Evaluation of the Immunosafety of Cucurbit[n]uril on Peripheral Blood Mononuclear Cells In Vitro

Cucurbiturils (CB[n]s) are nanoscale macrocyclic compounds capable of encapsulating a molecule or part of a molecule by forming host–guest complexes. Integration of drugs with CB[n] is used for the following purposes: controlling clearance; protection of the drug from biodegradation; targeted delivery to specific organs, tissues, or cells; reduction of toxicity; and improving solubility. One of the major problems encountered in the application of new drug delivery systems is lack of knowledge of their biological properties. CB[n], unlike many other often toxic nanoparticles, has extremely low toxicity, even at high doses. However, many aspects of the biological actions of these nanoscale cavitands remain unclear, including the immunotropic properties. In this study, we investigated the immunotoxicity and immunomodulation properties of CB[n]. It was found that CB[7] and CB[6] did not decrease the viability of mononuclear cells at all tested concentrations from 0.1–1 mM. Overall, the results indicated an immunomodulatory effect of different concentrations of CB[n]. In the case of a longer cultivation time, CB[n] had an immunostimulating effect, which was indicated by an enhancement of the proliferative activity of cells and increased expression of HLA-DR on lymphocytes.


Introduction
The use of nanoscale drug delivery systems is one of the most promising areas in modern pharmaceutical science [1,2]. Drug delivery systems are used for a variety of reasons, including to control the drug clearance; to protect the drug from biodegradation; for targeted delivery to specific organs, tissues, or cells; to reduce toxicity; and to increase solubility. However, before the application in clinical practice, nanostructures and nanomaterials used as components for such drug delivery systems require a comprehensive and careful study of their properties and biological activity. This research includes a study of the immunotropic properties of these substances because the cells of the immune system are the most sensitive to the damaging effect of nanomaterials.
One of the possible ways to create systems for the delivery of drugs is the use of nanoscale cavitands capable of host-guest complexation with drugs. Complexes of drugs can be obtained with various cavitands, including cyclodextrins, calixarenes, cucurbit[n]urils (CB[n]s), crown ethers,  The effect of CB[n] on the proliferation of T-lymphocytes was evaluated on days 3 and 7. The study was performed both with and without stimulation of cell proliferation using anti-CD3 antibodies in combination with IL-2. It was found that CB[n] at all studied concentrations did not affect the proliferative activity of T-lymphocytes at day 3 ( Figure 2). However, CB [6] enhanced both spontaneous and anti-CD3-induced proliferation of T-lymphocytes at day 7 of cultivation. CB [7]   The effect of CB[n] on the proliferation of T-lymphocytes was evaluated on days 3 and 7. The study was performed both with and without stimulation of cell proliferation using anti-CD3 antibodies in combination with IL-2. It was found that CB[n] at all studied concentrations did not affect the proliferative activity of T-lymphocytes at day 3 ( Figure 2). However, CB [6] enhanced both spontaneous and anti-CD3-induced proliferation of T-lymphocytes at day 7 of cultivation. CB [7] increased the stimulated proliferation of T-lymphocytes at concentrations of 0.1 and 0.3 mM, but not 0.5 mM. It is known that another cavitand, methyl-β-cyclodextrin, can also enhance the proliferation of PBMCs [23].
Molecules 2020, 25, x 4 of 10 increased the stimulated proliferation of T-lymphocytes at concentrations of 0.1 and 0.3 mM, but not 0.5 mM. It is known that another cavitand, methyl-β-cyclodextrin, can also enhance the proliferation of PBMCs [23].    In addition, we investigated the effect of CB[n] on early (CD69) and late (HLA-DR) markers of lymphocyte activation (Figures 5 and 6). CB[n] did not lead to a change in the level of expression of CD69 on T-lymphocytes. However, it should be noted that significant differences (p < 0.05) in the expression of this molecule were observed in individual donors. CB[n] increased the expression of HLA-DR on CD3 + CD4 -cells and decreased the expression of HLA-DR on CD3 + CD4 + T lymphocytes   In addition, we investigated the effect of CB[n] on early (CD69) and late (HLA-DR) markers of lymphocyte activation (Figures 5 and 6). CB[n] did not lead to a change in the level of expression of CD69 on T-lymphocytes. However, it should be noted that significant differences (p < 0.05) in the expression of this molecule were observed in individual donors. CB[n] increased the expression of HLA-DR on CD3 + CD4 -cells and decreased the expression of HLA-DR on CD3 + CD4 + T lymphocytes   Surprisingly, CB [6] at all studied doses increased the expression of HLA-DR on B lymphocytes after seven days of cultivation ( Figure 7). These data indicate a possible effect of CB [6] on humoral immunity or the antigen presentation process. Other cavitands can also enhance the expression of in anti-CD3 stimulated culture. In addition, CB[n] increased the levels of HLA-DR on T-helper cells and cytotoxic T cells not activated with anti-CD3 antibodies.  Surprisingly, CB [6] at all studied doses increased the expression of HLA-DR on B lymphocytes after seven days of cultivation (Figure 7). These data indicate a possible effect of CB [6] on humoral immunity or the antigen presentation process. Other cavitands can also enhance the expression of Surprisingly, CB [6] at all studied doses increased the expression of HLA-DR on B lymphocytes after seven days of cultivation (Figure 7). These data indicate a possible effect of CB [6] on humoral immunity or the antigen presentation process. Other cavitands can also enhance the expression of CLA on antigen-presenting cells [24]. Thus, further research is required to identify the mechanism of enhancement of the HLA-DR expression.
Molecules 2020, 25, x 7 of 10 CLA on antigen-presenting cells [24]. Thus, further research is required to identify the mechanism of enhancement of the HLA-DR expression.

Materials
CB [6] and CB [7] were synthesized at the Nikolaev Institute of Inorganic Chemistry SB RAS (Novosibirsk, Russia) through the standard procedure described in [25]. The structures of CB [6] and CB [7] were verified with 1 H NMR in D2O at 25 ºC ( Figure S2 in Supplementary) using a 500 MHz Bruker Avance III spectrometer. Medium, phosphate-buffered saline, and L-glutamine were obtained from Biolot (Saint Petersburg, Russia). HyClone fetal calf serum was obtained from GE Healthcare (Chicago, IL, USA). WST-1 reagent was purchased from Takara Bio Inc. (Kusatsu, Japan). Anti-CD3 antibody was obtained from MedBioSpectr (Moskow, Russia). Recombinant IL-2 was obtained from Biotech (Saint Petersburg, Russia).

Isolation and Cultivation of Peripheral Blood Mononuclear Cells (PBMCs)
The venous blood of 22 healthy donors (mean age: 36.0 ± 2.48 years) was used in the study. Donor blood was obtained at the donor center of the State Budgetary Healthcare Institution of the Novosibirsk Oblast ʺCity Clinical Hospital №1ʺ. All the participants signed an informed consent form approved by the local ethics committee of the Research Institute of Fundamental and Clinical Immunology. PBMCs were isolated by the standard method by centrifugation of blood in a Ficoll-Urografin density gradient (ρ= 1.077 g/cm 3 ) [26]. Cultivation of PBMCs was carried out in flat-bottom well plates (Costar, USA) at a concentration of 1 million/mL in Roswell Park Memorial Institute (RPMI-1640) (Biolot, Russia) culture medium containing 0.3% L-glutamine (Biolot, Russia), 50 μg/mL gentamicin (DalChimPharm, Russia), 25 μg/mL thienam (Merck Sharp & Dohme, Netherlands), and 10% inactivated fetal calf serum (HyClone, USA) at 37 °C under 5% CO2 in air with high humidity.

Materials
CB [6] and CB [7] were synthesized at the Nikolaev Institute of Inorganic Chemistry SB RAS (Novosibirsk, Russia) through the standard procedure described in [25]. The structures of CB [6] and CB [7] were verified with 1 H NMR in D 2 O at 25 • C ( Figure S2 in Supplementary) using a 500 MHz Bruker Avance III spectrometer. Medium, phosphate-buffered saline, and L-glutamine were obtained from Biolot (Saint Petersburg, Russia). HyClone fetal calf serum was obtained from GE Healthcare (Chicago, IL, USA). WST-1 reagent was purchased from Takara Bio Inc. (Kusatsu, Japan). Anti-CD3 antibody was obtained from MedBioSpectr (Moskow, Russia). Recombinant IL-2 was obtained from Biotech (Saint Petersburg, Russia).

Isolation and Cultivation of Peripheral Blood Mononuclear Cells (PBMCs)
The venous blood of 22 healthy donors (mean age: 36.0 ± 2.48 years) was used in the study. Donor blood was obtained at the donor center of the State Budgetary Healthcare Institution of the Novosibirsk Oblast "City Clinical Hospital №1". All the participants signed an informed consent form approved by the local ethics committee of the Research Institute of Fundamental and Clinical Immunology. PBMCs were isolated by the standard method by centrifugation of blood in a Ficoll-Urografin density gradient (ρ = 1.077 g/cm 3 ) [26].

Cell Viability
The cell viability was evaluated after incubation with different concentrations of CB [6] and CB [7] (1, 0.5, and 0.1 mM) using the WST-1 method. PBMCs were seeded at 10 5 cells/well into a flat-bottomed, 96-well plate. After 72 h of cultivation, 10 µL of WST-1 (Takara Bio, Kusatsu, Japan) stock solution was added into each well containing 100 µL of cell suspension. The absorbance was directly read at 450 nm, and the reference was read at 620 nm.

Cell Cycle Analysis
Analysis of the PBMC cell cycle distribution was performed after 72 h of incubation with or without different concentrations of CB[n] (0.5, 0.3, and 0.1 mM). After cultivation, the PBMCs were washed from the medium with phosphate-buffered saline (Biolot, St Petersburg, Russia). Then, cells were fixed overnight at −20 • C in 70% ethanol and stained with propidium iodide (100 µg/mL) in the presence of RNAse A (100 µg/mL) for 30 min at 37 • C. Cell cycle analysis was conducted by evaluating DNA histograms. Samples were analyzed on a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and ModFit 3.2 software (Verity Software House, Topsham, ME, USA). The relative amounts of cells with diploid (cells in G 0 /G 1 phases of the cell cycle) and hyperdiploid (cells in S and G 2 /M phases of the cell cycle) DNA sets were determined. Cells with fragmented DNA formed a characteristic hypodiploid peak.

T Cell Analysis
PBMCs were cultivated with different concentrations of CB [6] and CB [7] (0.5, 0.3, and 0.1 mM) in the presence or absence of anti-CD3 antibody (1 µg/mL) and recombinant human IL-2 (100 units/mL). The proliferation of PBMCs was assessed by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE) stained cells. Cells were labeled before culture with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (4 µM) (Invitrogen, Eugene, OR, USA), mixed well and incubated for 15 min in darkness, stirring occasionally. Then the reaction was stopped by adding 2 mL of PBS with 5% FCS, followed by centrifugation. To evaluate the proliferation of lymphocyte subsets, PBMCs after cultivation were stained with monoclonal anti-human antibodies (CD45-PE/Cy7, CD3-APC, CD4-PerCP/Cy5.5, and CD19-APC/Cy7) all from BioLegend, (San Diego, CA, USA). Analyses were performed using a FACSCanto II (Becton Dickinson, USA) and FACSDiva software (Becton Dickinson, USA). The analysis of proliferating activity was carried out after three and seven days of cultivation.

Expression of Activation Molecules
Cells were treated with CB[n] at different concentrations and cultured for 24 h or 1 w to evaluate early (CD69) and late (HLA-DR) activation markers, respectively. After cultivation, cells were stained with fluorochrome-labeled antibodies (CD69-PE or HLA-DR-PE, all from BioLegend).

Statistical Analysis
All data from experiments were expressed as median (25th-75th percentile). ANOVA analyses were performed using GraphPad Prism, with post-hoc comparisons carried out by Fisher's protected least significant difference tests. A p-value < 0.05 was regarded as the minimum criterion for statistical significance.

Conclusions
Our results showed that CB[n] (n = 6 and 7) did not affect the viability of PBMCs. In the case of a short cultivation time (1-3 days), CB[n] did not have an appreciable effect on the proliferation and phenotypic characteristics of the PBMCs. After a longer cultivation time, CB[n] had an immunostimulating effect, enhancing the proliferative activity of cells and increasing the expression of HLA-DR on lymphocytes. It is important to note that CB[n] did not have an immunosuppressive effect, with the exception of a slight decrease in HLA-DR expression on T-helper cells in stimulated cultures.