Volum 18, Issue 5 (2026)

Introduction

Acridines (in particular, the derivative amsacrine, which is used to treat acute leukemia) are heterocycles consisting of two rings connected to a central pyridine ring. The planar structure of the compounds in this class enables them to interfere with various metabolic processes, including intercalation between DNA base pairs and inhibition of topoisomerases, telomerase, and protein kinases [1]. This accounts for their effectiveness as potential antitumor agents. The 1,2,3-triazole ring has attracted researchers’ attention due to its high dipole moment, metabolic stability, and ability to form hydrogen bonds [2].

Studying the structure–activity relationship of aza-acridine derivatives and tetracyclic acridones have shown that the optimal antitumor effect is observed when side chains contain two or three methylene units, whereas the structural modifications that reduce the ability of the amino group to form hydrogen bonds substantially diminish antitumor efficacy. Introducing a carbonyl group between the amino group of the side chain and the chromophore increases the flexibility and geometrical regularity of the target molecules, while the addition of a fused pyrazole group further enhances the antitumor effect of the compounds [3, 4].

For example, the imidazoacridinone derivative C-1311 demonstrated antitumor activity both on in vitro experimental models of murine and human colorectal cancer [5] and in a phase II clinical trial involving women with metastatic breast cancer [6, 7].

The high potential activity of C-1311 against bladder cancer was initially predicted using the COXEN bioinformatic approach. The genetic analysis stated C-1311 and paclitaxel to affect cellular processes in yeast through different, nonoverlapping mechanisms, suggesting that they may act synergistically when used in combination. In a bladder cancer model, the combination of C-1311 and paclitaxel achieved the maximal suppression of tumor cell growth at lower drug doses than those required for monotherapy with either agent [8].

The studies of the biochemical mechanisms of C-1311 have shown that the compound intercalates into DNA, inhibits topoisomerase II activity, reduces tumor angiogenesis by suppressing the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway [9], and inhibits FMS-like tyrosine kinase 3 with internal tandem duplications (FLT3-ITD) [10].

The antitumor potential of spiroacridine compounds is associated with the antiangiogenic activity of 9-aminoacridines and the enhancement of Th1-type responses, primarily through the increased levels of TNF-α and IL-1β in the tumor microenvironment [11]. Acridine-8-one derivatives inhibit microtubule assembly. The molecular modeling analysis of the synthesized acridine-8-one derivatives at the colchicine-binding site showed the acridone scaffold to have high structural complementarity with the hydrophobic pocket of tubulin forming stable bonds with the key amino acid residues Cysβ241, Leuβ248, Alaβ250, Leuβ255, and Asnβ258. Due to the resulting steric hindrance, the acridine-8-one molecule prevents tubulin from adopting the correct conformation, thereby disrupting microtubule polymerization. The resulting blockade of microtubule function leads to impaired mitotic spindle formation, cell-cycle arrest in the G2/M phase, and apoptosis induction [12].

The studies of the expression of ATP transporter genes and proteins including molecular docking analyses, in human colorectal cancer cells COLO 205 and COLO 320 treated with the acridine-based synthetic chalcone 1C showed that the compound 1C selectively suppresses cancer cell growth and significantly reduces ABCB1 protein levels by acting as an allosteric modulator of ABCB1 [13].

The studies of the ability of a series of 3,9-disubstituted acridines to inhibit topoisomerases revealed that the inhibitory effect of these derivatives decreased as the length of the linker chain between the acridine and benzene rings increased. At the same time, the presence of a substituent at the 4^th position of the benzene ring (whether a methyl or fluorine-containing group) was found to have no significant effect on the activity of these structures compared with the compound lacking a substituent on the benzene ring [14].

In recent years, numerous structurally diverse hybrid molecules have been constructed at the 9^th position of the acridine scaffold to enhance antitumor activity. The examples of such compounds include acridine conjugates with mycophenolic acid, thiazolidinedione, or chlormethine. A number of acridine–triazole and acridine–thiadiazole derivatives demonstrated high cytotoxic activity (compared with 5-fluorouracil and cisplatin) against human MGC-803 gastric cancer cells, BEL-7404 hepatocellular carcinoma cells, NCI-H460 large-cell lung cancer cells, and T24 bladder cancer cells. The high cytotoxicity of these hybrid compounds is due to their ability to effectively inhibit topoisomerase I at micromolar concentrations (the most potent derivative had an IC₅₀=4.21 μM) [15].

The study [16] investigated the effect of chemical structure on the biological activity of the compounds based on thiazolidine-2,4-dione, an acridine moiety, and an acetamide linker. The compounds containing such substituents as a 3,4,5-trimethoxy group, a 4-nitro group, and a 2-trifluoromethyl group, as well as an acridin-9-yl moiety, exhibited lower IC₅₀ values, indicating the higher activity. Some compounds demonstrated exceptional selectivity toward the HeLa cell line and high activity against the HCT116 cancer cell line, whereas the others were effective against pancreatic PATU cells.

The findings of the studies described above indicate the enormous potential of acridines and their derivatives as antitumor agents. Consequently, these compounds are being synthesized and investigated by scientists both internationally and in Russia.

For example, a research group in Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences led by Professor Yu.V. Shklyaev obtained several potential pyrrolo[3,2-l]acridinones, including Mob-155 S (Figure 1), which served as the basis for the development of a liposomal drug formulation.

Figure 1. Structural formula of the substance under code Mob-155 S

The aim of the study was to develop the liposomes based on pyrrolo[3,2-l]acridinone derivative Mob-155 S and study their antitumor activity regarding leukosis.

Materials and Methods

Liposome synthesis. The following reagents were used to prepare the liposomes: egg phosphatidylcholine E PC S (96%) and MPEG-2000-DSPE (both manu­factured by Lipoid, Germany), and cholesterol with purity of ≥99% (Sigma-Aldrich, Japan). The solvents used were 96% ethanol (Konstanta-Pharm M LLC, Russia) and chloroform (KhimMed, Russia); lactose, mannose, and sucrose (KhimMed, Russia) were used as cryoprotectants. The liposomes were prepared using a conventional procedure [17] in two stages: stage 1 involved the evaporation of the organic solvents and washing of the resulting film with water; and stage 2 involved the extrusion [18] of the liposomal dispersion by sequential passage through nylon filters with different pore diameters (1.2, 0.45, and 0.22 µm; Pall Eurasia LLC, Russia).

Five-milliliter samples were dispensed into 20-ml flasks and lyophilized using an Edwards Minifast DO.2 laboratory freeze-dryer (Ero Electronic S.p.A., Italy).

Liposome characteristics. The mean liposome size and polydispersity index (PDI) in the samples were determined by dynamic light scattering [19] using a He–Ne laser (wavelength, 633 nm) at a scattering angle of 173°; the zeta potential was determined by the electrophoretic method. The measurements were performed using a Zetasizer Nano-ZS 3600 analyzer (Malvern, United Kingdom) according to standard measurement protocols [20, 21].

Mob-155 S concentration was determined spectrophotometrically at a wavelength of 307±3 nm.

In vitro cytotoxicity assay. Cytotoxicity was studied using a model of Jurkat T-cell lymphoblastic leukemia obtained from Cell Line Bank of N.N. Blokhin National Medical Research Center of Oncology in accordance with the procedures of International Standard ISO 10993-5:2009. The cell lines were cultured using a standard method [22] in RPMI-1640 medium containing 10% fetal bovine serum (ICN Biomedicals, USA), 10 mM HEPES (Sigma-Aldrich, Germany), 2 mM L-glutamine (Sigma-Aldrich, Germany), 40 ng/ml gentamicin (ICN Biomedicals, USA), amino acids, sodium pyruvate, and a vitamin solution (PanEco, Russia) at 37°C in an atmosphere containing 5% CO₂. The cells were maintained in the logarithmic growth phase by routinely subculturing the culture every 3–4 days [23]. The MTT assay was performed according to a standard procedure. Jurkat cells were incubated with the liposomal formulations under study at various concentrations for 72 h. After this period, an MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well to a final concentration of 0.5 mg/ml, and the cells were incubated with the dye for more 4 h at 37°C. After the resulting formazan crystals were dissolved in dimethyl sulfoxide, the absorbance of the solutions was measured using a Multiscan EX microplate spectrophotometer (Thermo Labsystems, Germany) at a wavelength of 540 nm. The cell viability was calculated as the percentage ratio of the absorbance in the experimental wells to that in the wells of the intact control.

Statistical analysis. The results were analyzed using Microsoft Excel and GraphPad Prism 7.0 (GraphPad Software, Inc., USA). All experiments were conducted in three independent replicates. The results are presented as the mean ± standard deviation. The data distribution was assessed using Shapiro–Wilk test. The homogeneity of variances was evaluated using Levene test. Statistical significance among multiple groups was assessed using one-way analysis of variance (ANOVA) followed by multiple comparisons using Tukey method. The threshold for statistical significance was considered equal p<0.05.

Results

Study of liposome stability during storage. During the initial studies, a formulation was selected with a lipid-component ratio of E PC S/MPEG-2000-DSPE/cholesterol of 72.0:7.2:1.0, respectively, and Mob-155 S concentration of 0.5 mg/ml [24]. The sample stability was monitored for 6 weeks at two temperature conditions: 6–8°C and 21–23°C (Figure 2). After 3-week storage at 6–8°C, Mob-155 S concentration in the liposomal dispersion decreased by 5.9% (p>0.05); the mean liposome charge changed only slightly, from −17.7 to −19.8 (p>0.05); and the particle size varied within 5%. 6 weeks later, the most pronounced change was a further decrease in Mob-155 S concentration. At room temperature (21–23°C), the liposomes underwent marked changes in their characteristics: the charge varied from −7.4 to −29.6, the particle size increased by 47%, and Mob-155 S concentration decreased to 0.3 mg/ml (p<0.01).

Figure 2. Monitoring of Mob-155 S liposome stability at two temperature conditions. Mean values of three independent measurements are represented in the diagrams. PDI — polydispersity index

Thus, the samples stored at room temperature were less stable, most likely due to an increase in the hydrolysis rate constant at elevated temperatures [25]. The between-group differences between the two temperature conditions became statistically significant by day 40 of the experiment (p<0.05). The gradual changes in the principal quality attributes during storage indicate the limited stability of the liposomes and the need for their stabilization. In addition to excipients protecting liposomes from oxidation or fusion, lyophilization is an attractive approach for extending shelf life and simplifying storage conditions throughout the supply chain [26].

Effect of cryoprotectants on liposome characteristics. Lyophilization was performed to stabilize the liposomes. The effect of cryoprotectants on the principal characteristics of the liposomes was studied. Three types of saccharides — lactose, mannose, and sucrose — were used as cryoprotectants at two concentrations (5% and 10%) in the liposomal dispersion. The control involved the initial liposome quality attributes before the cryoprotectant added (the initial Mob-155 S concentration was set at 100%; the particle size was 194 nm, zeta-potential was −20.5, and PDI was 0.21).

When evaluating the data obtained (Figure 3), the mean liposome size was found to have increased by 1.3–2.3-fold (p<0.01), the polydispersity increased by 1.9–3.4-fold, and the decrease in Mob-155 S concentration reaching 12% (p<0.05) in the samples containing sugars at a concentration of 5% and lactose at a concentration of 10%. The liposome quality parameters observed after incorporating 10% sucrose and 10% mannose were similar; however, the samples containing sucrose showed a statistically significant increase in polydispersity (p<0.05). Therefore, mannose is the preferred cryoprotectant.

Figure 3. The effect of a cryoprotectant on the physical characteristics of the liposomes and on the active substance concentration after lyophilization. Mean values of three independent measurements are represented in the diagrams. PDI — polydispersity index, L — lactose, M — mannose, S — sucrose

Cytotoxic activity of liposomes. To assess the antitumor potential of the liposomes, in vitro cytotoxic activity was evaluated using the Jurkat lymphoblastic leukemia cell line, in comparison with the native Mob-155 S substance and unloaded (“empty”) liposomes containing no active substance (Figure 4).

Figure 4. Cytotoxic activity of the liposomes on Jurkat lymphoblastic leukemia model

The “empty” liposomes had no antiproliferative effect, whereas the native substance and Mob-155 S liposomes demonstrated high cytotoxic activity even at low concentrations compared with the unloaded liposomes (p<0.001). Thus, at a concentration of 1.4 µg/ml, cell growth inhibition was 48±5 and 38±5% for the native substance and Mob-155 S liposomes, respectively; at a concentration of 2.8 µg/ml, the proportion of viable cells was 28±5 and 20±2%, respectively. No statistically significant differences were detected between the Mob-155 S liposomes and the native substance (p>0.05).

Discussion

The acridine scaffold structure has been extensively studied as a potential basis for developing new anticancer drugs. 9-aminoacridine and its derivatives are weak bases, with a pK_a of approximately 9.9; they are poorly soluble in water and soluble in alcohols [11, 27, 28]. The structural analysis of the compound under study (see Figure 1) showed the presence of a nitro group to decrease the basicity of the nitrogen atom, despite the weak electron-donating properties of the additional alkyl substituent. Accordingly, the pK_a value of this compound should be closer to 7.0–8.0. However, this has no significant effect on the compound solubility. Like other aminoacridines, Mob-155 S is insoluble in water, sparingly soluble in chloroform and acetone, and soluble in alcohol. Considering this, liposomes were selected as the formulation for this compound.

A number of physicochemical parameters critically affect liposome membrane fluidity and, consequently, their physical stability. These parameters include temperature, pH, ionic strength, phospholipid composition, and the presence of stabilizing agents in a lipid bilayer. In addition, such factors as vesicle size and surface charge determine the membrane integrity, overall formulation robustness, the efficiency of cellular uptake, and the interactions with biological systems [28]. Liposomes themselves are prone to oxidation and hydrolysis during storage [29].

The size and charge of liposomes affect not only their stability in the bloodstream but also their ability to accumulate at target sites. At the same time, larger particles are recognized and cleared more rapidly by the reticuloendothelial system cells [30]. Immediately after preparation and during short-term storage, Mob-155 S liposomes had particle sizes of approximately 200 nm. After 40-day storage at 21–23°C, a decrease in Mob-155 S concentration was observed, along with an increase in the mean particle size to 300–320 nm and the PDI increase to 0.45–0.55. The monitoring revealed not only the changes in the primary liposome characteristics but also substantial visible changes including the thickening and a small amount of sediment formed. These manifestations are associated with the fact that liposomes, micro- and nanoparticles due to their inherent instability, tend to aggregate and sediment. However, they can be effectively stabilized through lyophilization [31].

Lyophilization may affect the active ingredient content and particle size distribution, as well as lead to the formation of usable sediment with a short reconstitution time. To protect the compounds from possible changes caused by the dehydration process, it is recommended to add excipients protecting them during lyophilization [32]. Since short-chain saccharides are capable of protecting membranes, they are preferred auxiliary molecules to minimize the instability of lipid and polymeric nanoparticles during freezing. They replace the bound water surrounding liposomes and form a viscous matrix, which reduces their mobility during the freezing process [33].

During lyophilization, we assessed the effects of three saccharides at two concentrations on the main qualitative and quantitative characteristics of the liposomes. All dried liposome samples, regardless of the cryoprotectant used, were readily resuspended. However, the required protective effect manifested by maintaining the active ingredient concentration and other characteristics was observed only with 10% mannose. The findings are consistent with literature data [34], according to which the stabilization degree provided by saccharides often depends on their concentration. Moreover, liposomes require a substantial amount of cryoprotectants to saturate their surface and promote the formation of a glassy matrix around their structure [35].

Several studies have reported the antiproliferative activity of acridine derivatives [36, 37]. For example, some screening studies demonstrated a pyrrolo[3,2-l]acridinone derivative to have high activity against Jurkat lymphoblastic leukemia cells, with an IC₅₀ value being 29.6 µM [38]. In the study [39], several analogs of 1-nitroacridine/4-methyl-1-nitroacridine and 4-nitroacridone conjugated to tuftsin/retro-tuftsin peptides were also active against Jurkat cells. According to the molecular docking results, the observed biological effect was due not only to binding to the epidermal growth factor receptor but also to the inhibition of the proto-oncogenic tyrosine kinase Src [39]. Thiazacridines inhibited the growth of Raji and Jurkat neoplastic cells; the IC₅₀ values for compound LPSF/AC-119 were 0.6 and 1.53 µM, respectively [40].

MTT analysis using the Jurkat lymphoblastic leukemia cell model demonstrated the high potential of Mob-155 S liposomes as a novel antitumor agent. The similar IC₅₀ values observed for the native substance (1.76±0.20 µg/ml) and Mob-155 S-based liposomes (1.46±0.10 µg/ml) indicate that the active substance retains its antitumor activity after incorporation into the dosage form, as well as the safety of the excipients.

To further characterize the liposomes, there has been planned the research on investigating the release kinetics of Mob-155 S in various media, the expanded cytotoxicity studies using a panel of different tumor models, and the development of methods to identify Mob-155 S in biological fluids.

Conclusion

We developed the liposomes with pyrrolo[3,2-l]acridinone Mob-155 S derivative, which are characterized by a uniform particle size, good rehydratability after lyophilization, and antiproliferative activity against leukemia cells.

Study funding and conflict of interest. The authors declare no study funding and no conflict of interest.