Article pubs.acs.org/Biomac
Progress in Targeting Tumor Cells by Using Drug-Magnetic Nanoparticles Conjugate Anna M. Nowicka,* Agata Kowalczyk, Anita Jarzebinska, Mikolaj Donten, Pawel Krysinski, and Zbigniew Stojek Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
Ewa Augustin and Zofia Mazerska Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland ABSTRACT: To limit cytotoxicity of anticancer drugs against healthy cells, an appropriate carrier should be synthesized to deliver the drug to the tumor tissue only. A good solution is to anchor a magnetic nanoparticle to the molecule of the drug and to use a properly directed external magnetic field. The synthesis of the conjugate of doxorubicin with magnetic nanoparticles (iron oxide) modified by us resulted in a substantial depression of the aggregation process of the nanoparticles and therefore allowed the correct examination of cytotoxicity of the modified drug. It has been shown, by performing the electrochemical microbalance measurements, that the use of magnetic field guaranteed the efficient delivery of the drug to the desired place. The change in the synthesis procedure led to an increase in the number of DOX molecules attached to one magnetic nanoparticle. The release of the drug took place at pH 5.8 (and below it), which pH characterizes the cancer cells. It has also been found that while the iron oxide magnetic nanoparticles were not cytotoxic toward human urinary bladder carcinoma cells UM-UC-3, the tumor cell sensitivity of the DOX-Np complex was slightly higher in comparison to the identical concentration of doxorubicin alone.
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INTRODUCTION Chemotherapeutic agents are widely used in cancer treatment.1−3 However, they are also highly toxic. Doxorubicin (DOX) is especially toxic to the heart and the kidneys, which limits its therapeutic applications.4−7 The way it is delivered (intravenous shots) promotes the toxic action. Hence, novel drug delivery systems are urgently needed. The binding of a magnetic nanoparticle with doxorubicin might be a good alternative to such traditional forms of the drug-like liposomes and micelles.8−14 Some of the nanomaterials, including magnetic nanoparticles, are electroactive,15 and some particles can be transported across cell membranes into mitochondria.16 The magnetic nanoparticles based on iron oxide (Fe3O4) are not toxic, this was proved using the rat liver derived cell line (BRL 3A).17,18 The results for membrane leakage of lactate (LDH assay) in the case of Fe3O4 did not produce cytotoxicity up to the concentration of 100 μg/mL, but it produced a significant effect at the concentration 250 μg/mL.18 Different sizes of Fe3O4 were compared, and no substantial difference in the toxicity was found. Additionally, the nanoparticles are suspected to be easily transported through the cell membranes, maybe also by using the way of endocytosis. They became an attractive material for pharmacology not only as a tumortargeted drug delivery system.19 It has been demonstrated that © 2013 American Chemical Society
the cell-penetrating magnetic nanoparticles can be efficient carriers for siRNA20,21 and antisense oligodeoxynucleotides22 into tumor cells for intracellular gene knockout. In addition, they have a great potential in transmission electron microscopy (TEM) and magnetic resonance imaging (MRI) as the imaging tool.23 It should be possible to transport the modified drug into a selected body part by applying a magnetic field. The modified drug molecules are expected to stay in the tumor cells as long as the magnetic field is on till the drug is released. Interestingly, most of magnetic nanoparticles have a natural tendency to accumulate in a cancer tissue.24 Recently we have demonstrated that doxorubicin covalently bound to magnetic nanoparticles (DOX-Np) is still capable of intercalating the dsDNA helices. The intercalation behavior of DOX molecules is only slightly affected by their attachment, via a flexible adipoyltether, to the surface of magnetic nanoparticles.25 However, the tendency of nanoparticles to agglomeration made the examination of cytotoxicity of the conjugate (DOX-Np) impossible. Also, this agglomeration did not help in the examination of movement of the modified drug Received: December 5, 2012 Revised: January 16, 2013 Published: January 17, 2013 828
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in an external magnetic field. Herein, we report how the application of an external magnetic field may help in directing doxorubicin with magnetic Np selectively to appropriate places. We have done experiments to prove that our way of modification of doxorubicin does not affect the cytotoxicity of DOX against tumor cells. We have also done experiments with electrochemical quartz crystal microbalance (EQCM) to obtain evidence that the magnetized drug is more efficiently accumulated in the selected places. The last aim was to examine whether the pH of cancer cells is appropriate for releasing of doxorubicin from its conjugate.
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Scheme 1. Structure of DOX-Np Conjugate
EXPERIMENTAL SECTION
Materials. All chemicals were of the highest quality available. Iron(II,III) oxide magnatic nanoparticles solution, particle size 5 ± 1 nm (p.a. Sigma), adipoyl chloride (p.a. Sigma, 98%), dimethyl sulfoxide (p.a. Sigma), citric acid (p.a., POCH, Poland), acetonitrile (dry, Aldrich, 99.8%), triethylamine (p.a. POCH, Poland), N,N,N′,N′tetramethyl-O-(N-succinimidyl)uroniumtetrafluoro-borate (p.a. SigmaAldrich), NaOH (p.a., POCH, Poland), KH2PO4 (p.a. POCH, Poland), K2HPO4 (p.a. POCH, Poland), KCl (p.a. POCH, Poland), NaCl (p.a. POCH, Poland), NaClO4 (p.a. POCH, Fluka), doxorubicin hydrochloride (p.a. Sigma), poly(allylamine hydrochloride) (PAH; p.a. Sigma) were used as provided by manufacturers. All solutions, except for the PAH solution, were prepared in 0.02 M PBS buffer, pH 7.4. PAH was dissolved in 0.5 M NaClO4. Calf thymus (ct) doublestranded DNA (dsDNA) was purchased from Fluka; it was sufficiently pure and virtually free of protein. Good criteria for DNA purity are the values of the DNA absorbance ratios: Aλ=260 nm/Aλ=280 nm in the range 1.7−2.0 and Aλ=260 nm/Aλ=250 nm in the range 1.4−1.7.26 The ct dsDNA samples chosen by us gave an absorbance ratio in the middle of the indicated ranges. dsDNA solutions of 1 mg DNA per 1 mL of phosphate buffer (pH 7.4) were prepared at least 24 h before experiments to get full renaturation. The concentration of dsDNA was determined from the value of the absorbance at λ = 260 nm; ε = 13200 M−1 cm−1.27 All solutions were prepared with Milli-Q water. Synthesis Procedure. The iron oxide(II,III) magnetic nanoparticles with average particle size 5 ± 1 nm (TEM) exhibit superparamagnetic properties at ambient temperature with magnetization value of ≥30 emu/g (manufacturer data, Sigma-Aldrich) and were used as a doxorubicin drug carrier. The synthesis procedure given in the literature25,28,29 was modified by us. First, to limit the aggregation of nanoparticles, we sonicated the nanoparticles solution before and during the addition of the linker. Next, in the functionalization of the nanoparticles, triethylamine was used instead of 4-methylmorpholine, and the DOX solutions contained citrate buffer. The carboxyl groups were activated with N,N,N′,N′tetramethyl-O-(N-succinimidyl)uroniumtetrafluoro-borate. The structure of the DOX-Np conjugate is presented in Scheme 1. Cell Culture. UM-UC-3 cells (human urinary bladder carcinoma cell line) were obtained from ATCC (American Type Culture Collection). Cells (mycoplasma free) were maintained as a monolayer culture at 37 °C in a humidified 5% CO2 atmosphere in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (SigmaAldrich, St. Louise, CA, U.S.A.). Under these conditions, the celldoubling time was 24 h. Cytotoxicity and Morphology Assessment. Cytotoxicity of Np, DOX-Np, and DOX was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded in 24-well plates (2 × 104 /well) and treated with various concentrations of NP, DOX-Np, and DOX. The solutions of these agents were prepared in sterile water. After 72 h of incubation, the cell morphology and cytotoxicity were examined. Photos (magnification, ×125) of cells from each well were taken using light microscope TELAVAL 3 (Carl Zeiss, Jena, Germany). Then, 200 μL of MTT solution (4 mg/mL) was added to each well containing 2 mL of media and incubated for 4 h at 37 °C in a humidified 5% CO2 atmosphere.
After that, the media with MTT solution was removed and crystals of formazan from each well were suspended in 2 mL of dimethylsulfoxide (DMSO). The plate was left on shaking platform for 30 min. Finally, 200 μL of solution from each well was collected and transferred into a 96-well plate. The absorbance was recorded on a Microplate Reader (Bio-Rad, Hercules, CA, U.S.A.) at the length of 540 nm. A dose− response curve was plotted and used to calculate drug concentration that yielded 50% inhibition of cell growth (IC50). Characterization of DOX-Np Conjugate. The morphology and size of aqueous dispersions of the DOX-Np conjugate were characterized by using field emission scanning electron microscopy (FESEM; Merlin; Carl Zeiss Germany; images were taken at low primary electron beam energy: 2 keV), equipped with energy filter transmission electron microscopy (EFTEM; Libra 120 plus; operated at 120 kV), and atomic force microscopy (AFM; NanoScope IIIa instrument; Digital Instrument Santa Barbara; the cantilevers, type NP−S (Veeco), with a spring constant of ca. 0.12 Nm−1). In all experiments, glassy carbon plates were used as the substrates. The product (DOX-Np conjugate) of synthesis was identified by Raman spectroscopy (Labram HR800; Horiba Jobin Yvon confocal microscope system equipped with a Peltier-cooled CCD detector (1024 × 256 pixel), using a 20 mW HeNe (632.8 nm) laser). Electrochemical Measurements. An electrochemical quartz crystal microbalance (Autolab) with 6 MHz AT-cut quartz crystal resonators was used in the study. The piezoelectrically active surface area was 0.52 cm2. Cyclic voltammetry was performed an Autolab potentiostat, model PGSTAT 12. All voltammetric experiments were carried out in the three-electrode system consisting of a gold covered quartz electrode used as the working electrode, a reference electrode (Ag/AgCl) and a gold wire used as the auxiliary electrode. The working electrode was electrochemically pretreated first by cycling between 0 − 1.8 V (10 s treatment at 1.8 V) in a 0.1 M NaOH with a scan rate of 50 mV/s and then cycled between −0.3 − 1.5 V (vs Ag/ AgCl) in 0.1 M H2SO4 solution until a stable voltammogram typical for a clean gold electrode was obtained.30 As a source of magnetic field a neodymium magnet (345 mT) was applied. All synthetic experiments and electrochemical and biological tests were repeated at least three times.
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RESULTS AND DISCUSSION The sonication of the nanoparticles solutions led to successful limitation of the agglomeration process. Typical SEM, TEM, and AFM images of the residue after the evaporation of the aqueous solutions containing DOX-Np conjugate in concentration 10 nM of DOX are presented in Figure 1. The presented micrographs demonstrate that the process of 829
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of iron oxide nanoparticles at about 370 cm−1 (T1), 480 cm−1 (E), and 740 cm−1 (A1) are visible.31−34 Other Raman modes visible on the Np spectrum (at ca. 950, 1080, and 1450 cm−1) are due to the presence or traces of organic solvent and the stabilizer (toluene and oleic acid) of the commercial Np suspension. After the modification with adipoyl linker, these modes are enhanced by the presence of δ(CH2) aliphatic vibrations and the vibrations originated from the dissociated, in the aqueous environment, carboxylate groups ν(COO−, sym). Also, the iron oxide lattice vibrations are now more pronounced. After the covalent attachment of doxorubicin, several important features characteristic for this drug appeared on the Raman spectrum of DOX-Np conjugate. A broad band between 1210 and 1345 cm−1 can be assigned to the δ(C−O− H) vibrations35,36 (dihydroxyanthraquinone residue) and coupling of the ν(C−O) component and ring stretching ν(C−C), around 1310 cm−1 and 1340 cm−1, respectively. Another quite intense band located at 1580 cm−1 is due to the ν(CC) mode of aromatic ring.35,36 The presence of these bands confirms the successful attachment of doxorubicin. The amount of doxorubicin bounded to nanoparticle was estimated from the charge under the voltammetric cathodic peak at −0.6 V, yielding a value of 8.9 mg of DOX per 1 g of nanoferrites. The number of DOX molecules per one NP molecule increases from about 5% to about 35% of maximal 2D packing after application of sonication in the synthesis. The calculations were done according to the procedure described in our earlier paper.25 A better dispersion of the nanoparticles should lead to an increased available-for-modification surface area at the nanoparticles and to increase in amount of DOX on the surface. Cytotoxicity of magnetic nanoparticles (Np), doxorubicin with nanoparticles (DOX-Np), and doxorubicin (DOX) toward human urinary bladder carcinoma cells UM-UC-3 was evaluated by MTT assay after 72 h of incubation.37,38 We selected this cell line because urinary bladder cancer is one of the most common types of cancer in the world that is associated with poor prognosis. Our data showed that magnetic nanoparticles did not exhibit significant cytotoxic activity toward UM-UC-3 cells when used in a wide range of concentrations, including doses close to IC50 of DOX-Np, see Figure 3. In turn, treatment of UM-UC-3 cells with DOX-Np conjugates resulted in concentration-dependent growth inhibition of the cells (Figure 3). Interestingly, growth inhibition of UM-UC-3 cells caused by DOX-Np was even higher than in the case of free doxorubicin (IC50 = 0.018 ± 0.002 for DOX-Np and 0.076 ± 0.064 for DOX, respectively). Additionally, we performed morphological observation of the UM-UC-3 cells following incubation with nanoparticles, DOX-Np conjugate and doxorubicin, obtained photos are presented in Figure 4. These observations confirmed that magnetic nanoparticles did not affect cells growth at concentration, which was applied to reach IC50 of DOX-Np (A) and higher (B; Np at Figure 4 A,B). The morphology of these cells was similar to that of the control (untreated) cells. On the other hand, after the treatment with DOX-Np and DOX, the morphology of UM-UC-3 cells was changed compared to the control (untreated) cells. Namely, these cells were shrunken and a massive detachment from the culture plates was observed, what indicated that these cells may have undergone cell death (see DOX-Np and DOX micrographs in Figure 4 A,B). The influence of the magnetic field on the efficiency of DOX transport to the indicated place was examined by applying the
Figure 1. SEM, TEM, and AFM images of DOX-Np conjugate without and with sonication step in synthesis.
agglomeration was under control and the final size of the conjugate did not exceed 30 nm. So, the penetration of the cancer cells by the conjugate was possible. The attachment of the adipoyl linker (C6) to the magnetic nanoparticle and its consecutive functionalization by DOX was confirmed by Raman spectroscopy. The Raman spectra of the iron oxide nanoparticles (Np) and iron oxide nanoparticles after their grafting with adipoyl chloride linker (Np−C6) and after the covalent attachment of doxorubicin to such nanoparticles (DOX-Np) are presented in Figure 2. For the unmodified nanoparticles (Np), the characteristic lattice vibrations (Fe−O)
Figure 2. Raman spectra of magnetic iron oxide(II,III) nanoparticles (Np) and iron oxide nanoparticles after their grafting with adipoyl chloride linker (Np−C6) and after covalent attachment of doxorubicin to such nanoparticles (DOX-Np); λexc = 632 nm. 830
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Figure 3. Cytotoxicity of DOX-Np vs DOX on human urinary bladder carcinoma cells UM-UC-3 after 72 h of incubation. The growth inhibition of UM-UC-3 cells in increasing concentrations of equivalent doses of doxorubicin or Np was determined by MTT assay and is expressed as a percentage of the control value (untreated cells: C). Data are presented as mean ± SD of triplicate experiments. Inset: Cytotoxicity of Np on human urinary bladder carcinoma cells UMUC-3 after 72 h of incubation.
EQCM technique. To prevent the adsorption of the chloride ions present in the PBS buffer on the gold electrode surface, the surface was initially modified with PAH, a cationic polymer. Next, as a result of the electrostatic interactions between the positively charged polymer and the negatively charged phosphate-sugar backbone of ctDNA, the ctDNA strands are anchored at the electrode surface. The studies were done for free and modified with magnetic nanoparticles doxorubicin in the presence and absence of external magnetic field. The frequency changes obtained after each step of modification of the electrode surface are presented in Figure 5. The influence of magnetic field was seen only in the case of doxorubicin modified with magnetic nanoparticles. The resonant frequency change, Δf, corresponding to the mass accumulation, for DOXNp binding in the DNA layer with and without magnetic field are 273 ± 12 and 118 ± 4 Hz, respectively. After removal of the magnetic field, the frequency of the gold electrode increased by 64 ± 9 Hz; however, it has never reached the value obtained in the absence of magnetic field. It means that not all DOX-Np particles accumulated near ct dsDNA layer interacted with DNA. In our drug−carrier complex, DOX is conjugated with magnetic nanoparticles through an amide bond. Because the interior of the tumor cells is slightly acidic (pH 4−5),39 therefore, this pH should support the release of the drug from the carrier due to the hydrolysis of the amide bond. Finally, in the next step, we applied PBS buffer of lower pH (5.8). After more than 12 h, the EQCM frequency increased by 132 ± 7 Hz. This change is related to the removal of the magnetic nanoparticles. To be sure that only the carrier is removed in pH 5.8 and DOX is still bound to DNA, cyclic voltammetric curves were obtained, see Figure 6. The oxidation/reduction peaks of DOX (dashed green line) are well visible, which means that pH 5.8 caused only the removal of the carrier while the drug remained in the DNA−DOX complex. The presence of that peak and its position (compared to unmodified DOX) indicate that DOX is still bound to DNA. Hence, the corresponding value of the frequency shift (141 Hz) is related only to the DOX molecules
Figure 4. Morphological observation of UM-UC-3 cells, untreated (control) and treated with magnetic nanoparticles (Np), conjugate doxorubicin with Np (DOX-Np), and doxorubicin (DOX) at concentrations 0.01 μM (A) and 0.5 μM (B). Magnification: ×125.
accumulated in the ctDNA layer. We also want to stress here that the voltammogram for the DOX-Np complex differs significantly from the voltammograms obtained for the unbound DOX and liberated DOX. The observed mass increase related to the measured experimental frequency shift, for DOX binding to DNA, is mDOX = 4.3 × 141 = 606 ± 45 ng/ QC, which corresponds to about 1166 ng/cm2. In the case of unmodified DOX, the amount of the drug accumulated in the selected place was only 265 ± 26 ng/cm2.
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CONCLUSIONS We can conclude that the use of an ultrasound bath and the application of citric buffer in the synthesis of DOX-Np conjugates allowed us to limit the aggregation of the nanoparticles and to increase strongly the number of DOX molecules attached to one nanoparticle of the carrier. Finally, the amount of bound doxorubicin to 1 g of magnetic nanoparticles increased from 2.3 to 8.9 mg. The strong depression of aggregation allowed us to study the cytotoxicity of DOX-Np conjugates against tumor cells. We showed that Np itself were not toxic toward human urinary bladder carcinoma 831
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in particular places, before metastasis. Currently, the most popular carrier for cytostatic drugs from anthracyclines are liposomes.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Supports for this work by Grant No. IP2011 038871 from the Polish Ministry of Science and Higher Education and R&D Grant No. 020230/009 from the Gdansk University of Technology are gratefully acknowledged.
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Figure 5. Frequency shifts observed during the deposition at AuEQCM subsequent layers: (a) PAH; (b) PAH/ctDNA; (c) PAH/ ctDNA/DOX with (red line) and without (black line) magnetic field; (d) PAH/ctDNA/DOX-Np with (red line) and without (black line) magnetic field; (e) PAH/ctDNA/DOX-Np after removing magnetic field; (f) PAH/ctDNA/DOX-Np in pH 5.8 (cancer cell pH). The negative frequency shifts correspond to the apparent mass increases.
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Figure 6. Cyclic voltammograms obtained after each modification step of Au-EQCM. Experimental conditions: 0.02 M PBS buffer (pH 7.4), scan rate 50 mV/s, surface area of gold electrode 0.52 cm2; background, dashed black line; Au-EQCM/PAH/DNA, solid blue line; Au-EQCM/PAH/DNA/DOX, solid red line; Au-EQCM/PAH/ DNA/DOX-Np, solid green line; Au-EQCM/PAH/DNA/DOX-Np in pH 5.8, dashed green line.
cells UM-UC-3 at wide range concentrations, whereas nanoparticles carried doxorubicin DOX-Np reached cytotoxicity even higher than DOX alone. The application of liposomes as carrier is connected with some limitations. They are easily recognized by the immune system and quickly removed from the blood.40,41 The next parameter that limits the effectiveness of liposomes as drug carriers is their imperfect tightness which leads to too fast releasing of the drug. Additionally, the improper physicochemical properties, particularly high lipophilicity of liposomes and the shape of liposome capsules may slow down and even make impossible the penetration of the cancer cells.42,43 The experiments with magnetic field indicated that the DOX-Np complex can be directed to the requested place. This opens new possibilities for anticancer therapy of solid tumors, particularly in their first stage when tumor tissue is dominating 832
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