Antibacterial Therapy by Ag+ Ions Complexed with Titan Yellow/Congo Red and Albumin during Anticancer Therapy of Urinary Bladder Cancer

Abstract

According to the World Health Organization report, the increasing antibiotic resistance of microorganisms is one of the biggest global health problems. The percentage of bacterial strains showing multidrug resistance (MDR) to commonly used antibiotics is growing rapidly. Therefore, the search for alternative solutions to antibiotic therapy has become critical to combat this phenomenon. It is especially important as frequent and recurring infections can cause cancer. One example of this phenomenon is urinary tract infections that can contribute to the development of human urinary bladder carcinoma. This tumor is one of the most common malignant neoplasms in humans. It occurs almost three times more often in men than in women, and in terms of the number of cases, it is the fifth malignant neoplasm after prostate, lung, colon, and stomach cancer. The risk of developing the disease increases with age. Despite the improvement of its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. Hence, there is an urgent need to introduce innovative solutions that will prove effective even in the advanced stage of the disease. In our study, a nanosystem based on ionic silver (Ag⁺) bound to a carrier-Titan yellow (TY) was analyzed. The possibility of binding the thus formed TY-Ag system to Congo red (CR) and albumin (BSA) was determined. TY-Ag binding to CR provides for better nanosystem solubility and enables its targeted intracellular transport and binding to immune complexes. The binding of TY-Ag or CR-TY-Ag to albumin also protects the system against the uncontrolled release of silver ions. It will also allow the delivery of silver in a targeted manner directly to the desired site in the case of intravenous administration of such a system. In this study, the MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values of the TY-Ag or BSA-TY-Ag systems were determined in two reference strains (Escherichia coli and Staphylococcus aureus). The paper presents nanosystems with a size of about 40-50 nm, with an intense antibacterial effect obtained at concentrations of 0.019 mM. We have also discovered that TY-Ag free or complexed with BSA (with a minimal Ag⁺ dose of 15-20 μM) inhibited cancer cells proliferation. TY-Ag complex diminished migration and effectively inhibited the T24 cell viability and induced apoptosis. On the basis of the obtained results, it has been shown that the presented systems may have anti-inflammatory and antitumor properties at the same time. TY-Ag or BSA-TY-Ag are new potential drugs and may become in future important therapeutic compounds in human urinary bladder carcinoma treatment and/or potent antimicrobial factors as an alternative to antibiotics.

Keywords: Escherichia coli; Staphylococcus aureus; T24 cell line; apoptosis; bovine serum albumin; congo red; migration; minimum bactericidal concentration; minimum inhibitory concentration; necrosis; silver ions; titan yellow; tumor growth.

Figure 1

Agarose gel electrophoresis at pH 8.6 of TY, CR, Ag⁺, BSA and their combinations in different complexes (BSA-TY molar ratio was 1:10 and TY-Ag molar ratio was 1:1.6). (A): gel after electrophoresis in a veronal buffer; (B): reduction with sodium dithionite locating the presence of excess silver; (C): bromophenol blue-stained albumin; BSA free and in the complexes with CR, TY, or TY-Ag and CR-TY-Ag seen as migrating towards the anode; free Ag⁺ seen as migrating towards the cathode: (1) TY; (2) CR; (3) BSA; (4) BSA-TY (1:10); (5) BSA-CR (1:10); (6) BSA-CR-TY (BSA-CR = 1:10 and BSA-TY = 1:10; CR-TY = 1:2) visible additional streak of the complex CR-TY at the front; (7) TY-Ag (1:1.6); (8) CR-TY (1:1); (9) CR-TY-Ag (TY-Ag = 1:1.6); (10) BSA-CR-TY-Ag (BSA-CR = 1:10, BSA-TY = 1:10, TY-Ag = 1:1.6); (11) BSA-TY-Ag (BSA-TY = 1:10, TY-Ag = 1:1.6); (12) Ag⁺; (circles mark free silver moving towards the cathode (the dashed line indicates the smallest amount of free silver).

Figure 2

Agarose gel electrophoresis at pH 8.6 of TY, CR, Ag⁺, BSA and their combinations in different complexes (BSA-TY molar ratio was 1:10 and TY-Ag molar ratio was 1:0.8). (A): gel after electrophoresis in barbital buffer; (B): reduction with sodium dithionite locating the presence of excess silver; (C): bromophenol blue-stained albumin; BSA free and in the complexes with CR, TY or TY-Ag and CR-TY-Ag seen as migrating towards the anode; free Ag⁺ seen as migrating towards the cathode: (1) TY-Ag (1:0.8); (2) CR-TY-Ag (TY-Ag = 1: 0.8); (3) BSA-TY-Ag (BSA-TY = 1:10, TY-Ag = 1:0.8); (4) BSA-CR-TY-Ag (BSA-CR = 1:10, BSA-TY = 1:10, TY-Ag = 1:0.8); (5) BSA; (6) CR-Ag; (7) TY; a continuous line marks free silver moving towards the cathode, while the dashed line marks complexed silver.

Figure 3

Comparison of UV/VIS spectra of TY, TY-Ag, BSA, BSA-TY, and BSA-TY-Ag. Changes in the spectrum in the emerging complexes are visible.

Figure 4

Successive samples (0.5 mL) collected after passing through BioGel P-150: (A): TY-Ag, (B): BSA-TY, (C): BSA-TY-Ag, (D): TY.

Figure 5

Comparison of intensity autocorrelation curves, % of intensity and % of the mass of: (A): TY and (B): TY-Ag.

Figure 6

Comparison of intensity autocorrelation curves, % of intensity, and % of the mass of: (A): BSA and (B): BSA-TY.

Figure 7

Comparison of intensity autocorrelation curves, % of intensity and, % of the mass of: (A): BSA-TY and (B): BSA-TY-Ag.

Figure 8

Graph: Growth inhibition zones of E. coli and S. aureus after the addition of different concentrations of Ag⁺ in the TY-Ag complex; (A): photos of plates: no growth inhibition zones after adding 7.5 mM of TY, or (C): 0.03 mM of TY; (B): growth inhibition zones after adding 7.5 mM of Ag⁺ complexed with 7.5 mM TY or (D): 0.03 mM Ag⁺ complexed with 0.03 mM TY.

Figure 9

Determination of MIC by agar diffusion method for E. coli in the presence of complexes: (A): TY-Ag (1:0.5) (concentration No. 7: 0.019 mM; (B): BSA-TY-Ag (concentration No. 7: 0.019 mM; MIC determination for S. aureus in the presence of complexes: (C): TY-Ag (1:0.5) (concentration No. 7: 0.019 mM; (D): BSA-TY-Ag (TY-Ag 1:0.5; BSA-TY 1:10) (concentration No. 6: 0.039 mM). The ranges of silver concentrations (serial two-fold dilutions) were analyzed from 1.25 mM (No. 1) to 0.005 mM (No. 9). No. 10 is a control sample.

Figure 10

Determination of MBC on MHA agar plates for E. coli in the presence of complexes: (A): TY-Ag (1:0.5) (last of concentration No. 8: 0.036 mM); (B): BSA-TY-Ag (last of concentration No. 7: 0.07 mM); MBC determination for S. aureus in the presence of complexes: (C): TY-Ag (1:0.5) (above concentration No. 3: 1.12 mM—MBC not determined); (D): BSA-TY-Ag (TY-Ag = 1:0.5; BSA-TY = 1:10) (above concentration no. 3: 1.12 mM—MBC has not been determined). The ranges of silver concentrations (serial two-fold dilutions) were analyzed from 1.12 mM (No. 3) to 0.008 mM (No. 10).

Figure 11

(A): A histogram presenting a comparison of the size of growth inhibitory zones of two strains: Escherichia coli (E. coli)and Streptococcus aureus after adding the silver complexed with: TY, CR-TY, BSA-TY and BSA-CR-TY; growth inhibitory zones on plates from left to right for E. coli and for S. aureus (each probe in triplicate) after adding: (B): AgNO₃; (C): BSA-TY-Ag; (D): CR-TY-Ag, and (E): BSA-CR-TY-Ag (each probe in triplicate); concentration of silver in each probe: 4.7 mM. The graph shows the mean of the 3 experiments +/− standard deviation. The results were statistically analyzed using the Student’s T-test. Statistically significant differences (** p < 0.01; *** p < 0.001) in the size of growth inhibitory zones were observed between the AgNO₃ samples and those to which the silver complexes were added.

Figure 12

T24 cell proliferation expressed as % of control after addition of Ag⁺ (20 µM) (solid lines) complexed with TY (20 µM) (yellow), BSA-TY(20 µM) (blue), TY (40 µM) (red) and BSA-TY (40 µM) (green) separately; Ag⁺ (15 µM) (dashed lines) complexed with TY (20 µM) (yellow), BSA-TY (20 µM) (blue), TY (40 µM) (red) and BSA-TY (40 µM) (green) separately; Ag⁺ (10 µM) (dotted lines) complexed with TY (20 µM) (yellow), BSA-TY (20 µM) (blue) and TY (40 µM) (red) separately.

Figure 13

Caspase-3 activation after 24 and 48 h in T24 cells after TY-Ag stimulation.

Figure 14

The effect of TY and TY-Ag complex on apoptosis and necrosis of T24 cells. The images show flow cytometry analysis of Annexin-V and PI staining presented in a dot-plot graph; (A): negative control; (B): positive control with taxol (50 nM); (C): T24 cells were incubated with TY (20 µM concentrations), (D): TY-Ag (TY 20 µM and Ag⁺ 15 µM concentrations); (E): graphic representation of four cell states: alive - the lower-left square; cells undergoing necrosis—the upper left square; cells in early apoptosis - the right lower square; and cells in late apoptosis the upper right square; (F): Cumulative bar charts show the inter-relation between the state of T24 cells after 48 h exposure to TY (20 µM), TY(20 µM)-Ag(15 µM), and taxol.

Figure 15

The effect of TY(20 µM)-Ag(1 µM) on the motility of T24 cells (A): exposure to TY(20 µM)-Ag(1 µM) inhibited migration of T24 cells in a scratch assay observed after 24 h; (B): percentage of scratch reduction after 24 h (n = 3).

Figure 16

The effect of (B): taxol, (C): TY, (D): TY-Ag, (E): BSA-TY, and (F): BSA-TY-Ag on the cell cycle of T24 cells. (A): control; (G): percent of cells at G1, S or G2 phase of cell cycle.