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STUDY OF THE INFLUENCE OF AN ARTIFICIALLY FORMED PROTEIN CORONA ON THE SURFACE
OF UPCONVERSION NANOPARTICLES ON THE UPTAKE EFFICIENCY BY MOUSE PERITONEAL MACROPHAGES
D.K. Bausheva, A.O. Belotelov, E.L. Guryev*
Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, 23 Gagarin Ave., Nizhny Novgorod, 603950, Russia.
* Corresponding author: eguryev@ibbm.unn.ru
Abstract. The role of macrophages in nanomedicine is widely recognized, but systemically administered drug nanocar-riers are rapidly absorbed and eliminated by the mononuclear phagocyte system, consisting of resident macrophages, primarily in the liver. As a result, most encapsulated drugs are eliminated from circulation, resulting in unwanted side effects. Peritoneal macrophages, on the contrary, by ingesting nanoparticles and migrating to tumor cells, can promote the antitumor effect. This article describes the preparation of complexes based on upconversion nanoparticles (UCNP) coated with nitrosonium tetrafluoroborate (NOBF4) with a protein corona (PC) from native and denatured bovine serum albumin (BSA and dBSA). The efficiency of UCNP-protein complexes uptake by mouse peritoneal macrophages has been demonstrated. The use of this approach is a promising area of oncology, since instead of inefficient systemic intravenous delivery, peritoneal delivery is used, which can become the key to solving the problem of peritoneal cavity cancers.
Keywords: upconversion nanoparticles, protein corona, macrophages.
List of Abbreviations
UCNP - upconversion nanoparticles
NOBF4 - nitrosonium tetrafluoroborate
PC - protein corona
BSA - bovine serum albumin
dBSA - denatured bovine serum albumin
PL - photoluminescence
Introduction
At the moment, theranostics is an actively developing area of biomedicine. Many nano-medicines have been developed for the purpose of diagnosing tumor foci and targeting tumor cells, as well as for controlling drug distribution and assessing the therapeutic effect (Kim et al., 2009; Xie et al., 2010; Grebenik et al., 2016). However, general problems with the use of na-nomedicines require solutions. Thus, it turned out that most of the nanomedicines introduced into the blood accumulate in the critical organs of the mononuclear phagocytic system - the liver and spleen - leading to difficult-to-remove toxicity (Bertrand et al., 2014). There are also unwanted side effects caused by the basic effect of nanomedicines delivery - increased permea-
bility and retention (EPR effect) (Maeda, 2010; Danhier, 2016).
After nanomedicines enter the body, proteins of the internal environment are actively adsorbed on their surface, which leads to the formation of a protein corona (PC) (Corbo et al., 2016; Peng & Mu, 2016). The process of protein adsorption on the surface of nanomedi-cines is influenced by the affinity between proteins and nanomedicines, as well as between the proteins of the medium and proteins already adsorbed on nanomedicines (Monopoli et al., 2012). PC significantly changes the initial physicochemical properties of nanomedicines and their "biological identity", affects the interaction with cells and the effects of nanomedi-cines in the body.
To date, data have been obtained on the possibility of using a preformed protein corona to stabilize and increase the circulation time of nanoparticles using albumin, transferrin, apolipo-proteins and other proteins. Albumin is most often used for these purposes (Casals et al., 2010). It is biocompatible, biodegradable, non-toxic, non-immunogenic and capable of long-term
circulation in the bloodstream (15-19 days) (Sleep et al., 2013). The properties of albumin make it a potential stabilizer for nanoparticles, since it is adsorbed on their surface and forms a protein corona tightly bound to the surface of nanoparticles via electrostatic bonds (An & Zhang, 2013).
The two types of albumin most commonly used to create nanoparticle-based complexes are human serum albumin (HSA) and bovine serum albumin (BSA). HSA and BSA are homologous proteins. They share many characteristics, including molecular weight (65-70 kDa), high water solubility, and long half-life in the blood. Many cancer cells use albumin as a source of energy and nutrients (Stehle et al., 1997) and typically exhibit increased albumin uptake via macropinocytosis (Commisso et al., 2013).
When choosing a platform for creating theranostic complexes, special attention is paid to the use of upconversion nanoparticles (UCNPs). UCNPs are inorganic photoluminescent (PL) nanoparticles that have exceptional resistance to photo- and chemical degradation and excitation by wavelengths falling within the "transparency window" of biological tissue (Wang et al., 2010, Aires et al., 2015). These properties make it possible to create UCNP-based theranostic agents that have both diagnostic and therapeutic properties, which contributes to the search for new solutions in tumor therapy.
One of the solutions to the problem of insufficient efficiency of targeted delivery of nano-medicines in the case of tumors of the abdominal cavity and pelvic organs may be intra-peritoneal administration. In this case, nano-medicines will practically not enter the systemic circulation and be absorbed by liver macrophages. Their absorption by macrophages lining the peritoneal cavity, on the contrary, is desirable. In this case, peritoneal macrophages, activated by inflammation caused by the tumor environment, can deliver nanomedicines to peritoneal tumor sites. Therefore, it is important to evaluate the ability of different nanocarriers to be taken up by peritoneal macrophages depending on their surface properties and particle
sizes. In this work, we investigated the effects of an artificially formed protein corona of BSA and thermally denatured BSA (dBSA) on the surface of UCNPs on the efficiency of phagocytosis by peritoneal macrophages.
Materials and Methods
Obtaining and Characterization of UCNP
UCNP with core/shell structure NaY0,794Yb0,2Tm0,006F4/NaYF4 were synthesized by the solvothermal decomposition as described earlier (Guryev et al., 2020). The PL emission spectrum of UCNPs was studied using a spectrofluorimeter CM 2203 (SOLAR, Belarus). PL UCNPs were excited using a semiconductor laser generating radiation at a wavelength of 980 nm (Semiconductor Devices JSC, Russia).
Hydrophilic UCNPs were prepared by lig-and exchange method (Dong et al., 2011). Hydrophobic UCNPs in hexane (Kriochrome, Russia) were mixed with a solution of NOBF4 in dimethylformamide (PanEco, Russia) in a ratio of 60 mg NOBF4: 25 mg UCNP. The mixture was stirred overnight on a magnetic stirrer at room temperature. The mixture was centri-fuged at 10,000 g for 7 min, UCNP was resus-pended in dimethylformamide, and a hex-ane/toluene mixture (1:1) (Khimreaktiv, Russia) was added. The UCNPs were then washed with ethanol twice and resuspended in deion-ized water. The hydrodynamic diameter of the particles was measured by dynamic light scattering using a Zetasizer Nano ZS system (Mal-vern, UK).
Preparation of UCNP-protein complexes
UCNPs with stable PC from BSA and dBSA on the surface of nanoparticles were prepared by lyophilization as described earlier (Shanwar et al., 2021). The BSA solution was incubated for 30 min at 70 °C to obtain dBSA. The UCNP-NOBF4 suspension (0.5 mg/mL) was added dropwise to the BSA and dBSA solutions (100 pM) with stirring. The mixture was incubated at room temperature for 4 h. UCNP-NOBF4-BSA and UCNP-NOBF4-dBSA samples were lyophilized using a FreeZone 6L device (Labconco, USA). Samples were placed in
glass vials and frozen at -80 °C, then kept in a lyophilization chamber at -50 °C under high vacuum for 24 h until the water was completely removed. Lyophilized samples were stored at 4 °C.
Study of the circulation time of UCNP-NOBF4 and UCNP-NOBF4-BSA in the blood of laboratory animals
A series of dilutions of UCNP-NOBF4 and UCNP-NOBF4-BSA were prepared with concentrations of 5, 20, 40, 60, 80, 100 ^g/ml in heparinized blood. The PL signal intensity was measured using a UCNP imaging device DVS-02 as described previously (Guryev et al., 2019). Based on these data, calibration graphs of the dependence of the PL signal intensity on the UCNP concentration were obtained.
The studied particles and complexes in an amount of 200 ^g in 200 ^l of PBS were injected to Balb/c mice into the left lateral tail vein (the first two-thirds of the length of the vein from the tip of the tail). Blood samples were taken from the retrobital sinus at 0.5, 1.5, 2, 3, 4, 5, 10, 15 and 30 min after the administration of particles and complexes. Samples were diluted with heparin in the ratio 25 |il (blood) : 15 |il (heparin). The intensity of the PL signal of UCNP in the samples was measured using a DVS-02 device, the concentration of particles and complexes was calculated using calibration graphs.
Study of the complexes uptake efficiency by peritoneal macrophages
Macrophages were isolated from the peritoneal transudate of Balb/C mice, the complexes uptake was studied in vitro. Cells were seeded into the wells of a 96-well confocal plate and incubated for 12 h. After incubation of the peritoneal cell suspension in the wells of the plate, the culture medium was removed, which removed the majority of non-macrophage peritoneal cells. Wells with macrophages attached to the bottom were washed with PBS, 1 ml of a suspension of UCNP-NOBF4-BSA or UCNP-NOBF4-dBSA (0.2 mg/ml in PBS) was added to the wells, and incubated for 2 h. The suspension was removed and the wells were washed
with PBS. Cells were fixed with 4% formaldehyde for 30 min and washed with PBS and de-ionized water. The preparations were examined by confocal laser fluorescence microscopy. Quantitative data were processed using the program GraphPad Prism 6 (GraphPad Software, США)
Results
Obtaining and Characterization of UCNP
Using the solvatothermal decomposition method, UCNPs were obtained based on a NaYF4 matrix doped with ytterbium and thulium lanthanide ions. Inert NaYF4 shell reduces surface quenching effects and increases UCNP PL intensity (Grebenik et al., 2016). Such UCNPs exhibit anti-Stokes PL with pronounced emission maxima in the visible (around 474 nm) and near-infrared (IR) spectral regions (around 801 nm) when excited by light with a wavelength of 980 nm (Fig. 1). The main maximum of PL emission is in the IR region and falls within the transparency window of the biological tissue. This makes UCNPs doped with thulium ions effective agents for bioimag-ing at the level of tissues, organs and the whole body of small laboratory animals.
Nitrosonium tetrafluoroborate (NOBF4) coated UCNPs were prepared using the ligand exchange method (Fig. 2). Such a shell makes it possible to obtain a monodisperse suspension of colloidally stable biocompatible particles (Dong et al., 2011). The hydrodynamic diameter of the UCNP-NOBF4 particles was 54.80 ± ± 20.34 nm (Fig. 3), and the polydispersity index (PDI) was 0.229, indicating the homogeneity of the sample.
Preparation of UCNP-protein complexes
UCNPs with stable PC from BSA and dBSA on the surface of nanoparticles were obtained by lyophilization using previously developed technique (Shanwar et al., 2021). The use of native BSA in optimal concentration allows the formation of a stable protein layer that completely covers the surface of the particles, which makes the suspension colloidally stable (Fig. 2). An excess or deficiency of protein molecules leads to increased aggregation.
300
400 500 600 700 800
Wavelength, nm
Fig. 1. Emission spectrum of PL UCNPs with the composition NaYF4:Yb:Tm/NaYF4 when excited by light with a wavelength of 980 nm
UCNP UCNP-NOBF4 UCNP-NOBF4-dBSA
Fig. 2. Scheme for converting hydrophobic UCNP-Oleate into biocompatible UCNP-NOBF4 by exchanging ligands and obtaining UCNPs with an artificially formed protein corona
of BSA and dBSA
Fig. 3. Hydrodynamic diameter of UCNP-NOBF4 particles
Fig. 4. Hydrodynamic diameter of UCNP-NOBF4-BSA (A) and UCNP-NOBF4-dBSA (B) in deionized water
Fig. 5. Hydrodynamic diameter of UCNP-NOBF4-BSA and UCNP-NOBF4-dBSA
in phosphate-buffered saline
The use of denatured BSA allows for thinner and denser PC due to the unfolding of the BSA peptide chain. Also, epitopes of the BSA molecule, hidden inside the globule in the native state, are exposed.
The hydrodynamic diameter of UCNP-NOBF4-BSA particles resuspended in deion-ized water was 96.33 ± 27.25 nm (Fig. 4A). The PDI was 0.216, indicating that the sample was homogeneous. The hydrodynamic diameter of
UCNP-NOBF4-dBSA particles in deionized water was 195.4 ± 52.32 nm (Fig. 4B), PDI was 0.116, which also indicates the homogeneity of the sample.
The hydrodynamic diameter of UCNP-NOBF4-BSA particles resuspended in phosphate-buffered saline was 221.7 ± 11.59 nm (Fig. 5A), PDI 0.616. The hydrodynamic diameter of UCNP-NOBF4-dBSA particles in phosphate-buffered saline was 141.7 ± 19.46 nm,
and there was also a small peak at 851.9 ± 235.4 (Fig. 5B), and the PDI was 0.613, indicating sample heterogeneity and partial aggregation of particles.
Study of the circulation time of UCNP-NOBF4 and UCNP-NOBF4-BSA in the blood of laboratory animals
To assess the effect of the PC on the behavior of particles in the body upon systemic administration, a study of UCNP of removal rate from the mice bloodstream was performed. A suspension of UCNP-NOBF4 and UCNP-NOBF4-BSA was injected into the left lateral tail vein, and blood samples were collected from the retrobital sinus. Measuring the intensity of the PL signal of blood samples made it possible to identify a single-exponential dependence of the content of particles and complexes in the blood on time (Fig. 6). During the first minutes after administration, a rapid decrease in the concentration of particles and complexes in the blood was observed. The halflife of UCNP-NOBF4 particles calculated by interpolation was 2.621 min, UCNP-NOBF4-BSA complexes - 0.799 min. This value refers to the time required for the concentration of the test compound to decrease by half at any time point after administration. At the last time point
(30 min), the concentration of particles and complexes in the blood approaches zero.
Study of the complexes uptake efficiency by peritoneal macrophages
The macrophage fraction from the peritoneal transudate of Balb/C mice was isolated by sequential washing of peritoneal cells and morphological assessment. As a result of incubation of UCNP-protein complexes with peritoneal macrophages and microscopic examination, a greater accumulation of UCNP-NOBF4-BSA complexes in peritoneal macrophages was shown compared to UCNP-NOBF4-dB SA. The micrographs show a significant PL signal of UCNP in areas of the field of view occupied by cells incubated with UCNP-NOBF4-BSA complexes (Fig. 7). In the case of UCNP-NOBF4-dBSA complexes, the PL signal in the cell area is weakly expressed.
To confirm the obtained microscopy results, a quantitative assessment of the PL signal level in the areas of the visual field occupied by cells was carried out (Fig. 8). Data analysis using the Kruskal-Wallis test showed a statistically significant difference in the intensity of the PL signal of UCNP in the first group (UCNP-NOBF4-BSA) from the other two studied groups of images (p < 0.05).
Fig. 6. Concentration of UCNP-NOBF4 particles and UCNP-NOBF4-BSA complexes in mouse blood when administered intravenously at a dose of 10 mg/kg
UCNP PL signal in Luminal image the range 446-486 nm, Image overlay excitation 980 nm
Fig. 7. Micrographs obtained by confocal fluorescence microscopy: peritoneal macrophages incubated
for 2 h with UCNP-NOBF4-BSA and UCNP-NOBF4-dBSA complexes and peritoneal macrophages without UCNP. Scale bar: 50 ^m. The PL signal of UCNP is shown in red
50
UCNP-NOBF4 UCNP-NOBF4 Cells without -BSA -dBSA UCNP
Fig. 8. Median values of the intensity of the PL signal of UCNP-NOBF4-B SA and UCNP-NOBF4-dBSA complexes and peritoneal macrophages without particles. Error bars are represented by the interquartile range. n = 25
Discussion
While local drug concentrations in tumor tissue can be increased through the use of passive delivery, active delivery to a specific cell type
through targeting agents can in some cases further improve the effectiveness of the targeted drug. Typically, such strategies do not increase the total concentration in the target tissue, but
rather alter the distribution within the tissue, increasing the selectivity of the drug and minimizing side effects. It is expected that the intra-peritoneal administration method will overcome the lack of efficiency of targeted delivery of nanomedicines. In this case, nanomedicines will not be quickly removed from the bloodstream by cells of the mononuclear phagocytic system and accumulate in the liver and spleen.
In the present work, complexes of biocompatible UCNPs with BSA were prepared. The obtained results of measuring the hydrody-namic diameter of the complexes allow us to count on their effective use for delivery to tumor sites, since they remain fairly small in size and do not demonstrate significant aggregation. Such particles, when administered intraperito-neally, can be absorbed by macrophages and, with their help, delivered to tumor sites. It is obvious that the behavior of complexes in water or a buffer solution differs from that in the internal environment of the body. On the other hand, an increase in particle size due to possible aggregation enhances their uptake by macrophages rather than weakens it.
The coating of the particles with BSA was carried out in order to reduce the adsorption of proteins from the internal environment of the body. Several studies have shown that this coating increases the circulation time of particles in
the bloodstream. In contrast, our data showed faster clearance of BSA-coated particles compared to the original UCNP-NOBF4. A possible reason for the observed effect may be partial de-naturation of BSA molecules during lyophiliza-tion, which leads to increased uptake of particles by liver macrophages and reduces their circulation time in the bloodstream.
In the case of intraperitoneal administration of the resulting complexes, their increased uptake by macrophages, on the contrary, is desirable. The results of the study showed greater accumulation of UCNP-NOBF4-BSA in peritoneal macrophages compared to UCNP-NOBF4-dBSA. This may be explained by the larger size of UCNP-NOBF4-BSA complexes as well as the recognition of native BSA epitopes by peritoneal macrophages as signals to engulf objects bearing these epitopes. We hypothesize that these macrophages play a key role in the delivery of nanomedicines to tumor nodes, and our findings will contribute to the development of mechanisms for enhancing the therapeutic effect of antitumor therapy using macrophages.
Acknowledgments
This research was supported by the Ministry of Science and Higher Education of the Russian Federation, project No. FSWR-2023-0032.
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