Intraoperative staining of malignant lung tumors using bioorganic fluorescent gold nanoclusters bound to aptamers

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BACKGROUND

Currently, despite the development of the diagnostic and therapeutic methods of the lung cancer treatment methods, the problem of its recurrence, generalization, or metastatic spread remains relevant. The non-small cell lung cancer (NSCLC) recurrence rate in the late postoperative period is about 45% [1], and its localization varies depending on the disease stage. Thus, 73% of cases can show distant metastases, 19% can show locoregional ones, and 7% can show both types of metastases combined [2].

The primary treatment method for the localized lung cancer is surgery. The extent of resection is determined by the tumor localization, its spread into the adjacent tissues, and the lymph nodes’ lesion status. In cases of malignant lesions of the lobar or primary bronchi, bronchoplastic surgery is possible [3]. Nevertheless, even after resection of a large part of a lung, metastatic foci can remain in the healthy tissue because of their insufficient size and degree of malignancy to be visually different from it.

The malignant cells continue their mitosis after therapy, which leads to locoregional recurrencies and metastatic spread into distant organs. To improve the intraoperative diagnosing, the fluorescent dye-based fluorescence-guided surgery (FGS) can be used. This procedure allows surgeons to see small clusters of malignant cells at the low tumor stages.

To this date, many FGS studies using different molecular complexes of fluorescent dyes with specific ligands, such as antibodies, peptides, or aptamers, have been performed. Some products are already undergoing clinical or preclinical trials [4].

The infrared dyes have become especially popular in FGS due to their low background fluorescence and deep penetration of radiation. The limitations inherent to such dyes include the necessity of specialized equipment; besides, when using the infrared FGS mode, surgeons can visualize only the fluorescence of the dye, which makes the topography of tissues eye-imperceptible. The antibodies or peptides used in the available targeted dyes for fluorescence-guided diagnostics are immunogenic and can cause severe allergic reactions in patients [5].

Consequently, the development of the new targeted fluorescent complexes with the surgery-optimal optical characteristics — high specificity, high tumor fluorescence to background ratio, and low toxicity — is relevant.

In this study we present the liposome- and aptamer-based conjugates delivering in a targeted way the gold nanoclusters into tumor that can excite and emit in the range of porphyrin-IX (excitation: 400–410 nm, emission: 650–710 nm). Such dyes can be visualized using surgical fluorescent microscopes or by naked eye under ultraviolet excitation. A substantial advantage of the dyes emitting in this range is the absence of the tissues’ autofluorescence, which ensures a better contrast of the stained tumor areas.

The aptamers are the preferrable targeting agents due to their low immunogenicity, small sizes (favoring their penetration into tissues and rapid clearance), availability, and simple chemical synthesis and modifications [6].

The fluorescent gold nanoclusters (AuNCs) are ultra-small (1–3 nm) complexes of organic ligands-stabilized gold atoms. AuNCs have the properties that make them promising agents for the intraoperative fluorescent diagnostics. The advantages of AuNCs are the relatively simple synthesis, high photostability, the possibility of the fluorescence emission spectral tuning, and a large Stokes shift. AuNCs are less toxic than inorganic quantum dots. The best studied and characterized nanoclusters are the gold nanoclusters stabilized with glutathione (GSH-AuNCs) and bovine serum albumin (BSA-AuNCs), which have higher quantum efficiencies of fluorescence compared with other nanostructures of this type [7, 8].

Aim. To develop a product for FGS based on aptamers and fluorescent gold nanoclusters (fluorescence excitation wavelengths: 365–410 nm, emission wavelengths: 615–650 nm).

MATERIALS AND METHODS

Study design

The flow cytometry used primary cultures of lung adenocarcinoma. To visually evaluate the binding of complexes, postoperative tissues of lung adenocarcinoma were used.

 

Table 1. Postoperative tissue samples included in the study

Test	Sample type	Histological type	Stage
Flow cytometry	Primary culture	Adenocarcinoma	T4N3M0
		Adenocarcinoma	T3N2M0
		Adenocarcinoma	T4N2M0
Visual assessment	Postoperative tissue	Adenocarcinoma	Т3N0M0

 

The study included the samples from 4 patients with NSCLC. The detailed description is presented in Table 1, all the diagnoses are morphology-confirmed.

Study duration and setting

The gold nanoclusters were received and defined in the Institute of Biochemistry and Physiology of Plants and Microorganisms (IBPPM) of the Federal Research Center Saratov Scientific Center of the Russian Academy of Sciences. The studies were carried out from March 2021 to December 2023. The complexes of nanoclusters in aptamer-modified liposomes were received on the basis of the Laboratory of Numerical Controlled Medicines and Theranostics of the Federal Research Center Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences January to March 2024. The analysis of the complexes binding to the primary cultures and postoperative materials was performed from March 2024 to May 2024 on the basis of the Laboratory of Biomolecular and Medical Technologies of the Krasnoyarsk State Medical University named after Professor V. F. Voyno-Yasenetsky under the Ministry of Health of the Russian Federation.

Study object

The tumor tissues were obtained from NSCLC patients, who received radical surgeries in the A. I. Kryzhanovsky Krasnoyarsk Regional Clinical Oncology Dispensary. Before sampling, written informed consents to analyze the postoperative materials in the Research and Development Laboratory were obtained from all the patients. The tumor tissues were aseptically resected and placed into cooled DMEM with penicillin and streptomycin. The samples were delivered to the laboratory within two hours after resection for the further investigation and cultivation of cells.

The study used the LC-17 DNA-aptamer to the NSCLC tumor cells [9]. The aptamer’s specificity to the tissue cells, circulating tumors, and plasma proteins was pre-studied [10, 11].

Cell isolation and cultivation

The tumor tissues were washed in the DMEM. The tissues were minced, the vessels, necrotic foci, and thrombi were removed. The resulting tissues were dissociated using a dosage device. Then the suspension was filtered through a 70-µm filter and sedimented twice by centrifuging at 2000 rpm for 5 minutes. The sediment was resuspended in 2 mL of DPBS, then covered with 3 mL of lymphocytes separation media, and centrifuged for 10 minutes at 2000 rpm. The cell layer at the lymphocytes separation media/DPBS interface was collected and transferred into a sterile tube with DPBS, and then centrifuged for 5 minutes at 2000 rpm. The sediment was transferred into a cultivation tube or container with the medium (DMEM, 10% of plasma fetal calf serum, 20 μg/mL of insulin, 10 μg/mL of transferrin, 25 nmol/L of sodium selenite, 100 U/mL of antibiotics, and 1 ng/mL of epidermal growth factor) and cultivated in an atmosphere with 5% CO₂ at 37°C. When the cells reached confluence, they were dispersed with the Trypsin-Versene regenerant solution for subcultivation of cells. The cells were washed in a Ca²⁺ and Mg²⁺-containing phosphate buffer by centrifuging for 3 minutes at 2500 rpm and cultivated in the cultivation medium containing DMEM, 5% of plasma fetal calf serum, 20 μg/mL of insulin, 10 μg/mL of transferrin, 25 nmol/L of sodium selenite, and 100 U/mL of antibiotics.

Preparation of liposomes

To prepare the liposomes, a lipid mixture containing cholesterol, L-α-phosphatidylcholine, and stearylamine (kit for liposomes, Sigma-Aldrich, Germany) was used. The L-α-phosphatidylcholine/stearylamine/cholesterol molar ratio in the mixture was 63:18:9, respectively. The liposomes were prepared using the thin film hydration method with subsequent sonication. The lipids were initially dissolved in 5 mL of chloroform/methanol mixture (3:1). The solvent was evaporated in a rotary evaporator to obtain a dry film of lipids. Then the dry film of lipids was hydrated by adding 50 mL of DPBS (pH 7.4) at ambient temperature for 10 minutes. Following the hydration, the liposomes were ultrasonicated for 30 minutes at 25°C to ensure the correct mixing and formation of liposomes. The resulting liposomes were stored at 4°C for subsequent use.

Functionalization of liposomes

The liposomes were functionalized with the LC-17 aptamer, modified with cholesterol at the 3’-end, and methyl fluorescein f (FAM) at the 5’-end, at a final concentration of 0.5 µM. To incorporate the aptamer into the lipid layer of liposomes, the sample was incubated at 60°C for one hour. Once incubated, the mixture was immediately placed on ice for 5 minutes to restore the aptamer’s conformation.

Synthesis and characterization of fluorescent gold nanoclusters

All the synthesis glassware was pre-washed with aqua regia (HCl/HNO₃=٣:١), and then rinsed with ethanol and water. To synthesize the glutathione-based fluorescent AuNCs (GSH-AuNC), the following technique was used. 1.5 mL of 100 mM of reduced glutathione solution and 43.5 mL of H₂O were mixed in a glass vial and stirred. Then the resulting solution was spiked with 5 mL of 20 mM HAuCl₄. The resulting mixture was stirred for 2 minutes. After that the reaction mixture was incubated in a thermostat for 24 hours at 70°C without stirring. To synthesize the albumin-stabilized fluorescent gold nanoclusters (BSA-AuNCs), the following technique was used. 25 mL of 38.4-mg/mL BSA and 25 mL of 11.6-mM HAuCl₄ were mixed in a glass vial. The mixture was intensively stirred in a magnetic stirrer for 2 minutes. At the next stage, the reaction mixture was spiked with 2 mL of 1 M NaOH. Then the reaction mixture was heated at 90 °C for 45 minutes with intensive stirring.

The electron microscopy images of the synthesized nanoclusters were obtained using the transmission electron microscopy. The microscopic images were obtained using the Libra-120 (Carl Zeiss, Germany) microscope in the Simbioz Center for Collective Use of Research Equipment in Physicochemical Biology and Nanobiotechnology of the Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences (IBPPM RAS). The fluorescence excitation and emission spectra of the synthesized nanodyes were recorded using the Cary Eclipse (Agilent, USA) spectrofluorometer.

Introduction of nanodyes (GSH-AuNС and BSA-AuNС) into LC-17 aptamer-functionalized liposomes

The liposomes were incubated with nanoclusters in a shaker at 4°C for 12 hours. This allowed the fluorophores to enter into liposomes. After loading nanoclusters, the liposomes were transferred into a dialysis sac and put into a Ca²⁺- and Mg²⁺-containing phosphate buffer for 5 hours at 4°C to remove the excessive fluorophores that did not enter into liposomes. To destroy the existing aggregates and ensure the uniform distribution of liposomes, the suspension was placed in an ultrasonic bath for 30 minutes.

Analysis of efficiency of binding of tumor cells with nanoclusters-containing and aptamer-functionalized liposomes

The binding of the gold nanoclusters-containing liposomes with the lung cancer cells was analyzed using the FC-500 (Beckman Coulter Inc., USA) flow cytometer. The culture-derived lung cancer cells were pre-incubated with 1 ng/μL of yeast RNA for 30 minutes in a shaker at ambient temperature.

Then the cell mixture was incubated for 30 minutes at ambient temperature with the GSH-AuNС- and BSA-AuNС-containing liposomes bound to LC-17 aptamer. To avoid the non-specific binding of the lung cancer cells with liposomes, the mixture was washed by centrifuging at 3000 rpm for 5 minutes. The findings were analyzed using the Kaluza^® ١.١ (Beckman Coulter Inc., USA) software.

Postoperative tissue staining

To evaluate the efficiency of the fluorescent nanocomplexes, the Т3N0M0 adenocarcinomal tissue of the right lung middle lobe was used. The diagnosis was morphology-confirmed. The nanocomplexes were applied onto the lung tissue; to visualize the binding, an ultraviolet radiation source was used. Upon 3 minutes after staining, the tissue was three times washed with phosphate buffer; and the luminescence was evaluated.

Ethical review

The study was approved by the Ethics Committee of the Krasnoyarsk State Medical University (Confirmation No. 37/2012 of 01/31/2012).

Statistical analysis

The morphometric parameters of nanoclusters were obtained by calculating the ensemble of 200 nanoclusters in the electron microscopy images. The findings were presented as the arithmetic mean and its standard deviation (M±SD).

The findings were statistically processed using the Statistica ٧.٠ software. The hypothesis of the statistical significance of differences between samples was checked using the Mann–Whitney test.

RESULTS

Characteristic of synthesized nanodyes

Various methods are used to obtain gold nanostructures, including the traditional chemical techniques of condensation and the environment-friendly “green” approaches, when gold is regenerated using plant extracts [12, 13]. The AuNC synthesis involved using organic ligands (L-glutathione and bovine serum albumin), which acted simultaneously as regenerating and stabilizing agents. Figure 1, a, b shows the transmission electron microscopy images and the fluorescence spectra of the synthesized nanoclusters. The estimate of the sizes of at least 100 particles in the transmission electron microscopy images found that the respective BSA-AuNC and GSH-AuNC diameters were 1.8±0.5 and 1.5±0.3 nm. At the excitation with a 365-nm wavelength light, the maximal emissions of fluorescence were 655 nm for BSA-AuNC and 613 nm for GSH-AuNC. The calculated quantum efficiencies of fluorescence for BSA-AuNC and GSH-AuNC were 6 and 14% respectively.

 

Fig. 1. a — transmission electron microscopy image of the BSA-AuNCs (scale bar is 20 nm); b — fluorescence spectra of the BSA-AuNCs; c — BSA-AuNCs suspension under ultraviolet lamp irradiation (365 nm); d — transmission electron microscopy image of the GSH-AuNCs (scale bar is 20 nm); e — fluorescence spectra of the GSH-AuNCs; f — GSH-AuNCs suspension under ultraviolet lamp irradiation (365 nm).

 

Creation of liposomes with aptamer-based incapsulated dyes

In this study, the BSA-AuNC and GSH-AuNC gold nanoclusters were incapsulated into liposomes with LC-17 aptamer. This allowed ensuring the delivery of the nanoparticles to the lung cancer cells. This technique has several advantages compared with the direct binding of aptamers to nanoclusters via functional groups. First, this enhances the intensity of fluorescence, as each liposome can carry several fluorophore molecules. Second, the toxicity reduces, as the dyes are not in direct contact with the patients’ blood or tissues. Third, the complexes’ circulation time elongates, their stability improves, and their targeted delivery is ensured.

To visualize the aptamer-to-liposomes binding, the tertiary structure of the LC-17 aptamer was constructed (Figure 2, a). The complete aptamer–liposome–gold nanocluster complex is shown in Figure 2, b.

 

Fig. 2. The tertiary structure of the aptamer. a — the LC-17 aptamer model; b — a diagram depicting the LC17-liposome–gold nanoclaster.

 

Evaluation of efficiency of binding of nanodyes incapsulated in aptamer-functionalized liposomes with tumor cells and tissues ex vivo

The efficiency of the binding of the nanodyes incapsulated in aptamer-functionalized liposomes was evaluated by flow cytometry using FC-500 (Beckman Coulter, USA) cytometer. The BSA-AuNC and GSH-AuNC nanoclusters demonstrated fluorescence in the red spectral range, while the FAM-labeled aptamer showed it in the green spectral range. Hence, the fluorescent signal was detected in two channels: FL1 for FAM, and FL4 for BSA-AuNC and GSH-AuNC.

 

Fig. 3. Flow cytometry of lung cancer cells stained with liposomes containing GSH-AuNСs or BSA-AuNСs associated with the LC-17 aptamer.

 

To evaluate the efficiency of the nanoclusters’ binding with biological samples, the primary lung cancer cultures from postoperative material were obtained.

Figure 3 shows that the fluorescent signals from the lung cancer culture cells themselves were not detected; however, when stained with the complexes that contained the LC-17 aptamer and the GSH-AuNС and BSA-AuNС nanodyes, they showed fluorescence in the FL1 and FL4 channels.

To evaluate the binding of the resulting nanostructure with the lung cancer tissue, the ex vivo tumor was divided into several parts and stained with one of the liposomes/nanodyes complexes or phosphate buffer for control (Figure 4).

Figure 4 shows that no autofluorescence of tumor was observed. The GSH-AuNС-containing LC-17 aptamer-functionalized liposomes were bound to the tumor tissue; this was confirmed by the

 

Fig. 4. Ex vivо staining of lung cancer tissue: a — without staining; b — stained with GSH-AuNСs in liposomes containing LC-17 aptamer; c — stained with BSA-AuNСs in liposomes containing LC-17 aptamer.

 

visually red luminescence under ultraviolet light. The BSA-AuNС-containing LC-17 aptamer-functionalized liposomes stained the tumor tissue as well.

DISCUSSION

FGS has not, yet, become wide-spread when resecting the main and metastatic tumor foci. Nevertheless, there are studies that confirm the promising outlook of this method, such as using the methylene blue, indocyanine green, or molecule specific to the lung adenocarcinoma folate receptor alpha [15, 16].

Thus, for example, C.H. Li et al. used the AS1411 aptamer-functionalized GSH-AuNC with diatrizoic acid (AS1411-DA-AuNC) to stain tumors in animal models [17]. In the above study, the aptamer was directly conjugated with AuNC using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). However, this approach to conjugation has its limitations due to the rapid hydrolysis of EDC in aqueous medium and to the fact that many gold nanoclusters are subject to biodegradation and aggregation in biologic medium.

The novelty of the approach proposed in this study consists in the original design of the complex consolidating the following three components:

• lung cancer cells-specific aptamer
• liposomes
• albumin- or glutathione-based fluorescent nanoclusters characterized by large Stokes shifts and relatively high quantum efficiencies of fluorescence.

From an oncology and health economics perspective, using the fluorescent methods of tumor visualization —at the diagnostic stage and during the radical resection of tumor — will allow avoiding the early recurrences of disease and repeated surgeries, reducing the number of the adjuvant antitumor therapy courses by increasing the radicality of the surgical treatment. Such approach to treatment will result in substantial savings on the healthcare costs and better quality of patients’ lives. Noteworthy is that the greater diversity of the fluorescence inducers will extend the FGS range to other malignant neoplasms surgery areas and achieve more beneficial surgery outcomes for patients.

CONCLUSION

One of the most vital problems of surgery in lung cancer patients is visualization of not only the tumoral foci but also of the micrometastases as the malignant cells remaining in tissue continue proliferating, inducing recurrencies and disease progression. This study has investigated the possibility to use the BSA-AuNC and GSH-AuNC dyes-loaded and LC-17 aptamer-modified liposomes to identify tumoral foci during surgery. The possibility to visualize the tumor tissue during surgery was simulated with the postoperative material of a patient with lung adenocarcinoma using an ultraviolet radiation source. The potential possibility to integrate the BSA-AuNC and GSH-AuNC nanoclusters at the preoperative diagnostic stage to identify the metastatic foci with FGS was shown. Overall, the approach being developed has the potential to alter the treatment strategy for patients with NSCLC, considering both the criteria for radical surgery and alternative non-invasive treatment methods such as antitumoral and radiation therapies.

ADDITIONAL INFORMATION

Funding source. Research and publication were supported by the Ministry of Science and Higher Education of the Russian Federation FWES-2022-0005 (A.S.K.). The production and characterization of gold nanoclusters was carried out within the framework of the state assignment of the Ministry of Education and Science of the Russian Federation for the Federal Research Center “Saratov Scientific Center of the Russian Academy of Sciences”, topic No. 1022040700960-1.

Competing interests. The authors declare that they have no competing interests.

Authors’ contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work. Zamay GS, Zamay TN — conducting research, analyzing and interpreting data, writing the text and editing the article; Chumakov DS — conducting research, analyzing and interpreting data, editing the manuscript; Sidorov SA, Khlebtsov BN — conducting research, analyzing and interpreting data; Krat AV, Shchugoreva IA — conducting research, analyzing data; Koshmanova AA — conducting research; Zukov RA — manuscript editing; Khlebtsov NG — concept of the work; Kichkaylo AS — research, data analysis, article editing.

About the authors

Galina S. Zamay

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Federal Research Center “Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences”

Author for correspondence.
Email: galina.zamay@gmail.com
ORCID iD: 0000-0002-2567-6918
SPIN-code: 6501-0371

Cand. Sci. (Biology)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; 50, Akademgorodok st., Krasnoyarsk 660036

Tatiana N. Zamay

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Federal Research Center “Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences”

Email: tzamay@yandex.ru
ORCID iD: 0000-0002-7493-8742
SPIN-code: 8799-8497

Dr. Sci. (Biology)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; 50, Akademgorodok st., Krasnoyarsk 660036

Daniil S. Chumakov

Federal Research Centre “Saratov Scientific Centre of the Russian Academy of Sciences”

Email: laik2012@yandex.ru
ORCID iD: 0000-0003-1658-2562
SPIN-code: 8491-2313

Cand. Sci. (Biology)

Russian Federation, Saratov 410028

Semen A. Sidorov

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: sidorov.syoma2014@yandex.ru
ORCID iD: 0000-0001-6676-6656
SPIN-code: 8740-5259
Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; Krasnoyarsk 660133

Aleksey V. Krat

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: alexkrat@mail.ru
ORCID iD: 0009-0006-5357-2637
SPIN-code: 2846-8592

MD, Cand. Sci. (Medicine)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; Krasnoyarsk 660133

Boris N. Khlebtsov

Federal Research Centre “Saratov Scientific Centre of the Russian Academy of Sciences”; Saratov State University

Email: khlebtsov_b@ibppm.ru
ORCID iD: 0000-0003-3996-5750
SPIN-code: 5274-8718

Dr. Sci. (Physics and Mathematics)

Russian Federation, Saratov 410028; Saratov 410012

Irina A. Shchugoreva

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Federal Research Center “Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences”

Email: shchugorevai@mail.ru
ORCID iD: 0000-0003-4207-1627
SPIN-code: 8826-6672

Cand. Sci. (Chemistry)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; 50, Akademgorodok st., Krasnoyarsk 660036

Anastasia A. Koshmanova

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: koshmanova.1998@mail.ru
ORCID iD: 0000-0001-7339-8660
SPIN-code: 2217-2229
Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022

Ruslan A. Zukov

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Krasnoyarsk Regional Clinical Oncology Dispensary named after A.I. Kryzhanovsky

Email: zukov_rus@mail.ru
ORCID iD: 0000-0002-7210-3020
SPIN-code: 3632-8415

MD, Dr. Sci. (Medicine)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; Krasnoyarsk 660133

Nikolai G. Khlebtsov

Federal Research Centre “Saratov Scientific Centre of the Russian Academy of Sciences”; Saratov State University

Email: khlebtsov@ibppm.sgu.ru
ORCID iD: 0000-0002-2055-7784
SPIN-code: 4377-1257

Dr. Sci. (Physics and Mathematics)

Russian Federation, Saratov 410028; Saratov 410012

Anna S. Kichkailo

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University; Federal Research Center “Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Sciences”

Email: annazamay@yandex.ru
ORCID iD: 0000-0003-1054-4629
SPIN-code: 5387-9071

Dr. Sci. (Biology)

Russian Federation, 1 Partizan Zheleznyak street, Krasnoyarsk 660022; 50, Akademgorodok st., Krasnoyarsk 660036

References

Supplementary files