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THE ASSEMBLY OF A PHOTOLUMINESCENT NANOCOMPLEX BASED ON UPCONVERSION NANOPARTICLES
S. Shanwar1, L. Liang1, A. V. Nechaev3, I. V. Balalaeva1, V.A. Vodeneev1,
A.V. Zvyagin11 4, E.L. Guryev1
1 Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, 603950, Russia;
2 ARC Centre of Excellence «Nanoscale BioPhotonics», Department of Physics and Astronomy, Macquarie University, Sydney 2109, Australia;
3 Department of Chemistry and Technology of Biologically Active Compounds, Medical and Organic Chemistry, M.V. Lomonosov Institute of Fine Chemical Technologies, MIREA - Russian Technological University, 119571 Moscow, Russia;
4 The Institute of Molecular Medicine, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia.
Abstract. Over the past two decades, developments in the field of nanobiomedicine have come a long way despite the unresolved hindrances. The creation and development of effective theranostic agents based on nanomaterials are urgent needs of modern medicine. Upconversion nanoparticles (UCNP) appear to be the most promising agents for developing theranostics due to their unique optical properties. There has been extensive research on new approaches to obtain stable colloids capable of prolonged circulation in the bloodstream, particularly with bovine serum albumin (BSA). The present work contributes to solving the problem of obtaining stable agents based on UCNP by coating water-soluble UCNP-NOBF4 with a stable protein corona layer of BSA. The assembled nanocomplex is promising for usage as a diagnostic agent and is set for further investigation.
Keywords: Upconversion nanoparticle, Protein corona, Bovine serum albumin.
List of Abbreviations
UCNP - Upconversion nanoparticles UCNP-OA - Oleic Acid capped UCNP UCNP-NOBF4 - Nitrosonium tetrafluorob-orate coated UCNP NIR - Near Infrared PL - Photoluminescence EPR effect - Enhanced permeability and retention effect
PC - Protein Corona
BSA - Bovine serum albumin
^M - Micromolar
nm - nanometer
DMF - Dimethylformamide
TEM - Transmission electron microscope
DLS - Dynamic light scattering
ELS - Electrophoretic light scattering
PDI - Polydispersity Index
Introduction
Nowadays, the application of nanoparticles in biomedicine is an actively and rapidly developing research area. They are considered promising candidates, especially for optical imaging (Deyev & Lebedenko, 2017). Their successful
biomedical application is bound by specific requirements, including biocompatibility, bio-degradability, colloidal stability, and prolonged blood circulation (Aires et al., 1015).
Upconversion nanoparticles (UCNP) particularly have emerged amongst other nanoparticles for their unique and superior optical properties. The homogenous core and shell crystal structure of UCNP significantly increases the upconversion rate and upconversion coefficient in aqueous solutions (Wang et al., 1010; Zhou et al., 1011). Moreover, the trivalent lanthanide ion dopants (Yb3+, Tm3+, Er3+) of the core grant these UCNP the capability of excitation in the near-infrared (NIR) range and emission in the NIR, visible or ultraviolet regions (Li et al., 1015). Their exceptional features, alongside the prolonged photoluminescence (PL) lifetime (up to 1 ms), facilitate the avoidance of autofluorescence interference and deep tissue penetration up to several centimeters (Chatterjee et al., 1008; Song et al., 1011; Zheng et al., 1016).
The methodology of their synthesis has been improving to achieve well-formed and monodisperse nanoparticles with high PL intensity.
However, the outcome is lipophilic nanoparti-cles such as oleic acid (OA) coated nanoparti-cles and, hence not biocompatible. Further modifications of their surfaces are necessary to obtain hydrophilic nanoparticles, for example, using the ligand exchange procedure (Chen et al., 2017). One of the most successful modifications is with the inorganic compound nitro-sonium tetrafluoroborate (NOBF4) with a molecular weight of 116.81 g/mol by the ligand exchange method resulting in hydrophilic nano-particles and a relatively unaltered size as the compound is positively charged and very small (Dong et al., 2011).
Generally, nanoparticles take advantage of the passive delivery route, facilitated by the enhanced permeability and retention (EPR) effect. The EPR effect is observed in solid tumors where nanoscale materials tend to passively diffuse through the large pores of abnormal tumor vasculature and accumulate in tumor tissues more than normal tissues (Matsumura et al., 1987; Barua & Mitragotri, 2014).
Upon entering biological media, nanopar-ticles are quickly covered with a dynamic layer of proteins called protein corona (PC) (Cedervall et al., 2007; Lundqvist, 2013). The protein corona affects nanoparticles' physico-chemical properties, such as size and charge, and determines their interaction with biological objects (Nel et al., 2009; Dominguez-Medina et al., 2016). Protein adsorption onto the nanoparticle's surface is mediated by ionic and van der Waals forces, hydrogen bonds, and hydrophobic interactions depending on the nanoparticles' characteristics (Hadjide-metriou & Kostarelos, 2017). In the last decade, there have been several studies investigating the possibility of manipulating PC to stabilize nanoparticles, and the most intensively considered protein is bovine serum albumin (BSA) due to its abundance in blood, biocompatibility, biodegradability, and long circulation time (Bern et al., 2015; An & Zhang, 2017). In this work, we have assembled a diagnostic nanocomplex based on UCNP modified with NOBF4 and coated with BSA capable of up-converting NIR light into higher energy NIR and visible light.
Materials and Methods
Synthesis of UCNP-OA and UCNP-NOBF4.
Hydrophobic UCNP-OA core/shell structure (NaY0.794F4: Yb0.2Tm0.06/NaYF4) were synthesized in Federal Research Center «Crystallography and Photonics» RAS, Russia, by solvato-thermal decomposition method as described in (Mai et al., 2007, Guryev et al., 2020). The lig-and exchange reaction was carried out to remove OA with NOBF4 (Sigma-Aldrich, USA). A suspension of UCNP-OA in hexane (Cryo-chrom, Russia) (5 mg/ml) was mixed with NOBF4 solution in dimethylformamide (DMF) (Sigma-Aldrich, USA) (0.01 M) at a ratio of 1:1 (v:v) and kept stirring on a magnetic stirrer overnight at room temperature. The obtained UCNP-NOBF4 were pelleted by centrifugation (10000 x g, 7 min) and re-dispersed in DMF and flocculated with toluene (Himreaktiv, Russia) and hexane mixture (1:1, v:v). After floc-culation, the resulting UCNP-NOBF4 were collected by centrifugation (10000 x g, 7 min) and washed twice with ethanol and once with deion-ized water. Finally, they were dispersed in de-ionized water and stored at 4°C.
Characterization of UCNP-OA and UCNP-NOBF4
The TEM image of UCNP-OA was obtained using a transmission electron microscope LEO-912 ABOMEGA (Carl Zeiss, Germany) for size and shape confirmation. The hydrody-namic diameters of UCNP-OA and UCNP-NOBF4 were determined by dynamic light scattering (DLS), and the Z-potential of UCNP-NOBF4 was measured by electrophoretic light scattering (ELS), using a Zetasizer Nano ZS system (Malvern Instruments Ltd., UK) according to the manufacturer's recommendations. The PL properties of the UCNP-OA and UCNP-NOBF4 were investigated using an SM 2203 spectrofluorimeter (SOLAR, Belarus) and an ATC-C4000-200AMF-980-5-F200 external semiconductor laser module with a wavelength of 980 nm (Semiconductor devices, Russia). The PL emission spectra were recorded from 400 to 850 nm wavelengths in a quartz cuvette with an optical path length of 1 cm.
Optimization of BSA concentration for PC formation on UCNP-NOBF4
The BSA stock was prepared in deionized water by vigorous stirring and then filtered through a membrane filter (0.22 ^.m) to remove protein clusters. A dilution series of BSA was prepared and incubated with UCNP-NOBF4 (1:1, v:v), where UCNP-NOBF4 was added dropwise to the BSA suspension and mixed. The final concentrations were 0.25 mg/ml of UCNP-NOBF4 and 25, 50, 75, 100 and 120 ^M of BSA. The mixtures were incubated for 4 h at room temperature. The size and (^-potential were recorded 15 min and 4 h after incubation.
Results
Synthesis and characterization of UCNP-OA and UCNP-NOBF4
UCNP-OA core/shell structure NaY0.794F4: Yb0.2, Tm0.06/NaYF4 were synthesized with strictly controlled size and shape using the solv-atothermal decomposition method and subsequently converted into UCNP-NOBF4 by lig-and exchange (Fig. 1) (Mai et al., 2007; Dong et al., 2011). Coordinate stabilization of lanthanide and fluorine precursors in an OA solution, after being heated in an oxygen-free environment, yields nanosized cubic UCNP crystals (aphase crystals). The obtained hexagon-shaped and more stable UCNP (P-phase crystals) were formulated after additional heat treatment (Guryev et al., 2020). The transition of UCNP to the hexagonal phase and the subsequent formation of an inert shell from NaYF4 can significantly increase their PL brightness, which is useful for in vitro and especially in vivo imaging (Generalova et al., 2017). UCNP-OA investigation by TEM confirms obtaining hexagon-shaped nanoparticles with an average size of 36 ± 2.75 nm (Fig. 2A). The hydrodynamic diameter of UCNP-OA was 67.94 ± 0.38 nm because of formation of a hydration shell around the particles, with an average polydispersity since the PDI was 0.254 ± 0.011 (Fig. 2B) (Bhattacharjee, 2016).
The hydrophobic nature of the aforementioned UCNP-OA renders them unsuitable for biological applications. Hydrophilicity was achieved via the ligand exchange method where
980 nm 478 nm 980 nm 478 nm
Fig. 1. A scheme illustrating UCNP-OA core/shell structure and the ligand exchange process converting UCNP-OA into UCNP-NOBF4
Fig. 2. Characterization of UCNP-OA and UCNP-NOBF4 nanoparticles: A - TEM image of UCNP-OA core/shell structure (NY0.794F4: Yb0.2, Tm0.06/NaYF4), B - Hydrodynamic diameter distributions of UCNP-OA and UCNP-NOBF4 obtained by DLS in hexane and deionized water, respectively, C - PL emission spectrum of UCNP-OA and UCNP-NOBF4 under excitation at 980 nm
the OA residues were replaced with the small inorganic and ionic compound NOBF4. Surface modification of UCNP-OA with NOBF4 yielded hydrophilic nanoparticles with a hydro-dynamic diameter of 93.91 ± 1.03 nm and a moderate polydispersity since PDI was 0.171 ± ± 0.020 (Fig. 2B). Both PDI value were below 0.3 showing a high level of uniformity in size and a low level of aggregation. The particles of UCNP-NOBF4 were surrounded by NO+ ions on the surface of UCNP and BF4- as counterions (Fig. 1) (Dong et al., 2011). They had a positive C-potential of +57.2 ± 2.04 mV (Fig. 3B) that indicates high colloidal stability in suspension because the electrostatic repulsion between these positively charged nanoparticles over-weighs the gravitational attraction force between particles, hence lower aggregate formation (Bhattacharjee, 2016). The PL emission peaks of both nanoparticles are in the blue (478 nm) and NIR (800 nm) regions (Fig. 2C). The latter falls within the transparency window of biological tissue (700-1100 nm), rendering them ideal for bioimaging (Li et al., 2015). Although the PL intensity of UCNP was significantly reduced after NOBF4 coating, the exhibited intensity is still sufficient for bioimaging (Chen et al., 2017).
Optimization of BSA concentration for PC formation on UCNP-NOBF4
Protein corona of BSA is used to stabilize the obtained UCNP-NOBF4 prior to further investigation. The aim was to formulate a stable layer of BSA around UCNP-NOBF4 shielding the particles from each other and additional proteins in biological fluids as well as increasing their blood circulation time. PC formation is a protein concentration dependent phenomenon, therefore a dilution series of BSA concentrations was assessed to determine the finest concentration for this purpose (Zhang et al., 2017). Low concentrations of BSA caused relatively high aggregation of UCNP-NOBF4, whereas high concentrations led to the preservation of excess unbound protein molecules (Fig. 3A). The C-potential increases in absolute value with increasing albumin concentration (Fig. 3C) and higher values of C-potential tip the force scale
Fig. 3. Determination of the optimal concentration of BSA for the formation of PC on the surface of UCNP-NOBF4: A, B - Hydrodynamic diameter of BSA-UCNP-NOBF4 after the incubation of UCNP-NOBF4 (0.25 mg/ml) with BSA at different concentrations (25-120 pM) for 15 min and 4 h at room temperature acquired by DLS, respectively. C - The Ç-potential of UCNP-NOBF4, BSA, and BSA-UCNP-NOBF4 recorded by ELS. All measurements were carried out in deionized water
in favor of electrostatic repulsion resulting in even more stable colloids. Colloidal stability, size, and C-potential of all suspensions were
Table 1
The hydrodynamic diameter of UCNP-NOBF4 incubated with different concentrations of BSA (25 - 120 pM) for 15 min and 4 h
Sample / Incubation time Hydrodynamic diameter (nm) PdI
15 min 4 hours 15 min 4 hours
UCNP-NOBF4 (0.25 mg/ml) Incubated with BSA 25 pM 116.5 ± 1.361 118.5 ± 1.986 0.271 ± 0.009 0.279 ± 0.012
BSA 50 pM 120.6 ± 0.9849 119.4 ± 2.312 0.42 ± 0.006 0.435 ± 0.012
BSA 75 pM 121.7 ± 5.551 131.6 ± 8.9 0.342 ± 0.046 0.413 ± 0.026
BSA 100 pM 119.9 ± 0.7572 123.9 ± 1.553 0.322 ± 0.004 0.369 ± 0.055
BSA 120 pM 108.7 ± 2.946 108.9 ± 2.042 0.423 ± 0.071 0.488 ± 0.006
evaluated and sustained after 4 hours of incubation (Fig. 3B, C). The best BSA concentration capable of forming a single layer of BSA around UCNP-NOBF4 with minimum excess unbound albumin was 100 pM with a hydrodynamic diameter of 119.9 ± 0.9 nm, moderate polydispersity, and a relatively stable state in colloidal suspensions (Table 1). The absolute value of the Ç-potential in this case was the maximum among all samples, which confirms the greatest colloidal stability (Fig. 3C).
Discussion
Recently UCNP has become one the best candidates in nanobiomedical research for improved bioimaging and theranostic applications. Incubation of nanoparticles with albumin leads to the formation of PC on their surface. Several approaches have been developed to investigate albumin usage to stabilize nanoparti-cles and increase their blood circulation time (An & Zhang, 2017). Longer circulation facilitates their accumulation in tumor sites via passive diffusion through the large pores of tumor vasculature or otherwise known as the EPR effect (Barua & Mitragotri, 2014).
In this study, we assembled a new diagnostic nanocomplex based on UCNP coated with NOBF4 and stabilized with a PC layer of BSA. It was essential to determine the optimal ratio between BSA and UCNP-NOBF4 to stabilize the nanoparticles by forming a stable layer of
PC. The suspension of BSA with UCNP-NOBF4 promotes a protein concentration dependant formation of PC around the nanoparticles, a factor that was ignored for some time but established recently (Zhang et al., 2017). The results obtained showed the dependence of PC formation on BSA concentration to a certain level of saturation (Fig. 3). This approach takes advantage of the strong electrostatic interactions between positively charged UCNP-NOBF4 and negatively charged BSA (Fig. 3 C).
The present work encourages developing methods to exploit PC or reduce its impact to improve drug-carrying nanosystems' therapeutic effect. It was proven that it is possible to stabilize nanoparticles with a certain protein (albumin), which can prevent further adsorption of plasma proteins and increase nanoparticles' circulation time in the bloodstream, and consequently increase the efficiency of their passive delivery to tumor tissue via the EPR effect (An & Zhang, 2017). Moreover, these nanocom-plexes have a high PL intensity in the NIR region within the transparency window of living tissues. Therefore, they are now the subject of further intensive investigation for their potential diagnostic agents.
Acknowledgments
This research was supported by the Ministry of Education and Science of the Russian Federation (Agreement No. RFMEFI58418X0033).
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