Preparation of Fetal Bovine Serum-Copper Phosphate Hybrid Particles under Cell Culture Conditions for Cancer Cell Treatment.

Fetal bovine serum (FBS) particles, which mainly consist of bovine serum albumin, have the potential for biological and medical applications as drug carriers. The coacervation of albumin is a common technique for preparing albumin-based particles. The replacement of salt with novel metal salts such as Cu is an affordable way to embed the metal ion in the albumin-based particles. Further, increased Cu distribution is prevalent in many cancers. Here, we prepared adhesive cell-like FBS-copper phosphate hybrid particles [FBS-Cu3(PO4)2], which exhibited toxicity toward cancer cells, with a narrow size distribution under cell culture conditions for preventing tumor progression. FBS-Cu3(PO4)2 showed peroxidase-like activity. In addition, FBS-Cu3(PO4)2 was successfully loaded with rhodamine B and conjugated with fluorescein isothiocyanate as models of drugs by coincubation. Thus, we designed a simple preparation method for optimizing FBS-Cu3(PO4)2 synthesis under cell culture conditions. FBS-Cu3(PO4)2 has significant potential as an efficient reactive oxygen species generator and drug-delivery agent against cancer cells. Furthermore, the RhoB-loaded FBS-Cu3(PO4)2 successfully interacted with 4T1 mouse mammary tumor cells and were confirmed to exhibit toxicity.

Introduction

The biological origin of fetal bovine serum (FBS) particles affords them biodegradability, nontoxicity, nonimmunogenicity, and easy availability. Bovine serum albumin (BSA) is the major component of FBS and has been widely used in medical applications.[1] Generally, albumin-based particles are prepared by coacervation methods such as desolvation.[2] It has been reported that adding alcohols, such as ethanol, and salts to the albumin water phase induces phase separation by desolvation.[3] The replacement of salt with novel metal salts such as Cu is an attractive way to embed the metal ion in the albumin-based particles. Further, Cu is an essential element in biological systems. Several types of cancers exhibit increased intratumoral and altered systemic Cu distribution.[4] Thus, two approaches for combating cancer have been investigated: (1) the development of copper-specific chelators to prevent tumor progression, including growth, angiogenesis, and metastasis because Cu serves as a limiting factor for multiple aspects of tumor progression and angiogenesis growth and so forth[5] and (2) delivering Cu, which possesses anticancer properties, to cancer cells because of the increased intracellular Cu levels in cancer cells.[6] From the perspective of reactive oxygen species (ROS) generation, Cu-containing catalysts have garnered considerable attention.[7] Recently, some studies have reported that metal-containing catalysts of peroxidase mimic the chemical catalytic reaction that decomposes H2O2 into the highly toxic hydroxyl radical (•OH).[8,9] In biological systems, Cu-based ROS generation may amplify the therapeutic effect in the tumor tissue compared to the normal tissue. Using various proteins and Cu(II), protein–inorganic hybrid nanoflowers have been synthesized. They were formed by the coordination of copper phosphate [Cu3(PO4)2] and amide groups in the protein.[10] This has led to studies on protein–inorganic hybrid flowers that demonstrate high enzyme activity for the biosensor and the catalyst.[11−14]

In this study, we prepared adhesive cell-like FBS-Cu3(PO4)2 particles under cell culture conditions. The cell culture conditions are widely established for in vitro experiments; thus, this preparation method of FBS-Cu3(PO4)2 would widely spread as an easy and convenient way for anti-cancer materials in the biology laboratory. Optical microscopy revealed high transparency and narrow distribution of the particles. FBS-Cu3(PO4)2 also showed peroxidase-like activity. Further, the FBS-Cu3(PO4)2 particles were successfully loaded with rhodamine B (RhoB) and conjugated with fluorescein isothiocyanate (FITC) by coincubation. The RhoB-loaded FBS-Cu3(PO4)2 successfully interacted with 4T1 mouse mammary tumor cells and were confirmed to be toxic. This report presents a simple preparation method for optimizing the fabrication of FBS-Cu3(PO4)2 under cell culture conditions. FBS-Cu3(PO4)2 has potential as an efficient ROS generator and drug-delivery agent against cancer cells.

Results and Discussion

Preparation and Characterization of FBS-Cu3(PO4)2

We successfully prepared FBS-Cu3(PO4)2 determined by optical and electron microscopies under cell culture conditions.

Optical Microscopic Observation

Figure and Table show the optical microscopy images and the effect of FBS and CuSO4 concentrations on the diameter of FBS-Cu3(PO4)2. Initially, we focused on the effect of CuSO4 concentration at a constant FBS concentration. At 0% FBS, the diameter of Cu3(PO4)2 increased slightly as the CuSO4 concentration increased from 5.12 to 10.00 mM. It has been reported that the initial Cu concentration slightly affects the diameter of precipitated Cu(II) hydrous oxide particles.[15] With increasing Cu concentration (5.12–10.00 mM), the diameter of FBS-Cu3(PO4)2 increased at 2.5% FBS but decreased at 10% FBS. The diameter demonstrates opposite trends at low and high concentrations of FBS, suggesting the difference in the formation mechanism.[16]Figure S1 shows the optical microscopy images under different conditions without the Roswell Park Memorial Institute (RPMI) (+/+) medium. With 0% FBS in phosphate-buffered saline (PBS) (−), the particles were formed, and at 10% FBS in PBS (−), the particles formed, but were primarily inhomogeneous, suggesting that RPMI (+/+) contributed to the preparation of highly monodisperse FBS-Cu3(PO4)2.

Figure 1

Optical microscopy images of FBS-Cu3(PO4)2 in different concentrations of FBS and CuSO4. Scale bar = 10 μm.

Table 1

Effect of the Reaction Condition on the Size of FBS-Cu3(PO4)2

conditions		characterization
FBS (% (w/w))	Cu (mM)	diameter (nm)a
0.0	5.12	9.9 ± 0.5
0.0	6.40	9.9 ± 0.6
0.0	8.00	10.5 ± 0.5
0.0	10.00	12.5 ± 2.0
2.5	5.12	1.9 ± 0.4
2.5	6.40	3.5 ± 0.3
2.5	8.00	5.7 ± 0.2
2.5	10.00	14.6 ± 4.2b
5.0	5.12	no formation
5.0	6.40	5.5 ± 0.4
5.0	8.00	3.0 ± 0.5
5.0	10.00	5.1 ± 1.2
10.0	5.12	no formation
10.0	6.40	2.1 ± 0.3
10.0	8.00	1.0 ± 0.2
10.0	10.00	unmeasurable

Average diameters and S.D.s of FBS-Cu3(PO4)2 determined by optical microscopic images were calculated from more than 50 particles for each sample.

Less than 50 particles (formation of particles was less).

Electron Microscopy

Figure shows the electron microscopy images of FBS-Cu3(PO4)2 washed with PBS (−) and those after protein decomposition of FBS-Cu3(PO4)2. In the former, at 0% FBS, FBS-Cu3(PO4)2 formed a flowerlike structure,[12,13] whereas at 2.5, 5, and 10% FBS, it formed a spherical shape with a more smooth surface compared to the previously reported petal structure of hybrid nanoflowers composed of BSA and Cu3(PO4)2.[10] After protein decomposition at 0 and 2.5% FBS, flowerlike structures were observed, suggesting that the nucleation growth of Cu3(PO4)2 was prioritized under these conditions. These results indicate that the trend of the diameter increase with increasing Cu concentration is caused by the crystallization of Cu3(PO4)2 at low concentrations of FBS (Table ). Conversely, at 5 and 10% FBS, the structure of Cu3(PO4)2 was not observed; instead, a flat-shaped structure was observed in the scanning electron microscopy (SEM) images, which showed a high contrast in the scanning transmission electron microscopy (STEM) images. This implies that Cu3(PO4)2 was not crystallized, and FBS-Cu3(PO4)2 was constructed by the coordination of FBS and Cu3(PO4)2. Thus, the decreasing tendency of the diameter with the increasing Cu concentration at high FBS concentrations is caused by the coacervation of FBS by Cu(II) (Table ).

Figure 2

Electron microscopy images of FBS-Cu3(PO4)2 at different concentrations of FBS. The concentration of CuSO4 was fixed at 8.00 mM. Scale bars were inadvertently introduced into the figure. (A–D) FBS-Cu3(PO4)2 was washed with PBS (−). (E–L) Protein of FBS-Cu3(PO4)2 was decomposed using the cell lysis solution. (I,L) Enlarged SEM images. (K,L) Enlarged STEM images of the yellow parts of (G,H), respectively.

Selected Electron Diffraction Patterns, FT-IR Spectroscopy, and TGA

Figure A shows the TEM images and selected electron diffraction patterns of FBS-Cu3(PO4)2 for 0 and 10% FBS, namely, Cu3(PO4)2 and FBS-Cu3(PO4)2, respectively. Cu3(PO4)2 showed numerous diffraction spots from multiple crystals representing a concentric ring, suggesting that Cu3(PO4)2 particles prepared in 0% FBS are composed of multiple crystalline structures. Conversely, FBS-Cu3(PO4)2 showed a slightly broad pattern, called the halo ring pattern, which is indicative of its amorphous phase structure.[17]

Figure 3

(a) Selected electron diffraction patterns, (b) FT-IR spectra, and (c) TGA of Cu3(PO4)2 and FBS-Cu3(PO4)2 at different concentrations of FBS (0 and 10%). The concentration of CuSO4 was fixed at 8.00 mM.

Figure B shows the Fourier transform infrared (FT-IR) spectra of Cu3(PO4)2 and FBS-Cu3(PO4)2. For Cu3(PO4)2, the peak corresponding to the adsorbed water at 1635 cm–1 was observed. Additionally, the peaks at 1139, 1053, and 993 cm–1 correspond to the bending vibration of Cu–OH and asymmetric and symmetric stretching vibrations of PO43–, respectively. The peaks at 624 and 561 cm–1 correspond to the out-of-plane bending vibrations of the PO43– ions.[12,18] For FBS-Cu3(PO4)2, characteristic peaks ascribed to proteins were observed at the amide I (1641 cm–1, C=O stretching) and amide II (1541 cm–1, overlap of N–H bending and C–N stretching) bands, suggesting the presence of proteins in FBS-Cu3(PO4)2.[12,19] The peak at 1453 cm–1 may be attributed to the in-plane bending vibration of C–H and O–H bonds of the protein,[12] also suggesting the incorporation of proteins in FBS-Cu3(PO4)2.

Figure C shows the thermogravimetric analysis (TGA) data for Cu3(PO4)2 and FBS-Cu3(PO4)2. The initial weight loss of Cu3(PO4)2 and FBS-Cu3(PO4)2 below 150 °C is associated with the loss of physically adsorbed water.[12,20] FBS-Cu3(PO4)2 showed a weight loss at 150–350 °C, corresponding to the loss of low-molecular-weight compounds.[21] Above 350 °C, higher weight loss denoting the degradation of proteins originating from FBS and structurally bound water molecules occurred,[12,20] suggesting the presence of protein in FBS-Cu3(PO4)2.

Peroxidase-like Activity of FBS-Cu3(PO4)2

Figure A shows the peroxidase-like activity of FBS-Cu3(PO4)2. As compared to natural peroxidase enzymes, artificial enzymes possess the advantages of controlled synthesis at a low cost and tunability during catalytic activities. We evaluated the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate into ox-TMB (deep blue color, with a maximum absorbance at 652 nm) in the presence of H2O2 by FBS-Cu3(PO4)2. The absorbance (at 652 nm) increases as the concentration of FBS-Cu3(PO4)2 increases. Figure B shows the time-dependent absorbance changes with 25 μg mL–1 FBS-Cu3(PO4)2. An increase in absorbance is observed with time, which is indicative of the catalytic activity of FBS-Cu3(PO4)2. In contrast, under the same reaction conditions, the absorbance did not increase in the absence of the particles.

Figure 4

Peroxidase-like activity of FBS-Cu3(PO4)2. (A) Dose-dependent absorbance changes at 652 nm. Reaction conditions; 0.8 mM TMB, 50 mM H2O2, 10 mM NaAc buffer (pH 5.0), FBS-Cu3(PO4)2 = 0–400 μg mL–1 at 37 °C for 5 min. (B) Time-dependent absorbance changes at 652 nm of FBS-Cu3(PO4)2. The preparation condition for FBS-Cu3(PO4)2 was 10% FBS and 8.00 mM CuSO4. Reaction conditions for peroxidase-like activity were 0.8 mM TMB, 50 mM H2O2, 10 mM NaAc buffer (pH 5.0), and FBS-Cu3(PO4)2 = 0 and 25 μg mL–1 at 37 °C.

Preparation and Characterization of RhoB-Loaded and FITC-Conjugated FBS-Cu3(PO4)2

Albumin-based particles are commonly used for multiparticulate drug-delivery systems, owing to their advantage of broad functionalization, such as with organic compounds. We investigated two types of functionalization methods with organic compounds: RhoB for the loading method and FITC for the conjugation method were used as model drugs for observing and evaluating the drug-loading capacity (Figure A). RhoB and FITC were successfully embedded in FBS-Cu3(PO4)2 with the diameters of 2.3 ± 0.3 nm and 3.0 ± 0.5 nm, respectively (Table S1). The diameter of FBS-Cu3(PO4)2 was 1.0 ± 0.2 nm in the absence of the organic compound, suggesting that the addition of the organic compound in the reaction solution affected the size of FBS-Cu3(PO4)2. The functionalization with organic compounds also affected the size of the particles. These particles fluoresced when observed under fluorescent microscopy. RhoB as a cationic compound could interact via electrostatic interactions with FBS because BSA has an isoelectric point near pH ≈ 4.8, that is, it is negatively charged. FITC interacted with the amino residues of albumin because FITC possesses isothiocyanate (−NCS) groups.[22]

Figure 5

(A) Fluorescence microscopy images of RhoB-loaded and FITC-conjugate FBS-Cu3(PO4)2. The preparation conditions for FBS-Cu3(PO4)2 were 10% FBS, 8.00 mM CuSO4, and 10 μM RhoB or FITC. The fluorescence of RhoB and FITC are represented in red (Ex: 545/25 nm, Em: 605/70 nm) and green (Ex: 470/40 nm, Em: 525/50 nm), respectively. Scale bar = 10 μm. (B) Time-series images of fluorescent microscopy of the 4T1 cells coincubated with RhoB-loaded Cu3(PO4)2. The fluorescence of DAPI and Hoechst is represented in yellow (Ex: 360/40 nm, Em: 460/50 nm) and blue (Ex: 360/40 nm, Em: 460/50 nm), respectively. The 4T1 cells coincubated with RhoB-loaded FBS-Cu3(PO4)2 and in the absence of the particles were first stained with DAPI and observed, followed by staining with Hoechst. Scale bar = 50 μm.

Figure B shows the time-series of the fluorescent microscopy images of 4T1 cells coincubated with RhoB-loaded FBS-Cu3(PO4)2 and in the absence of the particles. The zeta potential of the RhoB-loaded FBS-Cu3(PO4)2 was −0.38 ± 0.56 mV [in PBS (−)], and this value shifted slightly toward the positive compared to the value −3.24 ± 0.19 mV [in PBS (−)] of FBS-Cu3(PO4)2 (Table S2). The loading quantity of RhoB for FBS-Cu3(PO4)2 was estimated from the fluorescent spectra measurements by FlexStation 3 (Figure S2); the number of RhoB that interacted with 1 mg of FBS-Cu3(PO4)2 was 1.14 × 1023. In Figure B, RhoB-loaded FBS-Cu3(PO4)2 gradually interacted with the 4T1 cells as time progressed. After 12 h of coincubation, the greater part of RhoB-loaded FBS-Cu3(PO4)2 interacted with the surface of the 4T1 cells, and the damaged 4T1 cell structure was observed after 24 h. In contrast, under identical incubation conditions, the 4T1 cells were not damaged in the absence of the particles. At a concentration of 25 μg mL–1, FBS-Cu3(PO4)2 reduced the viability of 4T1 cells to 5.7%, confirming its toxicity (see Supporting Information S3). These results indicate that RhoB-loaded FBS-Cu3(PO4)2 showed toxicity on contact with the surface of 4T1 cells because of their peroxidase-like activity. Among the active oxygen species, H2O2 is relatively stable and can diffuse to longer distances.[23] Therefore, it is considered that peroxidase-like activity occurred actively on the cell surface, thereby causing oxidative damage to the cells.

Conclusions

We have successfully prepared adhesive cell-like FBS-Cu3(PO4)2 hybrid particles and proposed a simple preparation method to optimize their synthesis under cell culture conditions. FBS-Cu3(PO4)2 demonstrated peroxidase-like activity as an artificial enzyme and was easily functionalized with organic compounds as model drugs. Our results clearly demonstrate the potential of FBS-Cu3(PO4)2 as an efficient ROS generator and drug-delivery agent for cancer cells. Therefore, FBS-Cu3(PO4)2 is highly promising for medical applications. Currently, a detailed study on its stability, drug release, and application in cancer therapy combined with radiotherapy for tumors is underway.

Experimental Section

Materials and Devices

Sulfate pentahydrate (CuSO4·5H2O) was purchased from Wako Pure Chemical Industries, Japan. RhoB and FITC were purchased from Sigma-Aldrich. PBS solution was prepared as follows: 8.0 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.2 g of sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O), and 2.9 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) were mixed, and distilled water (DW) was added to make 1 L. The cell lysis solution was prepared as follows: 2 g of sodium deoxycholate, 2 g of sodium dodecyl sulfate, 2 mL of NP-40, and 37.2 mg of EDTA disodium salt were dissolved in 100 mL of DW.

Optical and fluorescent microscopy images were observed using a BZ-X800 fluorescence and light microscopy system (Keyence, Japan). SEM images were obtained using a Quanta 3D FEG electron microscope (FEI, Hillsboro, Oregon, USA) operated at 30 kV. TEM images were obtained via a JEM-2100 electron microscope (JEOL, Japan) operated at 200 kV. FT-IR spectra were recorded using an FT/IR-4600 spectrometer (JASCO, Tokyo, Japan). TGA was conducted on powdered samples using a HITACHI STA7200 (HITACHI, Japan) system from 30 to 900 °C at a heating rate of 10 °C min–1 under airflow at a rate of 200 mL min–1.

Preparation of FBS-Cu3(PO4)2

FBS-Cu3(PO4)2 was prepared by adding 200 μL of the desired concentration of CuSO4 in RPMI medium containing 0–10% heat-inactivated FBS and antibiotics (100 μg/mL streptomycin and 100 units/mL penicillin) in a well of black 96-well plates (Greiner Bio-One, USA). The mixture was incubated at 37 °C in a 5% CO2 atmosphere for 2 d, yielding FBS-Cu3(PO4)2 at the bottom of the well. In the presence of FBS, the FBS-Cu3(PO4)2 adhered to the bottom of the well. The FBS-Cu3(PO4)2 was washed with PBS (−) and collected using trypsin treatments. The suspended FBS-Cu3(PO4)2 was centrifuged (1000g, 5 min) and purified by washing with PBS (−), and a blue FBS-Cu3(PO4)2 precipitate was collected. The protein was decomposed using the cell lysis solution. The blue FBS-Cu3(PO4)2 precipitate was mixed with the cell lysis solution at 37 °C for 30 min, centrifuged (1000g, 5 min), and purified by washing with PBS (−).

The peroxidase-like activity was induced by adding 400 μL of 10 mM buffer solution (NaAc, pH 5.0) in the order of certain amounts of FBS-Cu3(PO4)2, 800 μM TMB, and 50 mM H2O2, yielding immediate color reactions.[8] After incubation at 37 °C for 5 min in the dark, the mixtures were immediately centrifuged (30,000g, 5 min). The supernatant was measured using a FlexStation 3 (Molecular Devices, USA) reader. Time-dependent absorbance changes were monitored using FlexStation 3 prior to centrifugation.

Preparation of RhoB-Loaded and FITC-Conjugated FBS-Cu3(PO4)2

RhoB-loaded and FITC-conjugated FBS-Cu3(PO4)2 particles were prepared by adding 200 μL of 8.00 mM CuSO4 in RPMI medium containing 10% heat-inactivated FBS and antibiotics (100 μg/mL streptomycin and 100 units/mL penicillin) and RhoB and FITC adjusted to 10 μM, respectively, in a well of black 96-well plates (Greiner Bio-One, North Carolina). The mixture was incubated at 37 °C for 2 d. The RhoB-loaded and FITC-conjugated FBS-Cu3(PO4)2 particles were washed with PBS (−) and collected using trypsin treatments. The suspended FBS-Cu3(PO4)2 particles were centrifuged (1000g, 5 min) and purified by washing with PBS (−).

Cell Viability Assay Using WST-1

The viabilities of 4T1 cells were evaluated using the WST-1 cell proliferation reagent (Roche, Almere, Netherlands). The cells were seeded at a density of 1.0 × 104 cells/well in 96-well plates 24 h prior to being treated with FBS-Cu3(PO4)2. The cells were incubated with 0–100 μg mL–1 of FBS-Cu3(PO4)2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated FBS and antibiotics (100 μg mL–1 streptomycin and 100 units/mL penicillin) for 1 d at 37 °C. The absorbance of FBS-Cu3(PO4)2 coincubated solution was immediately measured, following the addition of the WST-1 reagent and incubation at 37 °C. To isolate the signals that were derived from the formazan dye (produced by viable cells), the former signal was subtracted from the latter.

Observation of 4T1 Cells Using RhoB-Loaded FBS-Cu3(PO4)2

4T1 cells were cultured overnight at 37 °C in a 5% CO2 atmosphere in 96-well plates in DMEM containing 10% heat-inactivated FBS and antibiotics (100 μg mL–1 streptomycin and 100 units/mL penicillin). Then, 25 μg mL–1 FBS-Cu3(PO4)2 was incubated in DMEM containing 10% heat-inactivated FBS and antibiotics (100 μg mL–1 streptomycin and 100 units/mL penicillin), and time-series images for 24 h at 37 °C in a 5% CO2 atmosphere were obtained.