Use of lectins as vector molecules for delivery of drugs to cells and tissues . Report 2

V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi © 2019 V. O. Antonyuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited Bioorganic Chemistry ISSN 0233-7657 Biopolymers and Cell. 2019. Vol. 35. N 1. P 54–63 doi: http://dx.doi.org/10.7124/bc.000994


Introduction
Various thiopyrano [2,3-d]thiazole derivatives with the anticancer, antiviral, anti-inflammatory, antimicrobial and antitrypanosomal activities were synthezized at the Department of the Pharmaceutical, Organic and Bioorganic Chemistry of Danylo Halytsky Lviv National Medical University [1]. A pharmaceutical potential of these derivatives can be significantly increased through the development of water soluble forms that also creates the conditions for their selective binding to the biological targets. In previous study [2], we have conjugated pea seeds lectin in the alkaline medium (pH 9.0) with the thiopyrano [2,3-d]thiazole derivative through the interaction of the aldehyde group of this compound with the amino groups of amino acids of the lectins. The biological activity of obtained conjugates towards the mouse leukemia cells of L1210 line was evaluated. It was shown that the obtained conjugates had a 2.5-fold increased antineoplastic effect (calculated as the amount of substance conjugated to the lectin) compared to the nonconjugated compound. It was assumed that such enhancement is caused by a selective interaction of lectin with certain types of cells in tissues. Thus, a targeted delivery of the substance to potential sites of its action is possible. The application of lectin conjugates with drugs might also have some disadvantages. The main one is a protein nature of the lectin carrier which can cause allergic reactions, especially after its direct administration into the bloodstream. At the same time, such conjugates could be useful at external application into the body cavities (oral or nasal) or as rectal suppositories. The advantage of lectins as drug carriers compared to the synthetic polymers is lectins biodegradability. There are numerous literature data describing commercially available lectins selectively binding to normal or pathological tissues. It has been established that peanut lectin (PNA) selectively binds the T-antigen present in 71 % of cancer cells and not detected by the monoclonal antibodies in healthy donors [3,4]. Our investigation on binding capacity of the PNA-E receptors with colon cancer cells revealed that PNA-E interacted with the adenoma cells and with adenocarcinomas of high degree of differentiation. This lectin possesses a higher selectivity to the adenoma cells compared to the tumor cells with a low degree of differentiation. Thus, the lectin binding can be used as a selective histochemical marker of these pathologies [5], as well as for the targeted delivery of drugs to the specific colon cells. Therefore, the next step of our work was to investigate the action of such conjugates on the cells of other lines, in particular, the T-lymphoblastic leukemia Jurkat cells, HCT 116 line of human colorectal carcinoma cells, and HEK 293 pseudonormal cells of human embryonic kidney. In this study, thiopyrano [2,3d]thiazole derivative (Les-1895) [6] was used as an adduct attached to the lectins.

Materials and Methods
In Fig. 1, one can see a schematic reaction of the thiopyrano[2,3-d]thiazole derivative (Les-1895) conjugation with lectins isolated from the pea seeds (PSA, Pisum sativum agglutinin), peanut seeds (PNA, peanut agglutinin), and erythroagglutinin of the common bean (PHA-E). In control, Les-1895 was conjugated to human serum albumin (HSA). These conjugations were accomplished through the aldehyde group of Les-1895 interacting with the amino groups of amino acids of lectins under the alkaline conditions (pH 9.0).
Conjugation. 10 mg of lectin or HSA were dissolved in 1.0 ml of 1 % aqueous solution of sodium bicarbonate with addition of 0.025-0.2 ml of 1 % solution of Les-1895 in DMSO. The obtained solution was stirred and kept for 3 h at room temperature. After stirring the turbid solution was gradually highlighted, 5 mg of sodium borohydride were added to the solution and the obtained mixture was kept for 12 h. To separate the unreacted Les-1895 from its conjugate, the mixture was dialyzed against 50 ml of 20 % aqueous solution of DMSO, and then against 50 ml of phosphate buffered saline (PBS).
Investigation of the obtained conjugate. The obtained conjugate was characterized for its lectin activity measured as an ability to bind specific carbohydrates and glycoproteins of the plasma membrane. The content of protein and Les-1895 attached to the lectin was also calculated. The lectin's activity before and after conjugation was measured by determining the agglutination titer of 2 % rabbit erythrocyte suspension in PBS. The concentration of Les-1895 in the conjugate was determined by measuring the absorbance at 361 nm wavelength, where this compound has a characteristic maximum of absorption (Fig. 2). The content of total protein was measured using Lowry method with an absorbance at 740 nm. Radiobiology, National Academy of Sciences of Ukraine (Kyiv). The HEK 293 cells were cultured in the Dulbecco's -modified Eagle's medium (DMEM, Sigma, USA) in the presence of 10 % of the decomposed blood serum of cattle embryos, using phenol red as an indicator of pH, and 50 μg/ml of gentamicin antibiotic (Sigma, USA). The cells of Jurkat and HCT 116 lines were cultured in the RPMI-1640 (Thermo scientific, USA) medium in the presence of 10 % decomposed blood serum of cattle embryos, phenol red, and 50 μg/ml of gentamicin (Sigma, USA). To evaluate cytotoxicity of the lectins (PSA, PNA, PHA-E), Les-1895, and the conjugates with these lectins, the appropriate samples were added in different concentrations to the medium in which the cells are cultured. After 24 h, the number of cells was counted in the hemocytometric chamber using the Trypan blue dye (DV-T10282, "Invitrogen", USA) that penetrates through the plasma membrane of the damaged (dead) cells. The cells colored in blue were considered as the necrotic ones. The number of cells in suspension was calculated according to the formula: с = 12500n, where: c -the number of cells in 1 ml of suspension, n -the average number of cells in 5 large squares of the hemocytometer chamber. The evaluation of cytotoxicity and antiproliferative activity of the tested compound was carried out after 72 h of incubation using the MTT assay. The cell viability was evaluated by measuring a reduction of the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, USA) to water-insoluble formazan which has a purple color.
Statistical analysis. Each experiment was performed in triplicate and average values were recorded. The data were evaluated statistically using Student's t-test, and a value of p ≤ 0.05 was considered to be statistically reliable.

Results and Discussion
Les-1895 is soluble only in organic solvents such as dimethyl sulfoxide or dimethylformamide. Here we demonstrated that the con-jugation of this compound with the lectin (see Materials and Methods section) led to creation of the water soluble product.
Comparing to the previously reported thiopyrano[2,3-d]thiazole derivative, Les-1351 [2], an increase of the amount of Les-1895 in the reaction mixture up to 1 mg per 10 mg of pea lectin (10 %) did not lead to the lectin inactivation. Noteworthy, Les-1895 present in the reaction mixture was not completely immobilized on lectin. According to our calculations, 10 mg of lectin bound 0.9 mg of Les-1895 (9 % of total weight of the conjugate). Similar results were obtained for PNA and PHA-E, where 10 mg of PNA bound maximum Unlike the murine leukemia L1210 cells [2] that were insensitive to the pea lectin (PSA), the T-lymphoblastic leukemia Jurkat cells were subjected to a cytotoxic action of the PHA-E and PNA lectins. IC 50 for PNA and PSA was 80 and 37 μg/ml, respectively, and for PHA-E -20 μg/ml. IC 50 for Les-1895 was 32 μg/ml. According to these data, PHA-E was more effective in targeting Jurkat cells than Les-1895. The conjugates of the lectins with this compound displayed a cytotoxic effect towards the T-lymphoblastic leukemia Jurkat cells -23 % inhibition for PSA-Les-1895, 34 % -for PNA-Les-1895, and 12 % -for PHA-E-Les-1895. IC 50 of the conjugates of lectins with Les-1895 was 10 μg/ml, whereas IC 50 of free Les-1895 compound was 30 μg/ml. Though the conjugation of Les-1895 to HSA increased a water solubility of Les 1895, it at the same time reduced by 40 % a cytotoxic effect of the obtained conjugate towards the T-lymphoblastic leukemia Jurkat cells (Fig. 3). Noteworthy, PNA and PHA-E significantly suppressed the viability of human colorectal carcinoma HCT116 cells comparing to the effects of intact HSA and pea lectin. The cytotoxic action of the Les-1895 conjugates with the PNA and PHA-E lectins was more potent than the action of free Les-1895 and nonconjugated lectins. IC 50 of the PSA-Les-1895 conjugate was 28 μg/ml, PNA-Les-1895 -4 μg/ml, and PHA-E-Les-1895 -3 μg/ml. IC 50 of Les-1895 was approximately 10 μg/ml, whereas the cytotoxic effect of the HSA-E-Les-1895 and the PSA-Les-1895 conjugates was weaker than that of free Les-1895 (Fig. 4).
Pseudonormal human embryonic kidney HEK 293 cells were insensitive to the action of PSA, PNA and PHA-E lectins, and at 30 mg/ml dose of these lectins there were ≈20 % of dead cells. The conjugation of Les-1895 with these lectins did not significantly enhance their cytotoxic effect as compared to the action of the Les-1895 free form (Fig. 5).
Taking into account the obtained results, one can suggest a positive correlation between the level of lectin binding with cells and a cytotoxic action of the Les-1895 conjugates with these lectins. This suggestion is in agreement with our preliminary results on the binding of specific lectins with histological specimens of the human adenocarcinoma colon cells compared to the cells of normal colon tissue [5].
Thus, the conjugation of Les-1895 with the pea and peanut lectins increased the antineoplastic activity of the resulting conjugates in the T-lymphoblastic leukemia Jurkat cells and human adenocarcinoma HCT 116 cells. At the same time, the pseudonormal HEK 293 line cells were insensitive to the action of tested lectins and their conjugates with Les-1895.
The human adenocarcinoma HCT 116 cells were 2-fold more sensitive to the action of studied conjugates of lectins and tested compound. According to the literary data [7][8][9], an increased toxicity of conjugates towards tumor cells could be caused by a selective binding of specific lectins to the carbohydrate-containing receptors of the plasma membrane of cells. The HSA-Les-1895 conjugate was not toxic for the T-lymphoblastic leukemia Jurkat cells and only slightly toxic for the human adenocarcinoma HCT 116 cells, comparing to a high toxicity of the conjugates of Les-1895 with the lectins. A cytotoxic action of conjugates Les-1895 with lectins towards the pseudonormal cells HEK 293 line was manifested after 72 h only at high doses of the conjugates. Thus, the action of the conjugates was more pronounced for the adenocarcinoma HCT 116 cells than for the pseudonormal cells HEK 293 line (Fig. 6).
We had no the pseudonormal colon cells in our collection, thus, we used the human embryonic kidney cells that possess a similar structure of the glycoconjugates on their surface [10,11]. Comparing to tumor cells, these cells have a significantly less amount of terminal sialic acids in the plasma membrane [12].
Lectins could interact specifically with the individual cell surface glycoconjugates creating an increased local concentration of bound substances. As noted in the Introduction section, using lectins as medicines has certain limitations. They can be used only externally at treatment of patients with diseases of skin, digestive tract, nasopharynx, lungs, or in the rinses and sprays, suppositoria and ointment bases. Lectin-containing medicines cannot be recommended for administration into the bloodstream because of possible allergic reactions. However, their inclusion in the ointment or utilizing in suppositories may be helpful for the treatment of colorectal cancer. formed conjugates in water, however, such conjugation does not guarantee an enhacement of the biological activity of the attached ligands.
2. Native lectins exhibit more pronounced cytotoxic action on specific tumor cells, than on the pseudonormal cells, and a degree of such action depends on the carbohydrate specificity. Conjugation of lectins with a low molecular weight ligand posessing antitumor activity leads to an increase in the cytotoxicity of the resulting conjugates.
3. The cytotoxic effect of Les-1895 conjugates with lectin, which selectively binds to the HCT 116 human intestinal cancers, was dose-dependent. At the same time the effect of the developed conjugates of lectins towards the pseudonormal human embryonic kidney HEK 293 cells was more pronounced after 24 h, although it was manifested only at high doses of the conjugates.