Deposition of Antibody Modified Upconversion Nanoparticles on Glass by a Laser-Assisted Method to Improve the Performance of Cell Culture
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A suitable surface is vital for maintaining or even promoting cells’ function and communication. Recently, studies show that nanostructured coatings could have a potential in improving cell adhesion. However, it hardly minimizes the contamination by using traditional solution-coating technology. Matrix-assisted pulsed laser evaporation (MAPLE) technique is a contamination-free process and demonstrates an efficient process to deposit biopolymer without damaging their backbone on the surface of various substrates. Here, upconversion nanoparticles (NaGdF4: Yb3+, Er3+) with/without immunoglobulin G (IgG) modification were produced by a one-pot synthesis method. The average size of the upconversion nanoparticles (UCNPs) is 50 ± 8 nm. IgG bio-conjugated on the surface of UCNPs can be directly observed by transmission electron microscope (TEM). MAPLE system utilizing a Nd:YAG laser (λ = 532 nm, ν = 10 Hz) is applied to deposit UCNPs with/without IgG modification on the glass bottom of culture dish. In addition, the behaviors of human umbilical vein endothelial cells (HUVECs) cultured on the culture dishes coated with UCNPs with/without IgG have been studied as compared to the control sample, glass coated with gelatin. No toxic effect is imposed on cells. The results of this work indicate that the deposition of UCNPs with/without antibody by the MAPLE technique could enhance the adhesion and proliferation of cells.
KeywordsCell culture Laser-assisted deposition Upconversion nanoparticles Antibody
Human umbilical vein endothelial cells
Matrix-assisted pulsed laser evaporation
Phalloidin–Tetramethylrhodamine B isothiocyanate
Epithelial cells can be found at the inner and outer surfaces of the human body, including the skin, intestines, airway, and reproductive tract. Epithelial cells not only provides a safety shell against the dirt and microbes, but they also exhibit important functions, e.g., stretch, tracks, etc. . Therefore, epithelial cells have been extensively used in tissue engineering and tissue regeneration. The interaction between epithelial cells and the surface of substrates is vital for maintaining cells’ function and communication. Normally, a protein-based coating, e.g., rat-tail collagen, is applied to allow the epithelial cells growing on the petri dish, or glass, for further studies. Recently, nanomaterials coated on a substrate demonstrate the potential for the control of the growth of cells by utilizing the fine morphologies, special textures/patterns of the nanostructured coating [2, 3, 4]. In addition, luminescent nanomaterials have shown the significant advantages over traditional organic dye in studying the interaction of cell-cell, and cell-surface because of their highly stable photoluminescence properties. It is interesting to find out the interaction of cells and a surface coated with protein-modified luminescent nanostructures.
The upconversion phenomenon, first investigated in 1959, is the sequential absorption of two or more photons to emit a light with high energy [5, 6]. The lanthanide-doped upconversion nanoparticles (UCNPs) consists of three different components including activator, sensitizer, and host matrix. The lanthanide ions such as Er3+, Ho3+, and Tm3+ could play a role as activators since they possess unique energy-level structures [7, 8, 9, 10, 11]. Yb3+ ion is the most common sensitizer which can be applied to transfer the energy from excited light to the activators [12, 13, 14]. Both oxidic materials and fluoride materials are normally used as the crystal host [15, 16, 17]. Upconversion nanoparticles, emitting light from the visible range to the near-infrared range under the excitation of the near-infrared (NIR) light, can be applied in deep tissue bioimaging because of the lower scattering coefficient of NIR light known as the “therapeutic window” . Recently, various surface modification of UCNPs have been developed for biological labeling/sensing [19, 20]. For instance, avidin was conjugated onto hexanedioic acid (HAD) modified on the surface of UCNPs to demonstrate the interaction with antibodies . ssDNA-modified core-shell UCNPs are developed for detecting specific oligonucleotides .
On the other hand, immunoglobulin G (IgG), an antibody found in blood and extracellular fluid, controls the infection of tissue. The interactions between IgG and nanoparticles have been studied, for instance, IgG can be used as a template to produce gold nanoparticles, and IgG modified magnetic nanoparticles to label bacterial cells [22, 23]. However, only few studies have been reported on modifying IgG onto UCNPs for cell culture or tissue culture.
Conventional methods such as sol-gel methods, spin coating, and solvent evaporation have been applied in deposit biomolecules modified nanoparticles on a substrate for biomedical assay [27, 28]. However, solution-coating methods for deposition of proteins or protein-based nanostructures hardly minimize the contamination. Laser deposition exhibits well-controlled thickness and avoids the operation contamination occurring in chemical depositions. Very few laser deposition techniques have been used for developing a protein-based surface for cell culture.
Unlike conventional physical deposition methods, matrix-assisted pulsed laser evaporation (MAPLE) technique does not directly ablate the target materials; instead, most energy of laser is absorbed by the frozen solvent (matrix) [24, 25]. In a MAPLE process, target materials dispersed in a highly volatile solvent (matrix) are introduced into the target holder cooled with liquid nitrogen [26, 27, 28, 29, 30]. Under the laser irradiation, the target materials can be transported to the substrate with evaporating solvent. APLE technique has been applied in various fields including sensors, organic electronic devices, drug delivery, implants coating, etc. [31, 32, 33, 34, 35]. However, very few work has been reported on the effect of the substrate with MAPLE-deposited biomolecule/protein nanoparticles on the performance of cell culture.
In this study, we produced UCNPs (NaGdF4: Yb3+, Er3+) and immunoglobulin G (IgG)-modified UCNPs (UCNPs-IgG) by hydrothermal method. Following that, UCNPs with/without modification of IgG were directly deposited on the glass bottom of a cell culture dish by using a MAPLE process with 532 nm Nd:YAG laser. The human umbilical vein endothelial cells (HUVECs) were seeded on the glass deposited with UCNPs to investigate the cytotoxicity of UCNPs’ coating, and the cells’ behaviors on the surface treated with UCNPs.
Gadolinium (III) nitrate hexahydrate (Gd (NO3)3·6H2O, crystals and lumps, 99.9% trace metals basis), ytterbium (III) nitrate pentahydrate (Yb (NO3)3·5H2O, 99.9% trace metals basis), erbium (III) nitrate pentahydrate (Er (NO3)3·5H2O, 99.9% trace metals basis), sodium fluoride (NaF, BioReagent, suitable for insect cell culture, ≥ 99%), branched polyethylenimine (PEI, average Mw ~ 800 by LS, average Mn ~ 600 by GPC), anti-human IgG (Fab specific)–FITC antibody produced in goat (affinity isolated antibody, buffered aqueous solution), 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), and Phalloidin–Tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) were purchased from Sigma-Aldrich. Ethylene glycol (EG) was purchased from Fisher chemical. Isopropanol (2-propanol) was purchased from Caledon laboratory chemicals.
Synthesis of UCNPs With and Without IgG by Using a One-Pot Process
The UCNPs (NaGdF4: Yb3+, Er3+) were synthesized by a modified one-pot method . Briefly, 720 mg of Gd (NO3)3·6H2O, 170 mg of Er (NO3)3·5H2O, 160 mg of Yb (NO3)3·5H2O, and 20 ml ethylene glycol were added into a three-neck flask. Then, 0.7 g of PEI was gently added to the flask. Then, 336 mg of NaF dissolved in 10 ml ethylene glycol was added dropwise to the flask. The mixture was heated to 200 °C and refluxed for 6 h in nitrogen protection. The reaction products were collected by centrifugation and washed several times with ethanol/distilled water. The product was dried at 60 °C.
Deposition of UCNPs With/Without IgG by MAPLE
Further, 532 nm Nd:YAG laser was applied with laser frequency at 10 Hz and the τfwhm ≅ 200 μs. Laser spot area size was 0.63 cm2 and laser fluence was 150 mJ/cm2. The glass substrates were treated with a 2 wt% solution of gelatin which was fixed on the substrate holder and the temperature of substrates was 25 °C during the whole experiment process. The laser deposition process was conducted under 1 × 10−6 Torr. As per our previous studies [42, 43], the deposition time was set to 2 h. The substrate-to-target distance was 4.5 cm (vertical configuration). The target holder and the substrate holder were rotating (target: 10 rpm, substrate: 25 rpm) during the laser irradiation period.
UCNPs and UCNPs-IgG before the MAPLE deposition were characterized by transmission electron microscope (TEM, Philips CM-10, operating at 80 kV). Fourier-transform infrared spectra (FTIR) of the UCNPs and UCNPs-IgG were obtained by a Bruker Vector 22 FTIR spectrometer (scan range 600 cm−1~4500 cm−1, resolution 4 cm−1, 64 scans). The photoluminescence properties of UCNPs were studied by using QuantaMaster™ 40 Spectrofluorometer (Horiba Canada—Photon Technology International Inc.). The growth of human umbilical vein endothelial cells (HUVECs) on the glass surface with/without the depositions of nanoparticles was investigated by a confocal microscope (Zeiss LSM 5 Duo Vario Microscope). Hitachi S-3400 N scanning electron microscope attached with INCA PentaFET-x3 EDX system (Oxford Instruments) was applied to perform the energy-dispersive X-ray spectroscopy (EDX).
Study on Cell Behaviors
Human umbilical vein endothelial cells (HUVECs, American Type Culture Collection) were applied to study the biocompatibility of UCNPs with/without IgG modification after the laser deposition treatment. HUVECs were seeded on the glass surface deposited with UCNPs and UCNPs-IgG. The glass substrate treated with 2 wt.% solution of gelatin (without UCNPs) is used as the control after the MAPLE deposition treatment. The deposited samples were soaked in MCDB medium (10% fetal bovine serum, 1% penicillin, and amphotericin B) for the seeding of HUVECs. The HUVECs were cultured at 37 °C for 24 h. HUVECs were fixed on substrates’ surface with 4% formalin for 2 h for obtaining the confocal imaging (Zeiss LSM 510 Duo). The cells were stained with Phalloidin-TRITC and DAPI. The samples were washed with PBS (pH 7.4) and treated with the anti-fade agent.
Study on Cytotoxicity
The HUVECs were cultured in MCDB medium (10% fetal bovine serum, 1% penicillin, and amphotericin B) on the surface of the deposited samples. After transfer to culture dishes, the cells were cultured for 24 h (37 °C, 5% CO2) on the surface of the samples. Approximately 100,000 cells were seeded on the surface of each sample (with/without UNCPs deposition). All samples including the control, the glass substrates with the deposition of, UCNPs, and UCNPs-IgG were measured in triplicate. Then the MTT agent (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyl tetrazolium bromide) was added to the cell media and cells were incubated for 3 h. The cell media was removed and the wells were rinsed two times with PBS. Then DMSO was added to each well to dissolve the formazan. The liquid was transferred to the 96-well plates and analyzed with the bio-kinetic reader at 490 nm (Bio-Tek Instruments EL340I Microplate Reader).
Results and Discussion
Characterization of UCNPs/UCNPs-IgG Before MAPLE Deposition
Characterization of UCNPs With/Without IgG After MAPLE Deposition
Compared to the spectrum of the bare glass (spectrum-3), the stretching vibration bands of methylene groups are around 2985 cm−1 and 2900 cm−1 in both spectrum-1 and spectrum-2 due to the carbon-based functional groups on the surface of UCNPs. It is noted that the peaks of coatings in Fig. 7 are significantly weaker than the peaks in Fig. 5 which are caused by the low quantities of nanoparticles deposited on the substrate surface, and the changes of spectra at 1250 cm−1 in Fig. 7 as compared to Fig. 5 stems from the effect of glass substrate.
Cell Behaviors on the Different Coatings
Effect of the Coating of UCNPs With/Without IgG on Cell Viability
In summary, the UCNPs and UCNPs-IgG were successfully synthesized by the one-pot method and have been deposited on culture dishes with glass bottom by MAPLE technique. The results of TEM and FTIR reveal the successful bioconjugation of IgG on the surface of UCNPs. The average particle size of the UCNPs is 50 ± 8 nm. The UCNPs modified with antibodies remained the cubic shape, and the average particle size is around 54 ± 8 nm. The bioconjugation of IgG on UCNPs can be observed directly by TEM which has the average size of 10 ± 5 nm. The FTIR spectra also confirmed the presence of the carboxyl group/peptide bond of the antibody modified on the surface of UCNPs. MAPLE deposition process was used to deposit UCNPs and UCNPs-IgG on glass substrate. The results of EDX, FTIR, and PL measures indicate the retention of structures and properties of UCNPs and UCNPS after MAPLE deposition. Our study demonstrates that the MAPLE process can achieve the retention of the properties and structures of UCNP with/without the modification of antibody. In addition, the performance of cell culture has been statistically studied by culturing HUVEC cell line on the different surfaces treated with UCNPs and UCNPs-IgG, respectively, made by MAPLE process. The cell area, cell length, and the length of connection are very important to support an ideal confluence and the formation of microvessel structures. The glass surfaces treated with UCNPs and UCNPs-IgG samples by MAPLE technique show no toxic effect to the HUVEC cell line. It is expected that MAPLE deposition of UCNPs and UCNPs-IgG could be applied in the fabrication of the new biological devices for tissue engineering and tissue regeneration.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).
JZ conceived and designed the study and revised and rewrote the paper. SY performed most of the experiments. WHT assisted in the work of cell culture and the characterization of cell behaviors. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
The authors declare that they have no competing interests.
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