carrier have received increasing attention due to their multifunctional properties, including their small size, low toxicity, chemical stability, and noncarcinogenicity (Liang et al. 2007; Ramanujan and Chong 2004). Modifications of the magnetic particles with monoclonal antibodies, proteins, nucleic acids, lectins, peptides, or hormones make them more efficient and also highly specific (Gu et al. 2007; LaConte et al. 2005). They have been used as contrast agents for magnetic resonance imaging (MRI) in the diagnostic evaluation of liver and spleen tumors (Alexiou et al. 2002). Furthermore, techniques based on the use of magnetic particles have found application in molecular biology, cell isolation and purification, site-specific drug delivery, radio-immuno assay, and hyperthermic treatment of malignant cells (Berry and Curtis 2003; Pankhurst et al. 2003; Saiyed et al. 2003). Many approaches have been employed to prepare superparamagnetic supports including coating macromolecules, monomer co-polymerization, activated swelling, and silanization. Silane coupling agents have been directly coated onto the surface of magnetite nanoparticles, providing the advantage of a high density of surface functional groups and simple operation (Liu 2004b). Such coating might also provide better protection against toxicity.
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Furthermore, it protects the magnetite core from oxidation (Cao et al. 2004). Various enzymes in tissues and body fluids play a role in the detoxification process and may influence the composition and availability of steroid hormones, toxins or carcinogens. One of the most important detoxification processes occurs via glucuronidation conjugation. Glucuronides of drugs often accumulate during long-term therapy. The hydrolysis of glucuronides can be catalyzed by the enzyme b-glucuronidase, which has already been proven to be useful in the tumor-specific bioactivation of the glucuronide prodrugs of anticancer agents (Biber et al. 2006; Ertay et al. 2006). Glucuronide prodrugs produce potent anti-tumor activity in antibody-directed enzyme prodrug therapy (ADEPT), in which specific antibodies are employed to deliver b-glucuronidase to cancer cells, as well as directly in prodrug monotherapy, which relies on the presence of elevated levels of b-glucuronidase in the tumor interstitial space (Chen et al. 2007). Glucuronide derivatives of pharmacologically active compounds can act as prodrugs. The active compound is released at the desired site of action by endogenous glucuronidase enzymes (David and Ulrike 1999). Magnetic nanoparticles can be labeled with radioactive materials, thus supplying radioactive properties to the particles (Hafeli et al. 2003). Magnetic micro and nanoparticles labeled with Re-188 (Wunderlich et al. 2005), In-111 (Hafeli et al. 2003), Y-90 (Hafeli et al. 1995), I-125 (Felinto et al. 2005), and I-131 (Dagdeviren et al. 2007) have been used in tumor targeting in clinical trials. Auger electron emitters (e.g., I-123, I-125) have been proposed as an attractive alternative to energetic b-emitters (e.g., 131 I) for use in cancer therapy (Semnani et al. 2005). In this study, glucuronide derivatives of uracil were firstly synthesized using enzyme fractions separated from human cancer cells. Magnetite nanoparticles were prepared and coated them with silica. Then, organosilane was covalently coupled to the surface of the magnetic silica nanospheres. The magnetic silica nanospheres were treated with the aminosilane coupling agent, which has active groups of NH2 that are transformed to aldehyde groups that can easily connect to uracil glucuronide derivatives via schiff base linkage. After immobilization of the uracil glucuronide derivatives to the silane-coated magnetic nanospheres, these compounds were labeled with I-125. Finally, the
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in vitro efficiency of these radiolabeled compounds was examined in cancer cells.
Materials and methods Materials Na125I was obtained from the Institute of Isotopes Co. Ltd., Budapest. Uracil was purchased from MP Biomedicals, France. Hutu-80, Caco-2, and Detroit 562 were obtained from the American Type Culture Collection, Rockville, MD, USA. Primary human intestinal epithelial cells (ACBRI 519) were obtained from the Applied Cell Biology Research Institute, Kirkland, WA, USA. Ferrous chloride (FeCl3) and ferric chloride tetrahydrate (FeCl24H2O) were supplied by Fluka. N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane and tetra ethyl orthosilicate were obtained from Merck Chemical Co. All other chemicals were supplied by Merck Chemical Company and Aldrich Chemical Company. Thin layer radiochromatography (TLRC) was performed using a Bioscan AR-2000 Imaging Scanner. Liquid chromatography mass spectrometry (LC/MS/MS) chromatograms were obtained using a TandemGold Triple Quadrupole LC/MS/MS instrument. Particle size and morphology were measured using a scanning electron microscope (SEM) (Phillips XL-30 S FEG) and a transmission electron microscopy (TEM) (Tecnai G2 F30). The crystalline structure of the nanoparticles was characterized by X-ray diffraction (XRD) (Phillips X’Pert Pro). Magnetic properties were analyzed using a vibrating sample magnetometer (VSM) (LakeShore 7407). Spectroscopic measurements were performed on a Fourier Transform Infrared (FTIR) (Perkin-Elmer Spectrum100 FT-IR) using KBr pressed discs. FT-IR spectra in the range 4000–450 cm-1 were recorded in order to investigate the nature of the chemical bonds formed. Enzymatic synthesis of uracil glucuronide Enzyme UDP–glucuronyl transferase (UDPGT)-rich microsomal fractions separated from Hutu-80 (Human duodenum adenocarcinoma cell line) were used for the glucuronidation of uracil. The microsomal fractions were separated from the cells as described by ZIHNIOGLU (Zihnioglu 1992).
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The cell homogenate was centrifuged at 12,000g for 10 min, and the resulting supernatant was separated and then centrifuged at 105,000g for 1 h at ?4 °C. The microsomal pellets were dissolved in a mixture of 0.2 M potassium phosphate, 2 mM mercaptoethanol, and 0.4% TritonX100 (pH 7) buffer. The suspension was stirred on ice for 30 min, and centrifuged for 105,000g for 1 h at ?4 °C to remove insoluble material. The resulting supernatant was stored at -80 °C. The protein content was measured using the bicinchoninic acid (BCA) method (Pierce). The protein content was approximately 153.11 mg/mL. The glucuronidation reaction was performed as reported previously (Alkharfy and Frye 2002). A scheme of the glucuronidation reaction is shown in Fig. 1. Briefly, 100 lL of microsomal enzyme preparation (153.11 mg protein/mL) was added to 5 mL of 50 mM Tris buffer (pH 8.0) containing 6 mM CaCl2, 10 mM UDPGA and 1 mM dithiothreitol at 37 °C. The reaction mixture was stirred at 37 °C in a water bath for 10 min. The contents were then sonicated in an ultrasonic bath for 30 s to disperse the microsomes. This solution was incubated by slow stirring at 37 °C for 18 h following the addition of 10 mg/mL of uracil in DMSO. The reaction was terminated after 18 h by adding 300 lL acetonitrile, and the precipitated protein was removed by centrifugation at 6,000g for 10 min. The supernatant was then analyzed by reverse-phase HPLC (Shimadzu 10 AVp). Two different glucuronide derivatives were obtained and the total reaction yield was 22.95 ± 2.4% (n = 4). The glucuronide ligands were identified as uracil-O-glucuronide (UOG) and uracilN-glucuronide (UNG) by LC/MS/MS. Preparation of magnetite nanoparticles coated with silica The magnetite nanoparticles were prepared accordingly to (Cao et al. 2004). Briefly, 12 mL of 2 M FeCl3 solution in 2 M HCl was added to a 500 mL three necked glass flask. Fifty mL of a freshly prepared 0.08 M Na2SO3 solution was slowly added to the flask with nitrogen gas. Forty mL of distilled water and 8 mL of NH3 solution (28%) were added to the mixture under nitrogen gas. The solution was incubated at 70 °C for 30 min and then cooled to below 45 °C. The black magnetite precipitate was
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Fig. 1 Synthesis of uracil glucuronide
N
OH
O H N
O
NH
O
+
N
O
OH
HO
O
O
HO
HO O
OH HO
OH
OH OH
Uracil
D-Glucuronic Acid
Uracil-O-Glucuronide
H N
O O O
N
HO O HO
OH OH
Uracil-N-Glucuronide
recovered using an external magnetic field and washed several times with distilled water. The particles were then washed with a water–ethanol (2:1) mixture. The precipitate was dispersed into a mixture of 80 mL ethanol and 20 mL distilled water. Five mL of tetra ethyl ortho silicate (TEOS) and 5 mL of NH3 (10%) were added. In order to remove unreacted TEOS and unbound silica, the solution was washed with methanol after 12 h of stirring at 40 °C. The precipitate was dried under vacuum prior to characterization. Surface modification with amino-silane N-(2-Aminoethyl)-3-aminopropyl-trimethoxysilane, a silane-coupling agent, was added to silica-coated magnetite nanoparticles to reach a concentration of 20 wt%. The solution was refluxed for 12 h at 60 °C, after ultrasonication for 5 min. The solution was washed several times with methanol to remove any unreacted silane-coupling agent. The resulting precipitate was dried under vacuum prior to characterization.
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Immobilization of uracil and uracil glucuronide derivatives Uracil, UOG, and UNG were immobilized on the particle surface using glutaraldehyde. One mL of silica-coated magnetite nanoparticles coated with a silane-coupling agent ([20 mg solid/mL concentration) in methanol was taken from the colloidal system and washed with a 0.1 M phosphate buffer solution (PBS, pH 7.0, 25 °C). Washed particles were dispersed in 1 mL of 2.5% glutaraldehyde in 0.1 M PBS by ultrasonication. They were incubated at 4 °C for 4 h. The suspension was centrifuged to remove the solvent. Centrifuged particles were then redispersed in 1 mL of uracil containing 0.1 M PBS, 0.15 M NaCl, and 0.005 M EDTA (pH 7.2). The last step was repeated separately for UOG and UNG. Particles were washed with 0.1 M borate buffer (pH 9.2) containing 0.5 mg/mL NaBH4 after 12 h incubation at room temperature and redispersed in the same buffer solution. In order to remove unreacted aldehyde groups and double bonds, the solutions were kept at 4 °C for 30 min. The particles were then
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washed with 0.1 M PBS (pH 7.0 25 °C) and redispersed in 1 mL of 0.5 M MES (pH 6.6).
labeling efficiencies were derived from the counts obtained in this way.
Labeling magnetic nanoparticles with I-125
Serum stability
Uracil-, UOG-, and UNG-conjugated magnetic nanoparticles were separately labeled with I-125 using the iodogen method (Enginar et al. 2005; Unak and Unak 1993). Briefly, 500 lL of magnetic particles containing 30 lg of UOG, 30 lg of UNG, and 30 lg of uracil were placed in iodogen-coated tubes to which 2–3 mCi of Na125I was then added. These reaction mixtures were kept at room temperature without stirring for 15 min. At the end of this time, 100 lL of 1 mM Na2SO3 was added to the mixtures and quality control was carried out as described below.
Three hundred lL of blood serum supplied by a healthy volunteer was added to I-125 labeled magnetic particles in triplicate and incubated at 37 °C. The samples were analyzed at four time points (30, 60, 240 min, and 24 h) by TLRC.
Quality control procedures HPLC A low-pressure gradient HPLC system (LC-10ATvp quaternary pump and SPD-10A/V UV detector and a syringe injector equipped with a 1 mL loop and 7-lm RP-C18 column (250 mm 9 21 mm ID, Macherey– Nagel)) was used for preparative procedures. The elute was collected using a FRC-10A fraction collector (Shimadzu). The flow rate was set at 9.0 mL/min. A 5-lm RP-C18 column (250 9 4.6 mm ID) (Macharey–Nagel) and a syringe injector equipped with a 20 lL loop was used for analytical experiments. UV detection was achieved at 254 and 280 nm. The column oven temperature was set at 30 °C. Ten lL of sample was applied to the column and eluted with 50% 3 9 10-3 M tetramethyl ammonium hydroxide and 50% 3 9 10-3 M formic acid (pH 8) at 0.4 mL/min.
Cell culture Hutu-80, Caco-2, Detroit 562, and ACBRI 519 were used in the cell culture studies. Hutu-80 cells were grown in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acid, and 1 mM sodium pyruvate. Caco-2 cells were cultured in Eagle’s minimum essential medium supplemented with 20% fetal bovine serum (FBS), 2 mM glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acid, and 1 mM sodium pyruvate. Detroit 562 cells were grown in Eagle’s minimum essential medium supplemented with 0.1% lactalbumin, 10% fetal bovine serum (FBS), 2 mM glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acid, and 1 mM sodium pyruvate. ACBRI 519 cells were cultured in complete serum-containing medium kits. In all experiments, cells were grown at 37 °C in an incubator with humidified air and equilibrated with 5% CO2. The cells were maintained in exponential growth by subculturing the cells using trypsin–EDTA (0.25% by w/v in Hanks’ balanced salt solution). Cells were pelleted and resuspended in cell medium. Cell culture studies were performed six times for each experimental condition.
Thin-layer radiochromatography (TLRC) TLC silica sheets (Merck, 20 9 20 cm code: 1.05554) were used for radiochromatography. In the TLRC studies, n-buthanol/H2O/acetic acid (4:2:1) was used as the bath solution. Each TLC sheet was covered by an adhesive band following development. These TLC sheets were then assessed using a Bioscan AR-2000 Imaging Scanner. The Rf values and
Incorporation of 125I-UOG-, 125I-UNG-, and uracil-conjugated the magnetic particles by adenocarcinoma cells
125
I-
Incorporation experiments were performed with three different human adenocarcinoma cell lines (Hutu-80 (duodenum intestinal), Caco-2 (colon), Detroit 562 (pharynx)) to determine the specificity of uracil
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glucuronide derivatives obtained using the enzyme UDPGT extracted from Hutu-80 cells. Thirty lg of each sample was labeled with 4 lCi/ mL of Na125I using the iodogen method. The trypan blue exclusion method was used to assess cell viability. Hutu-80, Caco-2, and Detroit 562 cells were seeded in 24-well plates at approximately 1.0 9 105 cells per well and cultured to confluence. The monolayers were then washed three times with PBS solution, and I-125-labeled samples (0.5 mL, 30 lg/mL in culture medium) and I-125 alone were added to the cells. The samples conjugated with magnetic particles were studied in two different 24-well plates. NdFeB magnets (DMEGC; 1.17–1.21 T) were placed under each well of the plate and the magnetic field was applied to each well of one plate whilst the second well was not treated with a magnetic field individually. A control consisting of 0.5 mL of labeled samples in a culture medium was used for radioactivity measurement. After 30 min incubation, the cells were washed three times with PBS solution. The cells were then suspended by treating with 200 lL of RIPA lyse buffer solution. Some of the lysed cell suspension (50 lL) was used to determine protein content via the bicinchoninic acid method, and 100 lL was collected for measurement of radioactivity using a Packard Tricorb-1200 liquid scintillation counter. Incorporation of 125I-UOG, 125I-UNG, and 125Iuracil-conjugated magnetic particles by human intestinal epithelial cells During this step, primary human intestinal epithelial cells (ACBRI 519) were used as a healthy cell line and the results were compared with the human duodenum adenocarcinoma cell line (Hutu-80). Following culture of the ACBRI 519 and Hutu-80 cell lines, I-125-labeled samples (0.5 mL, 30 lg/mL in culture medium) were added to the cells. The experiments were conducted under the same conditions according to the method described for adenocarcinoma cells. Specific-activity-dependent incorporation The effects of increasing specific activities of I-125 on incorporation were examined using five different specific activities of I-125. In this study, Hutu-80
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cells were seeded in isolation from each other 24-well plates. Approximately 1.0 9 105 cells per well were cultured to confluence for the labeled samples with different specific activities of I-125. 30 lg of each sample was labeled separately with 1, 3, 10, 30, and 100 lCi/mL of I-125 per well. Monolayers were washed three times with PBS solution. The labeled samples with different specific activities of I-125 (0.5 mL, 30 lg/mL in a culture medium) were added separately to the cells. After 30 min incubation, the cells were again washed three times with PBS solution. The cells were suspended by treating with 200 lL of RIPA lyse buffer solution. The lysed cell suspension was used to measure protein content and radioactivity according to the procedures described for the incorporation study of labeled samples by adenocarcinoma cells. Statistical analysis Statistical significance was assessed via one-way ANOVA and linear regression using the GraphPad program. P \ 0.05 was considered statistically significant.
Results and discussion Synthesis and structural analysis of uracil glucuronide HPLC analysis showed three peaks, the first two of which belonged to uracil glucuronide derivatives. These three peaks were separated using preparative HPLC conditions as described in the experimental section. The total glucuronidation yield was 22.95 ± 2.4% (n = 4), according to the HPLC chromatogram shown in Fig. 2. The peaks reflect the tautomeric shifts of uracil, which occur because uracil undergoes amide-imidic acid at pH 7 (Charlton and Booz 1981). The lactam structure is the most common form of uracil. Theoretical lipophilicities were compared with the experimental HPLC chromatograms. The first and second peak was thus identified, respectively, as uracil-O-glucuronide (UOG) and uracil-N-glucuronide (UNG). The third peak was identified as uracil. Structural analyses of the three separated peaks were carried out using LC/MS/MS (ZIVAK-TANDEM
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Fig. 2 HPLC chromatogram of uracil and uracil glucuronide derivatives
Fig. 3 SEM of magnetite silica nanoparticles
GOLD LC–MS/MS). The results confirmed the expected structures. The molecular fragments at m/z 142.11, 148.15, 150.13, 164.15, 172.13, 222.19, and 228.20 belong to fragments of UOG and UNG, and that the fragment at 112.08 belongs to uracil. Characterization of silica- and silane-coated magnetic nanoparticles Magnetite nanoparticles were synthesized by the coprecipitation method. This method produces a number of hydroxyl groups on the surface of the particles due to contact with the aqueous phase. In order to prevent the oxidation of ferrous ion, nitrogen gas was used. It has previously been reported that the magnetic properties of nanoparticles were rather poor in the absence of nitrogen gas (Cao et al. 2004). The magnetite nanoparticles were coated with silica to avoid rapid biodegradation. Naked magnetite damages the activity of biological substances (Liu 2004c). The amino-silane coupling agents can bind covalently to the silica-coated magnetite. The terminal amino groups of the coupling agents react with glutaraldehyde and produced a Schiff base. Aldehyde groups can bind with proteins, monoclonal antibodies, etc. Compared with uncoated magnetite Fe3O4 particles, the coupling of silane polymers to the magnetic silica nanospheres is much easier to achieve due to the production of Si–O–Si bonds (Liu 2004b). A SEM image of magnetite silica particles is shown in Fig. 3. The particles are regularly spherical. Figure 4 shows a TEM image for magnetite silica particles. From this image the average nanoparticle
Fig. 4 TEM of magnetite silica nanoparticles
diameter is determined to be about 50 nm, in agreement with the SEM results. The magnetic properties of bulk Fe3O4 and silicacoated magnetite nanospheres were analyzed by VSM. Figure 5 shows the magnetization curve of the nanoparticles. Their saturation magnetizations were 62.95 and 26.43 emu/g for bulk Fe3O4 and silica-coated magnetite nanospheres, respectively. In this experiment, the size of silica-coated magnetite nanoparticles ranged between 40 and 60 nm. As this was smaller than the superparamagnetic size (*25 nm) (Cao et al. 2004; Liu 2004a, 2006), the synthesized nanoparticles exhibited ferrimagnetic characteristics with a small
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Fig. 5 VSM magnetization curve of the bulk Fe3O4 and the magnetite silica nanoparticles
coercivity value. Whereas the saturation magnetization of bulk magnetite is 62.95 emu/g, the saturation magnetization of the synthesized silica-coated magnetite nanoparticles was about 60% lower, probably due to the silica layer. Xu et al. have pointed out that coating with non-magnetic materials influences magnitude of magnetization of the coated magnetic materials due to quenching of surface moments (Xu et al. 2007). Since the magnetite nanoparticles were coated with non-magnetic silica in this work, a similar mechanism could be considered for the decrease in saturation magnetization after silica-coating in the present work. This is favorably supported by the result of the EDX analysis for the silica coated nanoparticles. Weight% values of silica-coated magnetite nanoparticles with the EDX analysis were calculated to be 32.01 wt% O, 21.09 wt% Si, and 46.90 wt% Fe as seen Fig. 6. Therefore, the amount of SiO2 phase decreased the saturation magnetization of the coated nanoparticle sample. The magnetite core was characterized by XRD patterns, which were consistent with the crystalline characteristics of magnetite (ICDD No. 75-1609). Fig. 6 EDX of magnetite silica nanoparticles
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Fe3O4 crystals with a spinel structure have six diffraction peaks of {220}, {311}, {400}, {422}, {511}, and {440}, as shown in Fig. 7. The XRD patterns of the synthesized nanoparticles showed similarities with nanoparticles prepared by Cao et al. and Liu et al. XRD analysis was also used to determine the average diameter of the magnetite core using the Scherrer equation. The sizes of Fe3O4 crystals were calculated as 49.8 nm. Moreover we obtained a mean diameter about 40–60 nm for Fe3O4 from the SEM and TEM measurements.Therefore, it is reasonable to say that the results of the electron microscopy were in good agreement with those obtained using the Scherrer equation. Figure 8 shows the FT-IR spectra of (a) uracil glucuronides, (b) magnetic uracil glucuronide coated iron oxide nanocomposite. In the spectrum 7a the characteristic NH2 stretching frequencies of uracil glucuronide is observed at *3432 cm-1. In the spectrum for uracil glucuronides (Fig. 8a), the vibrations of asymmetric and symmetric stretching of carboxyl anion occurred at 1606 and 1360 cm-1 (Hong et al. 2009). The splitting between asymmetric and symmetric stretching of carboxyl group is higher than that present in the FT-IR spectra of magnetic uracil glucuronide coated iron oxide nanocomposite (Fig. 8b), indicating a unidentate coordination between the carboxyl anions and the metal ions (Nara et al. 1996). In Fig. 8b, the characteristic absorption of Fe–O bond is at 586 cm-1 (Lei et al. 2008). The presence of additional peaks between 478 and 1298 cm-1 were most probably due to the symmetric and asymmetric stretching vibration of framework and terminal Si–O– groups. Furthermore, four peaks at 2939, 2878, 2742, and 2613 cm-1 corresponding to C–H stretching absorptions in
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Fig. 7 XRD powder pattern of magnetic uracil glucuronide coated iron oxide nanocomposite
Fig. 8. The peak of the magnetic uracil glucuronide coated iron oxide was stronger at about 3401 cm-1, demonstrating that it carried some free Si–OH permitted by the incompleteness of tetraethoxysilane (TEOS) hydrolysis. According to these results, these FT-IR spectra provided supportive evidence that uracil glucuronides were successfully coated on the surface of iron oxide. Labeling and stability The labeling yield for I-125-labeled compounds is given in Table 1. The Rf values show that the nanoparticles remained at the point of application. The Rf values of coated magnetic nanoparticles differed from those of naked nanoparticles. The labeling yields of I-125 labeled UOG-conjugated magnetic nanoparticles and I-125 labeled UNG-conjugated magnetic nanoparticles were 87.8 ± 0.9% (n = 10) and 91.0 ± 1.4% (n = 10), respectively. The in vitro stabilities of the radiolabeled uracil glucuronide conjugated with nanoparticles in the blood serum were quite high. The labeling yields of I-125-labeled compounds were higher than 90% for the first 24 h. Each labeled compound was stable at least for 24 h at room temperature. The labeling efficiency of radioiodinated magnetic particles of coated uracil glucuronide derivatives was sufficiently high and the duration of stability was long enough to allow the cell culture studies. Cell culture The results showed that the incorporation of I-125labeled compounds was similar in each of the three
cell lines. Thus more I-125-labeled uracil-O-glucuronide was incorporated than I-125-labeled uracil-Nglucuronide, and the incorporation of both of these glucuronides was significantly higher than that of I-125-labeled uracil and I-125 in all cell lines, as shown in Table 2. The results show that O-glucuronide incorporation was about threefold higher than that of N-glucuronide. This may be due to greater hydrolysis of O-glucuronides compared to N-glucuronides. Both b-glucuronidases may be responsible for the hydrolysis of glucuronide drugs in cells (Bock and Kohle 2009). The glucuronide disposition may also be influenced by ER (endoplasmic reticulum)-localized b-glucuronidase. Table 2 shows that the incorporation of magnetic particle-conjugated ligands was higher than that of unconjugated ligands for all cell lines. The cellular incorporations of O-glucuronide-conjugated magnetic nanoparticles were higher than those of unconjugated O-glucuronides by 1.37 for Hutu-80, 1.26 for Caco, and 1.59 for Detroit cells. The rate of cellular incorporation of O-glucuronide under a magnetic field increased by 2.5-, 2.02-, and 2.6-fold for Hutu, Caco, and Detroit cells, respectively. Although the rate of incorporation of uracil and I-125 alone was rather low in all cells, the incorporation of magnetic particles conjugated with uracil and I-125 increased significantly due to the magnetic particle conjugation and magnetic field effect. These increases were similar for all applications of uracilO-glucuronide, and therefore the magnetic particles may not be tumor selective. The highest rates of incorporation were always achieved by magnetic
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Fig. 8 FT-IR spectra of a uracil glucuronides, b magnetic uracil glucuronide coated iron oxide nanocomposite
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adenocarcinoma cells since the enzyme b-glucuronidase was more abundant in tumor cells. These results indicate that glucuronidation increased the incorporation efficiency. O-Glucuronides might be more effective for targeted therapy. Franklin et al. (1996) were reported glucuronyl transferase, which was found to be common in colorectal cancer cells, plays a role in drug resistance. In order to demonstrate the damaging effect of increasing specific activities of I-125 on the cells, the incorporation of I-125 labeled UOG and I-125 labeled UNG with different specific activities was examined using Hutu-80 cells. The incorporation efficiencies of 125I-UOG and 125I-UNG were higher than those of 125I-uracil and I-125. The incorporation of 125I-UOG was higher than that of 125I-UNG in all experiments as presented in Fig. 9. The incorporation of I-125-labeled UOG and I-125-labeled UNG was not altered significantly by increasing the specific activity because of the specific decay characteristics of I-125. I-125 decays by electron capture (EC) that is a very effective Auger electrons emitter decay. Two successive Auger cascades occur for each decay of iodine-125, and consequently it emits an average of 21 Auger electrons in condensed matter per decay. The energy of Auger electrons ranges from 10 eV to 34 keV (Charlton and Booz 1981). It is also interesting to note that the range of Auger electrons emitted by an iodine-125 atom can reach 40–45 nm, which is a relatively short range for condensed materials. This means that the local absorption of these Auger electrons results in high energy
Table 1 % Labeling yield values for I-125 labeled compounds using TLRC-1 bath Compounds
% Labeling yield
125
93.86 ± 1.5
125
95.04 ± 1.1
125
94.19 ± 1.7
125
87.8 ± 0.9
125
91.0 ± 1.4
125
89.0 ± 1.7
I-UOG I-UNG I-uracil I-Magnetic particles coated UOG I-Magnetic particles coated UNG I-Magnetic particles coated uracil
conjugated O-glucuronides under a magnetic field. This result shows that magnetic particles are very good carriers and that they can be used more effectively with enzymatic modification. The incorporation of I-125-labeled samples in the adenocarcinoma cell line (Hutu-80) was also compared with that in a normal cell line (primary human intestinal epithelial ACBRI 519). 125I-UOG was incorporated more than 125I-UNG, and that both of these glucuronides were significantly more highly incorporated than 125I-uracil and I-125 in both cell lines. In addition, 125I-uracil and I-125 had quite low rates of incorporation in Hutu-80 and ACBRI 519. The percentage incorporation of samples increased with magnetic particle conjugation and the use of a magnetic field in both cell lines. However, the rate of incorporation of 125I-UOG by the normal cell line was significantly lower than that of the adenocarcinoma cell line (P \ 0.05). Compared to normal cells, UOG was more selective for Table 2 % Incorporation values of
125
I labeled samples on cell lines
Hutu-80
Caco
Detroit
ACBRI 519
125
I-UOG
14.6 ± 0.6
14.9 ± 0.5
12.6 ± 0.8
12.0 ± 0.7
M-125I-UOG
19.6 ± 0.6
18.7 ± 1.1
20.0 ± 0.8
16.9 ± 0.6
MA-125I-UOG
34.4 ± 0.8
30.1 ± 1.0
32.7 ± 1.5
30.7 ± 0.8
4.1 ± 0.2
3.4 ± 0.3
3.7 ± 0.3
3.4 ± 0.2
14.4 ± 0.7
15.7 ± 0.4
15.3 ± 0.8
12.0 ± 0.4
30.9 ± 1.0
31.2 ± 0.9
28.2 ± 1.6
28.5 ± 0.8
125
I-uracil
0.9 ± 0.0
0.6 ± 0.1
0.8 ± 0.0
0.7 ± 0.1
M-125I-uracil
6.9 ± 0.2
8.5 ± 0.6
7.0 ± 0.5
6.0 ± 0.1
17.8 ± 0.4
17.5 ± 1.2
19.1 ± 1.1
17.4 ± 0.8
0.5 ± 0.1
0.5 ± 0.1
0.4 ± 0.1
0.5 ± 0.1
125
11.7 ± 1.2
10.7 ± 0.3
13.0 ± 1.1
11.0 ± 0.7
MA-125I
21.6 ± 1.1
19.4 ± 0.9
19.0 ± 0.6
16.1 ± 0.6
125
I-UNG
M-
125
I-UNG
MA-125I-UNG
MA-125I-uracil 125
I
M-
I
123
4714
J Nanopart Res (2011) 13:4703–4715
References
Fig. 9 Incorporation effect of increasing specific activity of 125I
deposition in the vicinity of an iodine-125 atom. These electrons produce high cellular radiotoxicity if the I-125 is incorporated in the DNA but have less effect outside the nucleus.
Conclusions Magnetic nanoparticles were synthesized based on a magnetite/silica combination. These nanoparticles had an average size of 40–60 nm diameter and exhibited ferrimagnetic behavior. X-ray diffraction measurements indicated a spinel structure for the magnetite particles contained within the magnetic silica-coated nanoparticles. They could be activated by glutaraldehyde for immobilization to protein and affinity ligands such as glucuronide derivatives, which are selective for adenocarcinoma cells. Radiolabeled glucuronide derivatives could be used for therapy and tumor imaging because of their rapid and high incorporation by adenocarcinoma cells. This efficiency of glucuronide derivatives could be increased using magnetic particle conjugation and a magnetic field. Ultimately, radiolabeled magnetic particles conjugated with glucuronides might be used for targeted therapy as an effective magnetic drug carrier. Acknowledgments This work has been financially supported ¨ BI˙TAK (contract no 105 S 486), Ege University by TU Research Fund (contact no 06 NBE 03), and Ege University Research Fund (contact no 07 ILAM 01). The authors thank Adnan Menderes University Science Technology Research and Application Centre for conducting the cell culture experiments.
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