Metallo-Curcumin-Conjugated DNA Complexes Induces Preferential Prostate Cancer Cells Cytotoxicity and Pause Growth of Bacterial Cells

DNA nanotechnology can be used to create intricate DNA structures due to the ability to direct the molecular assembly of nanostructures through a bottom-up approach. Here, we propose nanocarriers composed of both synthetic and natural DNA for drug delivery. The topological, optical characteristics, and interaction studies of Cu2+/Ni2+/Zn2+-curcumin-conjugated DNA complexes were studied using atomic force microscopy (AFM), UV-vis spectroscopy, Fourier transform infrared and mass spectroscopy. The maximum release of metallo-curcumin conjugates from the DNA complexes, triggered by switching the pH, was found in an acidic medium. The bacterial growth curves of E. coli and B. subtilis displayed a prolonged lag phase when tested with the metallo-curcumin-conjugated DNA complexes. We also tested the in vitro cytotoxicity of the metallo-curcumin-conjugated DNA complexes to prostate cancer cells using an MTS assay, which indicated potent growth inhibition of the cells. Finally, we studied the cellular uptake of the complexes, revealing that DNA complexes with Cu2+/Ni2+-curcumin exhibited brighter fluorescence than those with Zn2+-curcumin.

DU145 (human prostate carcinoma isolated from brain metastasis), were used to determine the anti-bacterial and anti-cancer activity of the metallo-curcumin-conjugated DNA complexes. Finally, fluorescence microscopy was used to visualize the cellular internalization of metallo-curcumin-conjugated RDNA complexes in 22Rv1 cells, showing a substantial uptake of curcumin into the cells.

Results
Curcumin and metallo-curcumin binding to DNA. Typically, third-party molecules bind to DNA in three different ways: electrostatic interaction, intercalation, and groove binding. Curcumin (abbreviated hereafter as C) binds mostly to DNA via groove binding 43 . Groove binding is related to the lock-and-key model for drug binding to DNA (since the drugs adjust their structures to follow the groove as the DNA twists around its central axis, as shown in Fig. 1(c), which has been proven clinically as an anti-bacterial and anti-cancer agent 25 . We initially checked the structural stability of DNA structures (DNA rings without deformation and SDNA without precipitation) with the addition of a certain concentration of metallo-curcumin conjugates (to achieve significant function enhancement). We chose 4 mM of Cu 2+ , 2 mM of Ni 2+ , and 1 mM of Zn 2+ with 80 µM of C at a fixed concentration of either RDNA (500 nM) or SDNA (0.1 wt%) because these concentrations were sufficient to reveal the specific function of the metallo-curcumin conjugate (anti-bacterial and anti-cancer activity) without disturbing the DNA structure, which is discussed below 44 . The RDNA (SDNA) complexes with Cu 2+ and C, Ni 2+ and C, and Zn 2+ and C conjugates were named RDNA-Cu-C, RDNA-Ni-C, and RDNA-Zn-C (SDNA-Cu-C, SDNA-Ni-C, and SDNA-Zn-C), respectively.
Structural morphology of curcumin-and metallo-curcumin-conjugated RDNA complexes. AFM analysis was performed for the RDNA to verify its structural stability after doping with metallo-curcumin conjugates with [C] of 80 µM, [Cu 2+ ] of 4 mM, [Ni 2+ ] of 2 mM, and [Zn 2+ ] of 1 mM. We observed that the RDNAs doped with metallo-curcumin conjugates were well assembled with the expected geometry (Fig. 2). The RDNA formed a ring structure with an outer diameter of ~29 nm by hybridization of the two strands 45,46 . Spectroscopic evidence of curcumin-and metallo-curcumin, metallo-curcumin-conjugated SDNA complexes. The photo physical behaviour of the metallo-curcumin conjugates and their interactions with the SDNA were investigated using UV-vis and infra-red spectroscopy, as shown in Fig. 3. Figure 3(a) shows a UV-vis spectrum with an intense band for C at 435 nm due to π-π interactions. In the case of the metallo-curcumin-conjugated SDNA complexes, we observed absorption shifts of 1-6 nm and shoulder peaks at around 450 nm. The shoulder peaks contributed to the metal complexation with C and variation in the shoulder peaks depended on the type of metal ion 47,48 . To further study the physical interactions within the metallo-curcumin-conjugated SDNA complexes, we performed a qualitative analysis of the interactions between the DNA and the metallo-curcumin conjugates.
FTIR spectroscopy was performed to qualitatively investigate the interactions of the metallo-curcumin-conjugated SDNA complexes, as displayed in Fig. 3(b). The DNA signature peaks were divided into five regimes; their assignments are tabulated in detail in Supplementary Table S3. 3650-3000 cm −1 corresponded to -OH stretching, 1800-1500 cm −1 to in-plane base vibrations, 1500-1250 cm −1 to base-sugar vibrations, 1250-1000 cm −1 to sugar-phosphate vibrations, and finally 1000-700 cm −1 to sugar vibrations. Interestingly, the peak at 3330 cm −1 (the band attributed to the phenolic group) did not change compared to that of bare C even after chelating metal ions. This indicates that the phenolic -OH group was not involved in the metal complexation process. The peak intensities at 1016, 1058, and 1083 cm −1 in the phosphate band region (1250-1000 cm −1 ) were lower than those of pristine SDNA, which may be related to the specific binding of the metallo-curcumin conjugates to the DNA helix 48,49 . The SDNA peak at 1230 cm −1 (the PO 2 − antisymmetric stretching mode) shifted to a lower frequency of ~1224 cm −1 and decreased in intensity upon metallo-curcumin complexation. The B-form markers at 830 cm −1 for the metallo-curcumin-conjugated SDNA complexes indicated the presence of the B-form of DNA with little disturbance. With the help of the binding and interaction patterns obtained by UV-vis and FTIR measurements, we will test the drug release profile of the metallo-curcumin-conjugated DNA complexes.
For the detection and distribution of the molecular species exist in DNA and SDNA-Cu-C complexes, we performed the time-of-flight secondary ion mass spectroscopy (TOF-SIMS) (Fig. 4). DNA molecules were composed of deoxyribose sugar, phosphate groups, and nucleobases, which were detected as DNA molecular ion fragments using the negative ion mode TOF-SIMS. Figure 4   In vitro drug release kinetics. The cumulative Cu-C release profile from the RDNA nanostructures was measured at pH 7 and 5 at 37 °C for 2 days, as shown in Fig. 5. The free C profile was treated as a control. Cu-C (here, M 2+ enhances the solubility and therapeutic efficacy of C) is specifically discussed because similar patterns were observed for the release profiles of the other metallo-curcumin conjugates 53 . Free C displayed a similar pattern of burst release at both pH 7 and 5 (no noticeable differences with respect to pH), with ~95.8% and ~96.4% measured at 6 h, respectively. In contrast, the release of Cu-C from RDNA-Cu-C complexes was clearly different between pH 7 and pH 5. Two distinct phases (initial burst release and slower release regions) were observed. Around 64.7% of Cu-C was released from the RDNA at pH 5 after 6 h, whereas only 40.5% was released at pH 7. Finally, Cu-C release reached around 71.4% and 45.86% for pH 5 and 7, respectively. While DNA is stable in physiological conditions (e.g., blood pH of 7), pH-responsive DNA nanocarriers release their maximum payloads in acidic surroundings (e.g., lysosomal pH of 5) like an effective drug delivery system. Based on our results, metallo-curcumin-conjugated RDNA complexes also showed maximum release in acidic pH due to the disintegration of the phosphodiester bonds in the DNA structure, which releases the drug 24 . Sustained release of drugs helps improve drug efficacy, patient compliance, and toxicity, motivating the development of new nanomaterials.
Anti-bacterial assay of metallo-curcumin-conjugated DNA complexes against E. coli and B. subtilis. To confirm the penetration and drug delivery of the metallo-curcumin-conjugated DNA complexes, we investigated the effects of the complexes in bacterial cells. Representative bacterial population growth inhibition curves of E. coli and B. subtilis upon treatment with metallo-curcumin-conjugated RDNA and SDNA complexes are shown in Fig. 6. Examining the bacterial growth kinetics, the growth of E. coli continuously increased in the control samples (untreated RDNA and SDNA) after a lag phase of 2.5-3 h. Free C and DNA-C (i.e., RDNA-C and SDNA-C) had a lag phase of 0.5 h and a mild reduction in growth rate compared to the controls. On the other hand, the metallo-curcumin-conjugated DNA complexes had a strong-delayed lag phase of 5.5 h, and the total growth of E. coli was arrested at around 8.5-9 h with ~60% population reduction compared to the controls, Figure 5. In vitro release of curcumin and metallo-curcumin conjugates from RDNA under two different pH conditions. The release rate of C from the Cu 2+ -curcumin-conjugated RDNA complexes was analysed using 0.2 M phosphate buffered saline (pH 5 and pH 7) containing 0.5% Tween 20 for two days. The data represent the mean values of three measurements with error bars. as shown in Fig. 6(a,b). The Gram-positive bacterium B. subtilis showed a similar trend to E. coli, in which the lag phase was delayed by about 6 h for the metallo-curcumin-conjugated DNA complexes. The growth of the bacterium was completely arrested at ~9 h with ~50% reduction in the population, as depicted in Fig. 6(c,d). However, the B. subtilis treated with either RDNA-C or SDNA-C showed an extended lag phase until 9 h followed by a sudden log phase. This implies that free C was highly efficient at inducing a prolonged bacteriostatic effect on B. subtilis. Consequently, the metallo-curcumin-conjugated DNA complexes had promising bacteriostatic and bactericidal effects on both the Gram-negative and Gram-positive bacterial strains. Finally, we aimed to test the effects of the metallo-curcumin-conjugated DNA complexes in mammalian prostate cancer cells to elucidate their anti-cancer properties.
Cytotoxicity activity in human prostate cancer cell lines. Figures 7 and 8 show the cell viability profiles of the metallo-curcumin-conjugated RDNA and SDNA complexes, respectively, on five different prostate cancer cell lines (PC3, 22Rv1, TRAMP-C1, LNCaP, and DU145). Equivalent concentrations of C, RDNA-C (SDNA-C), RDNA-Cu-C, RDNA-Ni-C, and RDNA-Zn-C (SDNA-Cu-C, SDNA-Ni-C, and SDNA-Zn-C) complexes were assessed by MTS colorimetric assay for 24 and 48 h. The metallo-curcumin-conjugated DNA (both RDNA and SDNA) complexes had a positive impact on both androgen-sensitive (LNCaP, 22Rv1, and TRAMP-C1) and androgen-independent (PC3 and DU145) prostate cancer cells. Interestingly, the cytotoxicity of the metallo-curcumin-conjugated DNA complexes was higher in DU145 cells (derived from brain metastasis) at 48 h than in the other cell lines. Based on the data, the order of cytotoxicity of the metallo-curcumin-conjugated DNA complexes in the cell lines was DU145 > PC3 > TRAMP-C1 > 22Rv1 > LNCaP.
After treating with DNA-C with different M 2+ , the cells showed different characteristic toxicities. RDNA-Cu-C complexes showed better cytotoxicity in the five different prostate cancer cells (83-96%) than RDNA-Ni-C (49-84%) and RDNA-Zn-C (30-74%) complexes (Fig. 7). Similarly, the cytotoxicity in cancer cells was significantly greater upon treatment with the SDNA-Cu-C complexes (83-95%) than with the SDNA-Ni-C (58-79%) or    (Fig. 8). The decreased cytotoxicity of the DNA-Zn-C conjugates was due to the presence of strong O-H bonds in the O-methoxy phenolic group, which led to diminished scavenging activity compared to the other complexes which lose H-atoms more readily 39 . The synergistic effect of M 2+ and C binding to DNA is possibly made more effective in cancer cells by inhibiting NF-κB and AP-1 activity, thereby activating apoptotic genes. The cytotoxicity of the cells treated with bare M 2+ , C, and DNA-C was lower than that of the cells treated with metallo-curcumin-conjugated DNA complexes.

Cellular internalization of metallo-curcumin-conjugated RDNA complexes.
To validate the cellular uptake of the metallo-curcumin-conjugated RDNA complexes, we measured the in vitro cellular internalization in 22Rv1 cells treated with the complexes, as shown in Fig. 9. The complexes were internalized inside the cell through extrinsic pathways such as receptor-mediated endocytosis with vesicles known as early endosomes. These early endosomes mature and fuse with cytoplasmic vesicles to form late endosomes and lysosomes before activating apoptotic proteins, which subsequently leads to death 54 . RDNA-C was used as a comparison for the extent of uptake. C possesses a characteristic green fluorescence which was utilized to visualize the cellular uptake in the cells. Interestingly, RDNA-Cu-C and RDNA-Ni-C exhibited brighter fluorescence than RDNA-Zn-C. The lower uptake of RDNA-Zn-C coincided with the weak cytotoxicity observed in the MTS assay.

Discussion
To summarize, we constructed Cu 2+ /Ni 2+ /Zn 2+ -curcumin-conjugated ring DNA and salmon DNA complexes. We characterized their topology using AFM and their optical properties and interaction studies using UV-vis, FTIR, and mass spectroscopy. Metal ions improved the solubility of the curcumin and provided additional DNA binding ability. In addition, we evaluated the anti-bacterial and anti-cancer activity of the complexes in terms of growth inhibition, cell viability, and cellular uptake through the in vitro pH-controlled release of metallo-curcumin from the DNA complexes. RDNAs doped with metallo-curcumin conjugates were well assembled with the expected geometry. The chemical binding of the metallo-curcumin conjugates to DNA and the characteristic peaks of the DNA were studied using UV-vis, FTIR and mass spectra. Based on release profiles, the metallo-curcumin-conjugated DNA complexes released a maximum quantity of conjugates in acidic pH due to the breaking of the phosphodiester bonds in the DNA structure. The metallo-curcumin-conjugated DNA complexes exhibited a strong-delayed lag phase in both Gram-positive and Gram-negative bacteria. Metallo-curcumin-conjugated DNA complexes showed significant toxicity to prostate cancer cells compared to pristine DNA. Finally, we performed cellular uptake studies which revealed that RDNA-Cu-C and RDNA-Ni-C internalized more efficiently than RDNA-Zn-C, as measured by bright fluorescence. Our results prove that DNA nanostructures without any transfection agents can improve drug loading, drug release, and anti-bacterial and anti-cancer activity, warranting their use as novel and efficient nanocarriers for drug delivery applications.

Methods
Synthesis of DNA rings. Equimolar concentrations of two different DNA strands (Ring 1-1 and Ring 1-2) were added to 1 × TAE/Mg 2+ buffer (40 mM Tris(hydroxymethyl)aminomethane), 20 mM acetic acid, and 1 mM EDTA with 12.5 mM magnesium acetate). The final concentration of RDNA was 500 nM. The assembly of DNA rings in the test tube involved cooling from 95 °C to room temperature for 24 h to aid hybridization by placing the test tube in a Styrofoam box containing 2 L of boiling water. The annealed structures were incubated at 4 °C to stabilize the ring structure ( Supplementary Fig. S1, Supplementary Tables S1 and S2.  UV-vis spectroscopy. UV-vis spectroscopy of DNA, curcumin, metallo-curcumin, curcumin-conjugated DNA, and metallo-curcumin-conjugated DNA complexes was performed using Nano Drop 2000c (DE, USA) in the wavelength range of 200-800 nm. 2 µL of the sample was dispensed onto the pedestal before the absorbance measurement ( Fig. 3(a)).
TOF-SIMS measurement. Trace elemental and chemical fragment analysis on the surface of SDNA and SDNA-Cu-C complexes were performed using a reflection-based high-resolution TOF-SIMS-5 instrument (ION-TOF GmbH, Germany). Negative ion-based mass spectra and chemical mapping are acquired using a pulsed ion source with 25 keV and 1.3 pA primary Bi 3+ cluster in the high-current bunched mode for 150 × 150 µm 2 on the sample surface. (Fig. 4 and Supplementary Fig. S2).

Drug release.
Metallo-curcumin release rates were determined using a dialysis procedure. The metallo-curcumin-conjugated RDNA complexes were transferred into dialysis tubes that had been preconditioned with DI water for 15 min. Then, the metallo-curcumin-conjugated RDNA complexes in the dialysis tubes were dialyzed against 600 µL of 0.2 M phosphate-buffered saline (PBS) containing 0.5% Tween 20 in an incubator-shaker maintained at 37 °C and 200 RPM. At predetermined time intervals, 100 µL of the released medium was taken and analysed using fluorescence spectroscopy with an excitation wavelength of 420 nm (VICTOR Multi-label Plate Reader, Perkin Elmer 2030, Seoul, Korea) (Fig. 5).

Bacterial growth kinetics.
To assess the anti-bacterial activities of DNA, curcumin, curcumin-conjugated DNA, and metallo-curcumin-conjugated DNA complexes, pathological hospital bacterial strains-Escherichia coli (E. coli) for the Gram-negative and Bacillus subtilis (B. subtilis) for the Gram-positive category-were inoculated in LB medium. They were grown overnight in an orbital incubator-shaker at 37 °C. The culture was then diluted to an optical density (OD) of 0.1, and 100 µL of the culture was inoculated in the corresponding well of a 96-well plate. The OD absorbance of the culture medium was then recorded at 600 nm using an Epoch 2 Microplate spectrophotometer (BioTek Instruments, VT, USA) over a period of 20 h. The bacterial growth curve was found by optical density at 600 nm (OD 600 ). The measurement was conducted three times (Fig. 6).
Cell culture studies. (a) The prostate cancer cells (PC3, 22Rv1, TRAMP-C1, LNCaP, and DU145) were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% Antibiotic-Antimycotic solution. The cells were cultured and incubated in a 5% CO 2 environment at 37 °C in a CO 2 incubator. Once the cells attained 85% confluency, they were detached from the flask with 0.25% trypsin-EDTA and centrifuged at 1500 RPM for 5 min. The cell pellet was then resuspended in a complete growth medium. The cells were counted using a hemocytometer and used for further cell culture experiments.

Cell viability.
Cell viability studies were carried out on PC3, 22Rv1, TRAMP-C1, LNCaP, and DU145 cell lines by the CellTitre 96 Aqueous One Solution Cell Proliferation Assay (MTS), a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity, and chemosensitivity assays. The MTS was composed of solutions of a novel tetrazolium compound [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-c arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate; PMS). The MTS was bio-reduced by the cells into a formazan product that was soluble in tissue culture medium. The cells were seeded on a 96-well plate with a cell density of 5000 cells/cm 2 . Metallo-curcumin-conjugated DNA complexes with various metal ion concentrations (80 µM of curcumin with either 4 mM of Cu 2+ , 2 mM of Ni 2+ , or 1 mM of Zn 2+ ) were prepared. The final concentrations of RDNA and SDNA were 500 nM and 0.1 wt%, respectively. Appropriate controls such as DMSO, 1 × TAE/Mg 2+ buffer, and DI water were also tested in each case to compare the effect. After reaching 85% confluency, the media were removed and the cells were washed with DPBS. The metallo-curcumin-conjugated DNA complexes and fresh RPMI medium was added to a final volume of 100 µL in each well and incubated. The cells in medium alone (devoid of test samples) acted as a positive control and the cells treated with Triton X-100 acted as a negative control. The experiment was carried out for 24 and 48 h. After 24 and 48 h, 20 µL of MTS solution was added and the cells were incubated for 4 h to form the formazan product. The cell viability was then ascertained by measuring the OD of the culture at 490 nm using a BioRad iMark Microplate Reader (BioRad, CA, USA). This measurement was carried out in triplicate (Figs 7 and 8).
Cell uptake. 22Rv1 cells were seeded at a density of 5000 cells in a confocal dish attached to a cover slip and incubated for 24 h for cell attachment. After 24 h, the medium was removed and the cells were washed twice with PBS. Then, fresh RPMI medium was added to the cells along with the required concentration of metallo-curcumin-conjugated RDNA complexes. The cells were then incubated for 6 h at 37 °C in a 5% CO 2 incubator. After incubation, the cells were washed thrice with PBS and fixed in 3.7% paraformaldehyde solution for 10 min, before being permeabilized with 0.2% Triton X-100 for 5 min. The cells were then further washed with PBS. Finally, the cells were imaged using fluorescence microscopy (EVOS FLoid Cell Imaging Station, ThermoFisher Scientific, Korea) (Fig. 9).