The pW8-3C construct encoding the artificial tumor-targeting protein W8-3C with the addition of three residues of free Cys at the C terminus has been created and described for the first time. Using purified W8-3C protein, dispersions of nanoparticles 75.24 nm in diameter at a polydispersity index (Pdi) of 0.064 and Se content of 1.566 μg/mL were obtained and characterized for the first time. The dispersions remained stable during storage for 6 months at +4°C. For comparison, the maximum Se content in the nanoparticle dispersion obtained in the presence of W8-3C protein and Pluronic F-127 was 399 μg/mL. The cytotoxic activity of the obtained nanoparticles was studied on HeLa (cervical carcinoma), U87MG (glioma), MCF7 (breast carcinoma), and HCT116 (colon carcinoma) human tumor cell lines and compared with that on the diploid human fibroblast line WI-38 in vitro. It was shown that the IC50 of Se nanoparticles obtained using the W8-3C protein for tumor lines ranged from 5.25 to 8.37 μg/mL, while the IC50 for normal fibroblasts was 14.3 μg/mL (difference in values by a factor of 1.7–2.7 times).

99mTc is a well-known radionuclide that is widely used and readily available for SPECT/CT (Single-Photon Emission Computed Tomography) diagnosis. However, commercial isotope carriers are not specific enough to tumours, rapidly clear from the bloodstream, and are not safe. To overcome these limitations, we suggest immunologically compatible recombinant proteins containing a combination of metal binding sites as 99mTc chelators and several different tumour-specific ligands for early detection of tumours. E1b protein containing metal-binding centres and tumour-specific ligands targeting integrin αvβ3 and nucleolin, as well as a short Cys-rich sequence, was artificially constructed. It was produced in E. coli, purified by metal-chelate chromatography, and used to obtain a complex with 99mTc. This was administered intravenously to healthy Balb/C mice at an activity dose of about 80 MBq per mouse, and the biodistribution was studied by SPECT/CT for 24 h. Free sodium 99mTc-pertechnetate at the same dose was used as a reference. The selectivity of 99mTc-E1b and the kinetics of isotope retention in tumours were then investigated in experiments in C57Bl/6 and Balb/C mice with subcutaneously transplanted lung carcinoma (LLC) or mammary adenocarcinoma (Ca755, EMT6, or 4T1). The radionuclide distribution ratio in tumour and adjacent normal tissue (T/N) steadily increased over 24 h, reaching 15.7 ± 4.2 for EMT6, 16.5 ± 3.8 for Ca755, 6.7 ± 4.2 for LLC, and 7.5 ± 3.1 for 4T1.

Gold nanoparticles (GNPs) are actively used as dose enhancing agents in combination with X-ray irradiation. The synthesis of gold nanoparticles by laser ablation has a number of advantages over other methods (for example, the possibility of preparing a chemically pure solution and a relatively low production cost when synthesis is scaled up). An additional antitumor effect from the combined use of gold nanoparticles synthesized by laser ablation and X-rays was studied. Mice transplanted subcutaneously with Ca755 syngeneic adenocarcinoma were used for the study. The tumor was irradiated at a dose of 10 Gy in 30 min after injection of GNPs. As a result, 66% of complete regressions were found in the experimental group of mice within 210 days after transplantation, while there were no regressions of tumor nodes in the control irradiated group. The absorbed dose to the vascular walls with gold nanoparticles in the bloodstream was evaluated as 26.8 Gy owing to local energy release.

The ability of gold polyacrylate (aurumacryl) to increase the sensitivity of murine tumor (В-16/F10 melanoma) to radiation treatment has been explored. Radiation therapy employed in combination with aurumacryl leads to a moderate but significant inhibition of tumor growth by 81–90

Three artificial proteins that bind the gadolinium ion (Gd3+) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd3+-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd3+-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvβ3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.

Рассматривается возможность использования спектрофотометрического метода для оценки размера и концентрации золотых наночастиц, полученных методом лазерной абляции. Золотые наночастицы, синтезированные разными методами, имеют различные физико-химические свойства своей поверхности. Это может влиять на их оптические свойства в коллоидном растворе. В работе получены результаты, подтверждающие возможность использования спектрофотометрического метода для оценки размера и концентрации наночастиц, полученных методом лазерной абляции. Показано, что оценку концентрации наночастиц спектрофотометрическим методом оптимально проводить на длине волны 400 нм. Определение размера наночастиц по спектрам поглощения в ультрафиолетовой и видимой области возможно для частиц размером не менее 11 нм.

Abstract —The ability to use a spectrophotometric method to estimate the size and concentration of gold nanoparticles obtained by the method of laser ablation was considered. Gold nanoparticles synthesized by different methods have different physical and chemical properties of their surface. This can affect their optical properties in a colloidal solution. The results obtained in this work confirm the ability to use the spectrophotometric method to estimate the size and concentration of nanoparticles obtained by the method of laser ablation. It was demonstrated that it is optimal to estimate the concentration of nanoparticles using the spectrophotometric method at a wavelength of 400 nm. The determination of the size of nanoparticles by the absorption spectra in the ultraviolet and visible regions is possible for particles with a size of at least 11 nm.

Previously, a method of synthesis of trimethyl-substituted furodihydroquinoline (FDHQ), a putative photosensitizer for the therapy of psoriasis, was disclosed in patent RU #2614248. FDHQ was proposed for substituting derivatives of the psoralen (8-methoxypsoralen, 8‑MOP; 5-methoxypsoralen, 5‑MOP; and trimethylpsoralen, TMP) actually applied for this purpose. FDHQ exhibited an advantage over psoralens in safety for the patient. However, practical use of FDHQ was found to be impossible due to its insufficient solubility in any clinically permitted solvents. To solve this problem, a synthesis of six earlier unknown derivatives of FDHQ with side substitutes in position 5 of the benzene nucleus (carbamate, acetamide, and sulfuric moieties) were synthesized: N-(6,8,8-trimethyl-8,9-dihydrofuro[3,2-h]quinoline-5-yl)acetamide (W); tert-butyl (6,8,8-trimethyl-8,9-dihydrofuro[3,2-h]quinoline-5-yl)carbamate (K); tret-butylacetyl (6,8,8-trimethyl-8,9-dihydrofuro[3,2-h]quinoline-5-yl)-carbamate (WK); N-methyl-N-(6,8,8-trimethyl-8,9-dihydrofuro[3,2-h]quinoline-5-yl)acetamide (WС1); N-octyl-N-(6,8,8-trimethyl-8,9-dihydrofuro[3,2-h]quinoline-5-yl)acetamide (WС8); sodium acetyl (6,8,8-trimethyl dihydrofuro[3,2-h]quinoline-5-yl)sulfamate (S). In vitro testing on T-cell lympholeukosis (Jurkat), В-cell lymphoma (Raji) cell lines and immortalized human fibroblasts demonstrated optimal photosensitizing properties in compound S. The ratio between dark toxicity and phototoxicity of this compound (irradiation with UV light with λ = 302 nm) on the chosen model cell lines suggests it as a putative efficient and safe medicine for phototherapy of psoriasis.

At present, there is a need for a simple, noninvasive, highly specific and sensitive diagnostic test for hepatobiliary system disorders. Compounds labeled with carbon isotopes are widely used in various diagnostic breath tests; they are safe and can reliably detect a metabolic disorder or enzyme deficiency. The aim of this study was to synthesize 13С- and 14С-labeled linoleic acids suitable for use in hepatobiliary breath tests in terms of purity. In the synthesis of 13С-labeled linoleic acid, the chemical yield for 1-bromo-8,11-heptadecadien was 86.4% and the chemical yield for barium carbonate-13С, 96.0%. In the synthesis of 14С-labeled linoleic acid, the chemical yield for 1-bromo-8,11-heptadecadien was 87.39%; for barium carbonate-14С it was 97.1%. The specific radioactivity of 14С-labeled linoleic acids was 45.36 ± 0.02 mCi/g. The radiochemical yield of the reaction was 96.0%. The proposed method is suitable for batch production.

В настоящее время для диагностики заболеваний печени и билиарной системы требуется разработка простого неинвазивного теста с высокой чувствительностью и специфичностью. Соединения, меченные изотопом углерода, уже имеют широкое применение в диагностике различных заболеваний методами дыхательных тестов, безопасны и способны достоверно выявлять метаболические нарушения или дефицит специфичных ферментов в органах. Целью работы было получить линолевую кислоту, меченную 13С и 14С, по степени очистки пригодную для проведения дыхательных тестов в целях диагностики заболеваний гепатобилиарной системы. В предложенном способе химический выход реакции синтеза 13С-линолевой кислоты по 1-бром-8,11-гептадекадиену составил 86,4%, по 13С-карбонату бария — 96,0%. Химический выход реакции синтеза 14С-линолевой кислоты по 1-бром-8,11-гептадекадиену составил 87,39%, по 14С-карбонату бария — 97,1%. Удельная радиоактивность 14С-линолевой кислоты составила 45,36 ± 0,02 мКи/г. Радиохимический выход реакции — 96,0%. Способ удобен для серийного выпуска готового продукта.

Неинвазивные дыхательные тесты с применением изотопно-меченых соединений представляют собой новый высокоточный и безопасный метод функционального исследования печени и билиарной системы. Целью работы было провести биологические испытания острой и субхронической токсичности 13С-меченых линолевой и линоленовой кислот, синтезированных по оригинальной методике и предназначенных для проведения диагностических дыхательных тестов. При однократном внутрижелудочном введении изучаемых соединений лабораторным мышам линии BALB/c и крысам Wistar в дозах, превышающих диагностические в 500–2500 раз, образцы соединений не вызывали смертности экспериментальных животных. При проведении субхронического эксперимента на крысах при дозировках испытываемых соединений, в 5 и 25 раз превышающих терапевтическую дозу для человека, в течение 14 суток было выявлено отсутствие достоверных изменений у животных в экспериментальных группах по сравнению с контрольной по массе тела, гематологическим показателям (содержанию эритроцитов, лейкоцитов и тромбоцитов в крови) и биохимическим показателям сыворотки крови (уровню гемоглобина, общего белка, щелочной фосфатазы, аланинаминотрансферазы, аспартатаминотрансферазы, лактатдегидрогеназы, билирубина). Исследованные меченые кислоты безвредны в дозах, планируемых для перорального введения, и могут быть рекомендованы к доклиническим и клиническим испытаниям.

Noninvasive stable isotope breath tests allow highly accurate and safe estimation of liver and biliary tract function. The aim of this study was to test 13С-labeled linoleic and linolenic acids intended for diagnostic use for acute and subchronic toxicity. The acids were synthesized using the patented method. A single intragastric administration of the tested compounds to experimental BALB/c mice and Wistar rats in the amounts exceeding clinical doses 500 to 2500-fold did not cause animal death. In the subchronic toxicity test, the rats received 5 to 25 times higher doses than recommended for clinical use in humans. In a 14-day follow-up period, no significant differences were observed between the main and the control groups in terms of weight, blood count (red blood cells, white blood cells, platelets), and blood biochemistry (hemoglobin, total protein, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, bilirubin). The studied compounds are safe at doses intended for oral administration and are recommended for further preclinical and clinical trials.

Radionuclide therapy (RNT) is an effective modality for treating multiple metastases in patients with cancer. The list of malignancies that can be managed with RNT expands with the arrival of novel tumoritropic radiopharmaceuticals (RP). A versatile delivery platform capable of carrying various therapeutic and diagnostic radionuclides, as well as vector molecules needed to achieve sufficient specificity to tumor cells and ensure therapeutic efficacy may hold great promise for radiation therapy. The aim of this work was to assess the performance of a delivery system based on the original dendrimer. The dendrimer demonstrated low toxicity in mice (LD50 was 779 ± 111 mg/kg). To study the specificity of the dendrimer to tumor cells and its therapeutic efficacy, we used a nanostructure (NS) composed of the dendrimer itself, the RGD peptide and 188Re (188Re-NS). Lewis lung carcinoma LLC1 was used as a tumor model. The biodistribution analysis revealed that the compound effectively accumulated in the tumor demonstrating a tumor-to-normal ratio >1 (relative to healthy organs and tissues) and retention time of at least 6 hours. Injections of 185 MBq/kg 188Re-NS caused a statistically significant inhibition of tumor growth (p < 0.05) by day 7 following the injection (Т/С = 5%), which remained stable for 6 days. Our findings suggest that the proposed dendrimer is a promising platform for RP delivery.

BACKGROUND:Previously we demonstrated a high prevalence of hepatitis E virus (HEV) in domestic pigs and wild boars, the main reservoir and possible source of HEV infections in humans. But so far there are no reports about spread of HEV in Estonian human population.OBJECTIVES:The present study aimed to determine the prevalence and genotyping of HEV in different groups of the Estonian adult population.STUDY DESIGN:Totally 1426 human serum samples were tested (763 patients with clinically diagnosed nonA/B/C hepatitis, 176 hemodialysis patients, 282 patients with suspected HEV infection and 205 people who injected drugs (PWID)). Presence of anti-HEVantibodies was assessed by ELISA and confirmed by immunoblotting. All anti-HEV positive sera were analyzed for RNA by qPCR. Amplified ORF2 region was sequenced and used for phylogenetic analysis.RESULTS:Antibody assay revealed 49 samples from 1426 (3.4%) with acute (17) or past (32) HEV infection. HEV RNA was detected in 10 anti-HEV IgM positive samples, including 9 samples from patients with suspected HEV infection and 1 hemodialysis patient. Anti-HEV IgG were found in 7.8% patients with suspected HEV infection, in 4% hemodialysis patients, in 2.4% PWID and in 1.96% patients with nonA/B/C hepatitis. All groups demonstrated a trend to share of anti-HEV seroprevalence increasing with age. Phylogenetic analysis of 9 HEV RNA sequences revealed that 3 sequences belonged to HEV genotype 1; 6 ones to genotype 3 (1 sequence belonged to sub-genotype 3a, two ones - sub-genotype 3e, and three ones - to sub-genotype 3f).CONCLUSIONS:Despite the high seroprevalence among domestic pigs, no evidence of HEV transmission from Estonian pigs to humans was found. The results of our study suggest that HEV infections in Estonia are most likely associated with travel or with consumption of imported food products.

Peptide immunocortin sequence corresponds to the amino acid residues 11–20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. Since immunocortin was shown previously to inhibit phagocytosis in peritoneal macrophages and ConA-induced T-lymphocytes proliferation in culture, we suggested that immunocortin administering may be of use for patients with self-immune syndrome. Immunocortin in concentration 10 μM inhibited proliferation of both antigen (myelin)-induced and ConA-induced LN lymphocytes isolated from the lymph nodes of Dark Agouti (DA) rats immunized with chorda shear. The biological trials of the synthetic immunocortin were carried out on the DA rats with induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. These in vivo experiments have shown that intraperitoneal injections of immunocortin in a daily dosage 100 μg per animal reduced symptoms of EAE in DA rats.

Immunosuppressant peptide immunocortin for the first time was described in 1993. It corresponds to residues 11–20 of human Ig heavy chain (conserved motif of V H domain). There are no data about production of immunocortin by proteolysis of Ig in vivo. Synthetic immunocortin in concentration ~ 10 −9 M suppresses phagocytosis in peritoneal macrophages, ConA-dependent blast transformation of rat lymphocytes, exhibits ACTH-like neurotropic activity and was suggested as a potential drug for treatment of a multiple sclerosis (MS). Here, we report a sequence and method of synthesis of Abu-TGIRIS-Abu-NH 2 (Abu, alpha-aminobutyric acid), an artificial analogue of immunocortin. Biological trials of peritoneally injected Abu-TGIRIS-Abu-NH 2 gave an evidence of its better efficacy versus immunocortin in a test for suppression of the experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats.

Аурумакрил проявляет гемостатическую активность и обладает онколитическим и онкостатическим действием в отношении перевиваемых карцином in vivo. Образцы аурумакрила, полученные по известной методике, недостаточно стандартизированы по растворимости, что не позволяет изучить механизмы реакции и цитостатического действия. Целью работы было исследовать влияние на свойства аурумакрила следующих параметров проведения реакции его синтеза: молекулярная масса полиакрилатного полимера (в диапазоне 2–1000 кДа), наличие в качестве противоиона H+ или Na+, соотношение иона тетрахлораурата и звена полиакрилатного полимера (1 : 5 или 1 : 10), общая концентрация полиакрилатного полимера в момент синтеза (0,1 или 3%), а также влияние лиофильного высушивания аурумакрила. Сопоставление цитотоксической активности полученных образцов аурумакрила со значительно различающимся соотношением ионов золота и полиакрилатного полимера в отношении клеток карциномы молочной железы человека MCF-7 позволило установить, что удельное содержание в препарате полиакрилатного полимера и его молекулярная масса практически не сказываются на биологических свойствах конечного продукта. Методом УФ-спектроскопии показано влияние концентрации ионов Au (III) в аурумакриле на их цитотоксическую активность.

Aurumacryl is an incomplete metal salt of poly(acrylic acid) that exhibits hemostatic activity and inhibits the growth of transplantable carcinomas in vivo. The samples of aurumacryl synthesized following the original technique are insufficiently soluble, which complicates the study of the mechanisms involved in their synthesis and underlying their cytotoxic effect. The aim of this work was to study the impact of the following factors on aurumacryl properties: the molecular weight of the polyacrylate polymer in a range between 2 and 1,000 kDa, the presence of a counterion H+ or Na+, the molar ratio of AuCl– to the polyacrylate polymer (1 : 5 and 1 : 10), the total concentration of the polyacrylate polymer during synthesis (0.1 and 3%), and the type of drying (lyophilization). By comparing the cytotoxicity of aurumacryl samples with significantly different molar ratio of gold ions to the polyacrylate polymer against human breast carcinoma cells (MCF-7), we established that the proportion of the polymer and its molecular weight in the sample do not affect the biological properties of the synthesized substance. Using UV spectroscopy, we revealed that the concentration of Au (III) ions in aurumacryl determines its cytotoxicity.