ISSN 1866-8836
Клеточная терапия и трансплантация

Effect of the size of polymer particles bearing protein antigen on the in vivo T-cellular immune response

Rodion G. Sakhabeev1, Dmitry S. Polyakov2, Viktor A. Korzhikov-Vlakh3, Ekaterina S. Sinitsyna3,4, Galina A. Platonova4, Evgenia G. Korzhikova-Vlakh3,4, Mikhail M. Shavlovsky2

1 St. Petersburg Institute of Technology (Technical University), St. Petersburg, Russia
2 Institute of Experimental Medicine, St. Petersburg, Russia
3 Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russia
4 Institute of Macromolecular Compounds of Russian Academy of Sciences, St. Petersburg, Russia

Rodion G. Sakhabeev, St. Petersburg Institute of Technology (Technical University), St. Petersburg, Russia

Citation: Sakhabeev RG, Polyakov DS, Korzhikov-Vlakh VA et al. Effect of the size of polymer particles bearing protein antigen on the in vivo T-cellular immune response. Cell Ther Transplant 2024; 13(1): 42-48.

doi 10.18620/ctt-1866-8836-2024-13-1-42-48
Submitted 26 January 2024
Accepted 01 March 2024


In this work, we studied the effect of size of polymeric particles based on poly(D,L-lactic acid) bearing on the surface the fusion protein of human beta2-microglobulin with green fluorescent protein (β2M-sfGFP) on the levels of antigen-specific T-helper and cytotoxic T-lymphocytes. Microparticles with a diameter of about 1400 nm (MPs) and nanoparticles with a diameter of about 100 nm (NPs) were used as carriers for model protein. To evaluate the effect of different particle sizes on the immunogenicity of the model protein, two groups of mice were immunized so that the amount of β2M-sfGFP was equal. The identification of the antigen-specific interferon-producing T cells was carried out by the method of intracellular cytokine staining. For further analysis of T-lymphocyte populations, the method of flow cytofluorimetry was used. The number of antigen-specific CD4+ IFNγ-producing memory T cells (T-helpers) was not significantly different in the case of immunization with complex antigen MPs-β2M-sfGFP compared with the group immunized with antigen immobilized on the nanoparticles (NPs-β2M-sfGFP). However, the number of antigen-specific CD8+ T cells producing IFNγ was significantly higher (p = 0.031) in the case of immunization with complex antigen based on MPs compared to the group treated with the complex antigen based on NPs.


Cellular immune response, T helper cells, T cytotoxic lymphocytes, protein antigen, microparticles, nanoparticles, poly(lactic acid).


Currently, polymeric particles are of great interest as drug delivery systems [1-3], as well as for developing vaccines [4, 5] and trapping systems for viruses [6-8] and pathogens [9]. Polymeric particles can vary in size and properties. In particular, polymer particles <500 nm in size are classified as nanoparticles (NPs), while particles >1000 nm are considered microparticles (MPs) [10, 11]. Biodegradable (co)polymers are preferable for preparation of micro- and nanoparticles, since they can be totally eliminated from the body after degradation. Among these, aliphatic polyesters, e.g. poly(lactic acid) (PLA), or its copolymer with glycolic acid (PLGA), poly-ε-caprolactone, poly-3-hydroxybutirate, etc. are biodegradable and non-toxic polymers, which are promising for biomedical applications [12]. Moreover, they are approved by Food and Drug Administration (FDA) Agency [13]. Sometimes, the mentioned polymers are copolymerized with poly(ethylene glycol) (PEG; also FDA approved polymer) to prepare amphiphilic block- and graft-copolymers [14, 15].

The biological effect of particles is regulated by immobilization of the specific ligands such as peptides, antibodies, carbohydrates, and aptamers, etc., on the surface of polymer particles. To date, there are a number of studies on the evaluation of humoral immune response towards protein immobilized on the surface of micro- and nanoparticles [16-19]. For example, it was shown that the use of particles based on PLGA, carrying ovalbumin and oligonucleotide, and having a diameter of 300 nm led to a higher antigen-specific immune response in mice compared to particles of the same origin, but of different size (1, 7 and 17 μm) [18]. A similar trend for ovalbumin immobilized on the surface of polystyrene particles with diameters ranging from 40 nm to 2 μm has been shown by Fifis et al. [17]. In this case, the highest antibody induction was detected for the complex antigen immobilized on 40- nm NPs, and the lowest level was found for complex antigen based on MPs of 2-µm diameter.

Along with humoral immune response, the complex antigens consisting of polymer particle bearing protein antigen may also affect T-cellular immune response, based on activation of antigen- specific CD4+ and CD8+ T-lymphocytes. Induction of both humoral and also cytotoxic immune system is required, for example, for effective elimination of some viruses (HIV, hepatitis C virus, SARS-Cov2, etc.) from the host organism mediated by specially designed trapping systems. For effective activation of the immune response, the antigen uptake by macrophages or dendritic cells is required, followed by its presentation in a complex with molecules of the major histocompatibility complex. Recently, Uto et al. reported that immunization of mice with nanoparticles (NPs, 210 nm in diameter) based on poly(γ-glutamic acid) bearing immobilized ovalbumin induced significant activation of CD8+ T cells [20]. Similarly, Zhang et al. reported the suitability of ovalbumin-loaded NPs (about 250 nm in diameter) based on poly(glutamic acid) modified with phenylalanine ethyl ester for enhancing cellular immunity [21].

Recently, we studied the humoral immune response on the immunization of mice with PLA-based MPs and NPs bearing β2M-sfGFP as a model protein [16]. When administered intraperitoneally, the PLA-based MPs as carriers for protein were less effective in the production of specific antibodies against the immobilized protein than NPs. Finally, the particles covalently modified with model proteins cause a less pronounced humoral immune response compared to a mixture of particles with protein.

The aim of this study was to investigate the size effect of polymeric particles with covalently bound model protein (complex antigen) on the counts of antigen-specific T-helper cells and cytotoxic T-cells. As another type of polymer particles, the PLA microparticles and nanoparticles based on block-copolymer of PLA with PEG (PEG-b-PLA) were used as carriers for comparative studies. A fusion protein containing human beta2-microglobulin (bMG) with green fluorescent protein (β2M-sfGFP) was selected as a model antigen. The obtained complex antigen (β2M-sfGFP), loaded on micro- and nanoparticles (MPs or NPs), were physically characterized and used for immunization of mice. Finally, the antigen-specific interferon-producing T-lymphocyte populations were analyzed by intracellular cytokine staining and flow cytometry.

Materials and methods

Production and Characterization of Polymer MPs and NPs

MPs were obtained by the single emulsion method using PLA (Мw = 23200, Ð = 1.13). The details on PLA synthesis and microparticle preparation can be found elsewhere [16]. NPs were prepared by nanoprecipitation of PEG-b-PLA (Мw = 30300, Ð = 1.20) from a solution in acetonitrile into water under vigorous stirring as previously described [22]. The mean hydrodynamic diameter (DH) and polydispersity index (PDI) of MPs and NPs, as well as the surface ζ-potential were determined by dynamic and electrophoretic light scattering using the Zetasizer Nano ZS (Malvern, UK). The properties of MPs and NPs are summarized in Table 1.

Table 1. Physico-chemical characteristics of initial and modified with PEG-b-PLA protein particles


Protein Production and Its Covalent Immobilization on Surface of the Particles

Model protein β2M-sfGFP was produced in Escherichia coli (strain BL21(DE3)) transformed with plasmids containing corresponding genes as elsewhere described [23, 24]. E. coli cells were disrupted by ultrasound treatment, the soluble cell fraction was separated by centrifugation, and the target recombinant proteins were purified by column liquid chromatography using Ni-agarose (Ni-NTA Agarose, QIAgen, USA) according to the manufacturer's protocol.

The process of protein immobilization on the particle surface consisted of several steps. (1) Free carboxyl groups were generated on the surface of polymer particles by partial PLA hydrolysis with 0.1 M NaOH solution for 30 min at room temperature (25°С). The particles were separated and rinsed with distilled water followed by centrifugation; (2) At the second stage, free carboxyl groups were activated. Carboxyl groups formed on the surface of the particles were activated by a mixture of N-hydroxysuccinimide and water-soluble carbodiimide-N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride in order to obtain activated ester [16]. The amount of immobilized β2M-sfGFP was determined as a difference of protein contents in soluble phase before and after the reaction. The protein concentration was determined spectrophotometrically at 490 nm using a ThermoScientific NanoDrop 2000 (USA) spectrophotometer. The calculation was performed by a calibration curve previously plotted for the β2M-sfGFP.

Mice Immunization

Female F1 hybrids (CBA x C57BL) weighing 20-25 g (age 4-6 months) were used for immunization. The mice were kept in the vivarium at ambient temperature and 12/12 h light regime, with food and water ad libitum. To assess immune response to antigens, the mice were injected with test preparations. Both experimental and control groups (naive animals) included 15 mice. The amount of complex antigen was calculated in order to reach 1 μg of β2M-sfGFP per mouse. The preparations diluted in sterile saline solution (0.9% NaCl) were administered intraperitoneally at a volume of 0.4 mL/animal. All animal experiments were performed in accordance with International Recommendations for biomedical research using animals. Mice were immunized 4 times at an interval of 2 weeks.

Isolation and Cryopreservation of Mouse Splenocytes

Splenocytes were isolated from the spleens of mice 2 weeks after the last quadruple immunization. The spleens were placed in 1 mL of sterile DMEM nutrient medium (Gibco, 41965-039) with antibiotic/antimycotic (Gibсo, 15240-062), being gently homogenized. The cell suspension was washed with 10 mL of sterile PBS for 10 min (centrifugation at 200g, 10°C). The cell sediment was resuspended in 3 mL of buffer and, after lysis of erythrocytes, the nucleated cells were washed with excess sterile PBS (10 min at 200g, 10°C). The resulting cell precipitate was resuspended in 1 mL of fetal calf serum (HyClone, SH30071.02), then supplied with 1 mL of double cryopreserving solution (fetal calf serum with 20% DMSO) was added in cold bath (0°C) with constant stirring. 1 mL of the suspension was poured into cryotubes (Biolab, 028049) and stored at -70 °C followed by transfer to liquid nitrogen for the next day.

Intracellular Cytokine Staining

Splenocytes were characterized using antibodies to CD3 labeled with allophycocyanin (BioLegend, 100236), antibodies to CD4 labeled with PE (BioLegend, 100408), antibodies to CD8a labeled with PC7 (BioLegend, 100722), antibodies to IFNγ labeled with FITC (BioLegend, 505806). To identify antigen-specific T cells, the generally accepted method of intracellular cytokine staining (IFNγ) was used [25]. The cells (106/well) were cultured in round-bottomed 96-well plates in 200 µL of complete culture medium prepared on the basis of RPMI-1640 (Sigma-Aldrich, R8758-100ML) supplied with10% embryonic calf serum, HEPES aqueous solution (Biolot, 1.2.6.), antibiotics/antimycotics (Gibсo, 15240-062), 2-mercaptoethanol (Amresco 0482-0.1), ronkoleikin (106 U/mL, NPK Biotech, St. Petersburg). The cells were incubated in a CO2 incubator at 37°C for 12 h with an antigen. Five hours before the end of incubation, a 1x solution of GolgiPlug (BD Bioscience, 555028) was added, blocking the excretion of cytokines from the cell. To determine spontaneous production of IFNγ, an appropriate volume of RPMI-1640 nutrient medium was added, instead of stimulating agent. In the analysis, these data (negative control) were subtracted from the indices obtained for antigen-stimulated cells. Flow cytometry studies were performed with Beckman Coulter Navios device (Beckman, USA).

Statistical Data Processing

Newman-Keuls test was used for pairwise comparison of the groups. Normality was checked using the Shapiro-Wilk criterion. All statistical calculations and plotting were performed in the Rstudio 1.1.453 software. All "box plot" plots were presented as medians with confidence intervals. Each group of animals consisted of 15 mice. The immunoassays were done in 3 replicates for each sample.

Results and discussion

The chosen approach to β2M-sfGFP immobilization presumed formation of hydrolytically stable amide bonds between the protein and the surface of the polymer particle. The protein loading conditions were optimized in order to provide immobilization capacity of 10 μg of protein/mg particles. The protein binding to the surface of polymer particles led to a slight increase in their hydrodynamic diameter (DH) and a decrease in the surface ζ-potential (Table 1).

Two groups of mice were immunized in order to compare the immune effects of model protein loaded on the polymer particles. The first group was immunized with a complex antigen representing polymer nanoparticles bearing model protein (NPs-β2M-sfGFP), and the second group was treated with a complex antigen with polymer microparticles bearing the same protein. A third group represented the non-immunized intact (naive) mice. A four-time immunization of mice at 2-week intervals was performed, and spleen samples were collected 13 days after the last immunization. Mouse spleen cells were phenotyped using antibodies to CD3, CD4, CD8 and interferon γ (IFNγ). A common intracellular cytokine staining method was used to identify antigen-specific interferon-producing T cells followed by flow cytometry analysis of T cell populations.

The relative content of IFNγ+-lymphocytes specific to sfGFP protein was determined for the following T-cell subpopulations: CD4+ T cells (IFNγ+CD3+CD4+), i.e., type 1 T-helper cells, and CD8+ T cells (IFNγ+CD3+CD8+) as shown in Fig. 1.


Figure 1. Detection of antigen-specific T helper and cytotoxic T lymphocytes by multicolor flow cytometry

A, exclusion of adherent cells from the analysis area based on peak (x-axis) and integral (y-axis) signals of direct light scattering (single cells are located in the "Singlets FSAxFSH" area); B, exclusion of adherent cells from the analysis area based on peak width (x-axis) and integral (y-axis) signals of direct light scattering (single cells are located in the "Singlets FSAxFSW" area); C, identification of splenocytes on the basis of lateral (y-axis) and direct (x-axis) light scattering parameters (single splenocytes are located in the "Splenocytes" area and analyzed in the subsequent histogram); D, removal of dead cells from the analysis area based on the inclusion of Zombie Aqua dye (the "Live" area contains live single splenocytes); E, detection of T-lymphocytes based on CD3 expression (the "T cells" area contains T-lymphocytes); F, separation of T-lymphocytes into T-helper (phenotype CD3+CD4+, area "CD4+ T cells") and cytotoxic T-lymphocytes (phenotype CD3+CD8+, area "CD8+ T cells"); histograms G and H – IFNγ production by T-helper and cytotoxic T-lymphocytes in response to in vitro stimulation, respectively (area "IFNγ+ CD4+ T cells" in histogram G and area "IFNγ+ CD8+ T cells" in histogram H, respectively).

The flow cytometry method was used to assess relative contents of CD4+ and CD8+ T cells. Using the Shapiro-Wilk criterion, it was found that the distribution was not normal in each group (p<0.001). Therefore, the nonparametric Newman-Keuls statistical test was used for pairwise comparison of the three groups. The results of this comparison are summarized in Table 2.

Table 2. Results of statistical analysis of CD4+ and CD8+ T-lymphocytes in the groups of immunized and naïve mice (see text)


* MPs, microparticles; NPs, nanoparticles; MPs-β2M-sfGFP, mice immunized with complex protein antigen loaded on MPs (DH of 1427 nm); NPs-β2M-sfGFP, mice immunized with complex protein antigen immobilized on NPs (DH of hydrodynamic diameter 115 nm); naïve (intact) mice.

Fig. 2 shows that the number of antigen-specific IFNγ-producing memory T cells of CD4+ phenotype (T-helper cells), was not significantly different between the animals immunized with a complex β2M-sfGFP antigen bound to polymer microparticles and the group immunized with similar antigen loaded on nanoparticles. However, the number of IFNγ-producing antigen-specific CD8+ T cells was significantly (p=0.031) higher in the case of immunization with a complex MPs-β2M-sfGFP conjugate than in the group, immunized with a complex NPs-β2M-sfGFP antigen. It is also worth of note that the group of naïve (non-immunized) mice showed a significantly lower response compared with both immunized groups.


Figure 2. Relative contents of antigen-specific CD4+ T cells (A) and CD8+ T cells (B) responding to the model protein sfGFP in mice

***, P level of significance <0.005. ** P level of significance >0.005 but less than 0.05. NS, differences are insignificant (p>0.05). For the group designations, see footnote to Table 2.

The obtained results may be explained by different mechanisms of their interaction and entrance to the cells. In particular, it is known that the particles of <200 nm have been found to penetrate cells in an actin-independent manner (e.g., by clathrin-dependent endocytosis) [26]. The particles of larger size are usually engulfed by actin-dependent manner by phagocytosis. These features of particle uptake appear to play a role in the immune response to the particle-associated antigens. The same features distinguish the immune response to particulate antigens from the response to conventional classical vaccines [27].


Hence, one may conclude that polymer microparticles are more suitable, e.g., for preparation of trapping systems for viruses, since they promote a more pronounced activation of cellular immune response, being quite important for development of antiviral immunity. In turn, the PLA-based nanoparticles around 100 nm in diameter cannot be recommended for this purpose due to the fact that they cause mainly a strong humoral immune response, i.e., production of antigen-specific immunoglobulins [16]. Therefore, the latter type of particles is more preferable as adjuvants in vaccine development.

Compliance with ethical standards

All procedures involving animals complied with the ethical standards approved by the legal acts of the Russian Federation, the principles of the Basel Declaration and recommendations of the Local Ethical Committee of the Institute of Experimental Medicine.


The work was performed within the frame of State assignments of IMC RAS (124013000730-3) and IEM (FGWG-2022-0009, 122020300191-9).

Conflict of interest

The authors declare no evident and potential conflicts of interest related to the publication of this article.


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Volume 13, Number 1

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doi 10.18620/ctt-1866-8836-2024-13-1-42-48
Submitted 26 January 2024
Accepted 01 March 2024

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