Encapsulation of zinc-rifampicin complex into transferrin-conjugated silver quantum-dots improves its antimycobacterial activity and stability and facilitates drug delivery into macrophages

In order to improve the chemotherapy of tuberculosis, there is an urgent need to enhance the efficacy of existing agents and also to develop more efficient drug delivery systems. Here, we synthesized a novel anti-TB drug complex consisting of zinc and rifampicin (Zn-RIF), and encapsulated it into transferrin-conjugated silver quantum-dots (Zn-RIF-Tf-QD) to improve delivery in macrophages. Successful synthesis of Zn-RIF and Zn-RIF-Tf-QD was confirmed by UV/Vis-spectroscopy, TEM, FTIR, photoluminescence, XRD, XPS, and NMR. The sizes of silver QDs and transferrin-conjugated QDs were found to be in the range of 5–20 nm. Activity assays showed that Zn-RIF-Tf-QD exhibited 10-fold higher antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis-BCG as compared to Zn-RIF, RIF and Zn. Immunofluorescence studies showed that Zn-RIF-Tf-QD-conjugates were actively endocytosed by macrophages and dendritic cells, but not by lung epithelial cells. Treatment with Zn-RIF-Tf-QD efficiently killed mycobacteria residing inside macrophages without exhibiting cytotoxicity and genotoxicity. Moreover, the conjugates remained stable for upto 48 h, were taken up into the late endosomal compartment of macrophages, and released the drug in a sustainable manner. Our data demonstrate that Zn-RIF-Tf-QDs have a great potential as anti-TB drugs. In addition, transferrin-conjugated QDs may constitute an effective drug delivery system for tuberculosis therapy.

UV-Vis Spectra analysis of the Zn-RIF complex. UV-Vis spectrometry analysis did not show any difference in the spectra of RIF and Zn-RIF complex (Fig. 1C). The Zn-RIF complex exhibited only charge transfer transitions, from the ligand (RIF) to the metal and vice versa. Therefore, no d-d transitions are expected for d 10 Zn(II) complexes 27 . Both RIF and Zn-RIF showed strong absorption bands in the range 390-420 nm (Fig. 1C), which may be associated with a π → π * and n→ π * transitions originating mainly in the RIF chromophore (carbonyl, double bonds, imine, n→ π *transition).
Photoluminescence analysis of the Zn-RIF complex. We also performed photoluminescence experiments to confirm the formation of the Zn-RIF complex. The photoluminescence properties of the complex, which is a d 10 system, has been used in various biological applications such as in chemical sensors, photochemistry and inorganic LEDs 28 . The emission spectra of free RIF and the Zn-RIF complex at λ ex = 280 nm are depicted in Fig. 1D. It can be seen that the free RIF exhibited a much weaker emission band as compared to Zn-RIF (Fig. 1D). This could be due to an intra-ligand emission state, which is characteristic of fluorescent emission spectra of Zn complexes 29 .
X-ray photoelectron spectroscopy. The oxidation state of Zn was confirmed by high-resolution X-ray photoelectron spectroscopy (XPS). From the peak positions of the elements present in the sample their binding energies can be determined. As shown in Fig. 1E, the XPS spectrum of Zn showed two peaks corresponding to Zn 2p 3/2 at 1030 eV and to Zn 2p 1/2 at 1045 eV. The Zn 2p 3/2 peak is much higher than the Zn 2p 1/2 peak indicating the involvement of Zn 2+ in complex formation 30 (Fig. 1E).
X-ray powder diffraction analysis of the Zn-RIF complex. X-ray diffraction (XRD) analysis of Zn-RIF was carried out using CuKα at λ = 1.54 Å and Θ -2θ scans from 20° to 80° with a scan speed of 0.5°/min and an incidence angle of 1° at a step size of 0.05°. The powder XRD spectra of the Zn-RIF, free RIF and Zn(NO 3 ) 2 shown in Fig. 1F confirm the formation of the Zn-RIF complex. RIF is amorphous in nature, which reduces peak intensity. However, RIF complexed with Zn produces a sharp peak at an angle of 20°-25°. Another intense peak at angle of 15° showed the presence of the ligand i.e. RIF 31 .
NMR spectroscopy. 1 H NMR spectrum analysis of RIF and the Zn-RIF complex was carried out in DMSO-D 6 using a Bruker AVANCE 400 NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard. All peaks arising from unbound RIF ligand were clearly resolved ( Fig. 2A) with the phenolic hydroxyl protons appearing at 9-10 (2 H) ppm. In the Zn-RIF spectrum these peaks were absent due to replacement with zinc. To detect exchangeable protons, we performed deuterium exchange experiments with D 2 O. It was observed that all the protons with shifts between 8 and 16 ppm were exchanged with deuterium ( Fig. 2C). By comparing the splitting patterns, it can be concluded that the metal to ligand ratio is 1:1 32 .
The Zn-RIF complex showed enhanced antimycobacterial activity as compared to free RIF and zinc. We compared the antibacterial activity of Zn-RIF complex against M. smegmatis and M. bovis-BCG with Zn and RIF alone. M. smegmatis is a non-pathogenic mycobacterial strain, whereas M. bovis-BCG behaves like pathogenic mycobacteria. Exponentially grown bacteria were incubated with various concentrations of RIF, Zn and Zn-RIF, and surviving colonies were counted after 72 h for M. smegmatis and 3 weeks for M. bovis-BCG. Free RIF and free Zn in concentrations corresponding to those in Zn-RIF were used as controls. The results showed distinct differences in susceptibility of mycobacteria. As shown in Fig. 3, Zn-RIF exhibited significantly higher antibacterial activity in a dose-dependent manner when compared with Zn and RIF alone. After 6 h of incubation, approximately 53% and 83% (P ≤ 0.01 and P ≤ 0.001) of the M. smegmatis population was killed at 1 and 8 μg/ml concentrations of Zn-RIF, respectively; whereas after 24 h of incubation at similar concentrations about 91% and 99% (P ≤ 0.001) of M. smegmatis were killed (Fig. 3A,B). With RIF alone, only 14.2 and 67.5% killing was observed at 1 and 8 μg/ml after an 6 h incubation; whereas no bacterial killing at all was observed after exposure to Zn(NO 3 ) 2 at equimolar concentrations. Similarly, after 24 h of incubation, RIF and Zn(NO3) 2 at 1 μg/ml concentration showed no killing activity against M. smegmatis, whereas an exposure to 8 μg/ml RIF inhibited 94% of bacterial growth (Fig. 3A,B). In comparison to M. smegmatis, M. bovis-BCG was more susceptible to Zn-RIF action such that exposure to 1 μg/ml of Zn-RIF complex killed approximately 90% (Fig. 3C, P ≤ 0.001) of M. bovis-BCG, whereas no colonies at all were observed at 8 μg/ml after 6 h of incubation. After 24 h exposure to Zn-RIF at 1 and 8 μg/ml, approximately 82% and 98% of M. bovis-BCG cells were killed ( Fig. 3D; P ≤ 0.001). RIF alone at 1 and 8 μg/ml killed 46% and 86% of M. bovis-BCG cells, whereas Zn(NO 3 ) 2 alone had no effect after 24 h of treatment. These data indicate that Zn-RIF exhibits significantly higher antimycobacterial activity as compared to RIF and Zn(NO 3 ) 2 alone.

Zn-RIF kill mycobacteria residing inside macrophages. Fighting intracellular pathogens is a major
challenge as the drugs used must enter the cells to kill the bacteria. Since mycobacterium is an intracellular pathogen 33 , we checked the intracellular burden of M. smegmatis after treatment of macrophages with RIF, Zn and Zn-RIF. As shown in Fig. 4A, 6 h treatment with Zn-RIF at 8 and 20 μg/ml resulted in an approximately 3-fold decrease of the intracellular bacterial burden as compared to RIF and Zn alone. After an 24 h treatment with 1 and 20 μg/ml, the intracellular burden of M. smegmatis was reduced by 50% and 98%, respectively; whereas RIF alone killed 2.7% and 91% of bacteria under similar conditions (Fig. 4B).
Zn-RIF in bactericidal doses did not exert cytotoxicity on mouse macrophages. From the therapeutic perspective, it is important that drugs should kill intracellular pathogens without exhibiting cytotoxic effects on the host cells. In the present study, we compared the cytotoxic effects of Zn-RIF on mouse macrophages with those of RIF and Zn using the MTT assay. As shown in Fig. 4C, Zn-RIF had no cytotoxic effects on the macrophages at bactericidal concentrations, indicating that decrease in intracellular bacterial burden is not due to reduction in macrophage cell viability.
Characterization of Zn-RIF encapsulated in transferrin-conjugated quantum dots. As mentioned above, mycobacteria are intracellular pathogens. To improve the delivery of Zn-RIF into macrophages, we encapsulated Zn-RIF in QDs that was used previously as vehicles for drug delivery to various cell type 34 . To further increase the specificity of the system for macrophages, we conjugated the QDs to transferrin (Tf). The scheme for the synthesis of Zn-RIF-Tf-QD conjugates is shown in Fig. 5A. The resultant Zn-RIF-Tf-QD conjugates were characterized by various physico-chemical methods.
Transmission electron microscopy. The size and morphology of silver QDs, transferrin conjugated QDs (Tf-QDs) and Zn-RIF encapsulated Tf-QDs (Zn-RIF-Tf-QDs) were characterized using TEM. As shown in Fig. 5B (upper panel), mono-dispersed spherical Zn-RIF-Tf-QD nanoparticles were obtained with a mean diameter of 4.0 to 0.6 nm. The TEM analysis also confirmed the conjugation of transferrin to silver QDs. The size of Tf-QDs was about 5 nm, whereas silver QDs had diameters of about 20 nm. This decrease in size upon Tf-conjugation could be due to the fact that proteins act as a stabilizer and it also help in reducing the size of the particles and prevent their aggregation. However, encapsulation of Zn-RIF in Tf-QDs again increased their size. TEM images of Zn-RIF-Tf-QD conjugates showed the presence of a layer surrounding the QDs (Fig. 5B, right upper panel). We speculate that transferrin first bind to QDs to form a complex (Tf-QDs), which then facilitates the binding of Zn-RIF by covalent and non-covalent interactions (Fig. 5B). High resolution TEM images showed the presence of  UV-Vis Spectral analysis. The Zn-RIF-Tf-QD sols were yellow and showed a single visible band near 400 nm ( Fig. 5C), which is characteristic of silver QDs 35 . The sols were stable for several weeks at room temperature with no precipitation or change in color on standing. The Tf-QD complex showed a bathochromic shift with a maximum peak at around 450 nm. Moreover in comparison to QDs, the peak intensity of Tf-QD also decreased due the binding of transferrin. The Zn-RIF-Tf-QD exhibited two bands at 320-340 nm and 460-480 nm, which originate from QDs. This band was broadened due to interaction with the transferrin molecule (Fig. 5C). With increasing Zn-RIF and transferrin precursor ratio the absorption spectra and emission spectra of the Zn-RIF-Tf-QD was shifted towards longer wavelength (Fig. 5C).

Zn-RIF encapsulated transferrin-conjugated QDs (Zn-RIF-Tf-QD) had higher in-vitro bactericidal activity as compared to Zn-RIF. First, we compared the antibacterial activity against M. bovis-BCG
of Zn-RIF-Tf-QD with those of RIF and Zn-RIF under in vitro condition. As shown in Fig. 6A, exposure of M. bovis-BCG to 0.1 and 1 μg/ml of Zn-RIF-Tf-QD for 6 h inhibited growth by 58% and 84%, respectively, whereas no bacterial killing was observed by RIF and Zn-RIF at 0.1 μg/ml . Exposure to 1 μg/ml of RIF and Zn-RIF inhibited M. bovis-BCG growth by 61% and 71%, respectively. After 24 h, we observed an approximately 10-fold higher killing rate by Zn-RIF-Tf-QD as compared to RIF and Zn-RIF (Fig. 6B). Treatment with 0.1 μg/ ml Zn-RIF-Tf-QD killed 99% of the bacteria, which is equivalent to the killing observed after exposure to 1 μg/ ml concentration of RIF and Zn-RIF (Fig. 6B). No bacterial colonies were observed at 0.75 μg/ml and higher concentration of Zn-RIF-Tf-QD (Fig. 6B). These data indicate that Zn-RIF-Tf-QD are at least 10-fold more potent than RIF and Zn-RIF.
Zn-RIF-Tf-QDs are more efficient in killing intracellular mycobacteria in macrophages. Next we compared the intracellular bacterial killing efficacy of Zn-RIF-Tf-QD with those of Zn-RIF and RIF in Zn-RIF-Tf-QD is actively endocytosed and localized in the late endosomal compartment of macrophages. Next, we performed fluorescence microscopic studies to examine the internalization of Zn-RIF-Tf-QD by macrophages. Since particles of a certain size can be engulfed by macrophages 36 , Zn-RIF-Tf-QDs were labeled with FITC and then added to mouse macrophages. Our microscopic studies showed active endocytosis of FITC labeled Zn-RIF-Tf-QD by macrophages (Fig. 7C).

Intracellular stability of Zn-RIF-Tf-QDs in mouse macrophages and dendritic cells. Next, we
studied the intracellular stability of FITC labeled Zn-RIF-Tf-QD in RAW264.7 macrophages, dendritic cells and peritoneal macrophages isolated from mice. Our microscopic data showed that the complex remained stable up to 48 h in all cells (Fig. 8). Afterwards a gradual decrease of fluorescence intensity was observed, indicating that Zn-RIF-Tf-QDs are quite stable inside the cells.
Zn-RIF-Tf-QDs are selectively internalized by macrophages, but not by lung epithelial cells. We further compared the rates of uptake of FITC labeled Zn-RIF-Tf-QD by A549 lung epithelial, peritoneal macrophages and dendritic cells. As shown in the Fig. 9A, FITC-labeled QDs were actively internalized by peritoneal macrophages and dendritic cells, but not by A549 lung epithelial cells. These results indicate that Zn-RIF-Tf-QDs are indeed specifically delivered to the macrophages.
We also examined the genotoxicity of Zn-RIF-Tf-QDs against mouse macrophages using the micronuclei and comet assays. As shown in the Fig. 9B,C, no signs of micronucleated cells or comet tail formation were observed in macrophages treated with RIF and Zn-RIF-Tf-QD.

Discussion
The development of drug resistance and detrimental side effects of the administered drugs are of serious concerns in the treatment of many diseases. The current treatment regimen of tuberculosis is associated with multiple problems such as prolonged treatment duration, poor permeability of target cells for the drug with unpleasant side effects, difficulties in maintaining sufficiently high drug concentrations at the infected site, and premature degradation of the drug before it reaches the target site. Due to these circumstances, new drugs and novel drug delivery strategies are required to improve the TB therapy. Among them, one strategy aims at the development of new anti-TB drugs by re-engineering existing drugs to improve their antimycobacterial activity and bioavailability and reduced toxicity.
Transition metals can exhibit a wide variety of coordination properties, and reactivities, which can be used to form complex with drugs as ligands. Previously, several studies have reported improved therapeutic properties of several metal complexes against M. tb. RIF, which is considered the cornerstone in the short-course TB treatment regimen, exhibits detrimental side effects. To address these issues, we employed a strategy in which Zn was complexed with RIF to form a Zn-RIF complex, which was subsequently encapsulated in transferrin-conjugated silver QDs to yield the Zn-RIF-Tf-QD conjugate. Detailed physico-chemical analyses confirmed the formation  and encapsulation of Zn-RIF complex in transferrin-coupled QDs. We demonstrated that encapsulation of transferrin on the surface of quantum dots enhanced the binding efficiency of drug molecules. Then we showed that transferrin-conjugated and Zn-RIF encapsulated silver QDs successfully targeted to the macrophages, remaining stable for up to 48 h and significantly reducing the bacterial burden inside the macrophages.
A major factor that contributes to the drug resistance of mycobacteria is the presence of a multi-layered hydrophobic cell wall 37 , which reduces the permeability to anti-TB drugs. Therefore, it is of utmost importance to identify molecules that act directly on the cell wall of mycobacteria. First, we evaluated the antibacterial activity of Zn-RIF against M. smegmatis and M. bovis-BCG and compared it with that of Zn and RIF alone. In this way we confirmed that Zn-RIF exhibited more potent antibacterial activity against both mycobacterial strains than Zn or RIF. This may be due to better penetration of Zn-RIF through the bacterial membrane as the complex is more lipophilic than free RIF. Moreover, Zinc nitrate is known to disintegrate bacterial cell membranes 16 . This may have facilitated the diffusion of RIF into the bacterial cell and inhibit RNA synthesis.
Efficient clearance of intracellular mycobacteria is a major challenge. Mycobacteria are intracellular pathogens that can reside inside macrophages for extended periods of time. We therefore examined the intracellular killing efficacy of Zn-RIF in mouse macrophages infected with M. smegmatis. We found that Zn-RIF significantly reduced intracellular bacterial burden after penetrating into the macrophages.
Despite of the widespread use of RIF in the treatment of TB, toxic effects have been reported by many researchers 38 . RIF is known to induce hepatotoxicity and lipid peroxidation in the liver and bone marrow 39,40 . Here, we compared the cytotoxic effects of Zn-RIF with RIF and Zn(NO 3 ) 2 on mouse macrophages by MTT assay. In our previous studies, we have shown that zinc oxide nanoparticles kill intracellular mycobacteria without causing any toxic effect on macrophages 16 . In the present study, we also observed that Zn-RIF showed less cytotoxic effects against mouse macrophage, and that no cytotoxic effects were observed at bactericidal levels. These results indicate that Zn-RIF is a promising candidate for therapeutic applications.
Quantum dots (QDs) are nano scale semiconductor crystals with sizes of 1-10 nm. QDs provide excellent tools for sensing, imaging, drug delivery and therapy due to their optical properties, broad excitation range, well-defined emission wavelengths and their ability to attain different shapes thus providing an excellent structure for coating with various biomolecules 24,25,34,41 . To improve the delivery of the Zn-RIF inside the macrophages, we encapsulated it in transferrin-conjugated silver QDs. As macrophages express a Tf-receptor on their surface, we hypothesized that transferrin should facilitate the uptake of drugs by the macrophages through interactions with receptor 42,43 . Indeed, we found that the Zn-RIF-Tf-QD exhibits at least 10-fold higher in vitro and intracellular killing activity as compared to Zn-RIF and free RIF, indicating that the conjugate formation leads to enhanced bactericidal efficacy. This could be due to the targeted delivery of the conjugate to the mycobacteria-containing phagosomes followed by the slow release of Zn-RIF from the conjugate.
It is now well established that pathogenic mycobacteria survive inside the macrophages by preventing the phago-lysosome fusion. Therefore, from the therapeutic perspective it is important that drug molecules be trafficked via the endocytic pathway to achieve direct killing effects on mycobacteria. By fluorescence microscopy and immunofluoroscence staining we confirmed that FITC labeled Zn-RIF-Tf-QDs were indeed endocytosed by the macrophages and eventually co-localize with LAMP-1, indicating that that the drug composite taking the endocytic pathway of macrophages.
Many drugs are either prematurely degraded or pumped out from their target cells through efflux pumps, which impair their therapeutic efficacy. Therefore, in the therapeutic perspective it is important to improve drug stability and also to release the drugs in a sustainable manner inside the cells. Development of nanoscale drug delivery system that allows a slow release of drug over prolonged periods of time is important to thus avoid burst effects. Our results show that Zn-RIF-Tf-QDs meet these criteria. We further show that Zn-RIF-Tf-QD selectively target of Zn-RIF into macrophages and dendritic cells, but not into A549 lung epithelial. In summary, we conclude that 1. Zn-RIF-Tf-QDs have a great potential as anti-TB molecules with reduced side effects, and 2. Transferrin-functionalized QDs can be used as a novel and efficient drug delivery system in TB therapy.

Isolation of peritoneal macrophages and dendritic cells. Peritoneal macrophages and dendritic cells
were isolated from 6 to 8 week old BALB/c mice. All mice were maintained in high efficiency particulate air (HEPA) filter bearing cages under 12 h light cycles in our animal facility, and were given sterile chow and autoclaved water ad libitum. All animal experiments were performed in accordance with national guidelines for the care and handling of laboratory animals and have been approved by the Institutional Animal Care and Use committee of KIIT University (Approval Number: KSBT/IAEC/2013/MEET1/A11). Peritoneal macrophages were isolated by following a previously published protocol 44 . Similarly, dendritic cells (DCs) were isolated as described previously 45 . Bone marrow progenitor cells were stimulated with 20 ng/ml of granulocyte/macrophage colony stimulating factor (GM-CSF) for DC proliferation and maturation. Silver QDs were synthesized as described previously 35 . Briefly, silver QDs were synthesized by reduction of silver nitrate (1 mM) by addition of excess of ice cold sodium borohydride (2 mM) solution by vigorous stirring at room temperature. QDs were synthesized in less than a minute reaction time. The stoichiometric ratio of silver nitrate to sodium borohydride is very critical for the synthesis of QDs.

Synthesis of Zinc-Rifampicin complex and its encapsulation with transferrin coupled silver
To synthesize Zn-RIF encapsulated transferrin conjugated silver QDs, 1 mg/ml of transferrin and 500 μg/ml of Zn-RIF complex were added together with 1 mM concentration of silver nitrate. Then the reaction mixture was reduced by excess ice cold sodium borohydrate solution.

Characterization of Zn-RIF encapsulated transferrin conjugated QDs (Tf-QDs).
After encapsulation of Zn-RIF complex into transferrin conjugated QDs (Zn-RIF-Tf-QDs), the synthesized Zn-RIF-Tf-QDs conjugate was characterized by UV-Vis spectroscopy, FTIR analysis and transmission electron microscopy (TEM). For TEM, a drop of aqueous solution of Zn-RIF-Tf-QDs was placed on the carbon-coated copper grids. The samples were dried and kept overnight under a desiccator before loading them onto a specimen holder. TEM measurements were performed on JEM-2100, HRTEM, JEOL, JAPAN operating at 200 kV.
In vitro killing assay. To determine the antimycobacterial activity of Zn-RIF and Zn-RIF-Tf-QDs complexes, 4-5 × 10 5 bacteria were incubated with various concentrations of these complexes in 7H10 medium in 96-well round bottom plates in triplicates. Bacteria were harvested at the indicated time points and the number of colony forming units (CFUs) was assayed by plating suitably diluted cultures on 7H10 agar plates. All samples were plated in triplicate and values were averaged from three independent trials. Intracellular killing assay. RAW 264.7 macrophage cells were infected as described previously 16 . Briefly, RAW 264.7 cells (2 × 10 5 cells/well) were infected with M. smegmatis and M. bovis-BCG at a multiplication of infection (MOI) 10 for 2 h followed by the treatment with different concentrations of drug complexes. Extracellular bacteria were killed by addition of 20 μg/ml gentamycin. Cells infected with bacteria alone were used as control. After the incubation period, cells were washed, lysed with 0.5% triton-X-100 and intracellular survival was determined by plating serially diluted samples on 7H10 agar plates and the M. smegmatis and M. bovis-BCG colonies were enumerated after 3 days and 1 month, respectively. Cytotoxicity assay. RAW 264.7 cells (1 × 10 5 cells/well) were grown in 24-well plates for 24 h followed by treatment with different concentrations of drug complexes for another 24 h. Cell viability was determined by MTT assay as described previously 16 .

Endocytosis of drug encapsulated QDs by macrophages, dendritic cells and lung epithelial cells.
Zn-RIF-Tf-QD conjugate was labeled by addition of 5 μg/ml of FITC and the mixture was stirred continuously for 8 h in dark. RAW 264.7 cells, peritoneal macrophages, dendritic cells and lung epithelial cells (1 × 10 5 cells/ml) were grown on glass cover slips in 24-well plate. FITC labeled Zn-RIF-Tf-QD conjugate were added to the cells. DMEM media with FITC only was taken as control. Then the cells were incubated for 1 h, washed with 1× PBS, fixed with 4% paraformaldehyde for 30 min at 37 °C and mounted on a glass slide. The cells were observed under a fluorescence microscope (Nikon, Japan).

Intracellular stability of Zn-RIF-Tf-QD conjugate.
Zn-RIF-Tf-QD conjugate were labeled as described above. RAW 264.7 cells, peritoneal macrophages and dendritic cells grown on glass cover slips were treated with FITC labeled Zn-RIF-Tf-QD conjugate for 48 h. After the incubation period, cells were washed, fixed and mounted by DAPI containing mounting solution. The images were visualized by using fluorescence microscopy.
Immunofluoroscence studies. The immunofluorescence study was performed to evaluate the localization of Zn-RIF-Tf-QD conjugate in mouse macrophages. RAW 264.7 cell lines were grown on glass cover slips and treated with FITC labeled Zn-RIF-Tf-QD conjugate. After 3 h of incubation period, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% saponin and blocked with 5% BSA. Then the cells were incubated with 1:200 diluted rabbit LAMP-1 primary antibody ( Cell Signaling) for 1 h at room temperature, washed with 1× PBS and incubated with Alexa fluor conjugated secondary antibody (1:200) for 2 h. Then the cells were washed twice with 1× PBS and mounted with DAPI containing mounting solution (Invitrogen). Localization of FITC labeled Zn-RIF-Tf-QD conjugate complexes were visualized using the fluorescence microscope (Nikon, Japan).
In-vitro cytokinesis-blocked micronucleus assay. Micronucleus assay measures the DNA damage resulting from exposure to toxic agents 46 . In vitro cytokinesis-blocked micronucleus assay (CBMN) was performed by treating the mouse macrophages with 1 and 10 μg/ml doses of both Zn-RIF and Zn-RIF-Tf-QD conjugatefor 24 h. Then the cells were treated with cytochalasin B (8 μg/ml) (MP-BIO) and were incubated for another 22 h. Cells were harvested, centrifuged and the pellet was fixed with fixative (3:1 methanol/acetic acid). The cells were kept at 4 °C for at least 4 days, added drop wise on glass slides and dried. The slides were then stained with propidium iodide (4 μg/ml) and analyzed by fluorescence microscopy to visualize binucleated and micronuclei forming cells.
Comet assay. The alkaline comet assay was performed to check the effect of Zn-RIF-Tf-QD conjugate on DNA damage. Macrophages were treated with 1 and 10 μg/ml doses of both Zn-RIF and Zn-RIF-Tf-QD conjugatefor 24 h at 37 °C. Untreated cells were used as a control. Cell suspensions (400 μL) were mixed with 1% low melting point agarose (1 ml) and added to glass cavity slides (Blue Label Scientifics Pvt Ltd, Mumbai, India). The agarose was allowed to solidify and then the slides were submerged in lysis solution (1.2 M sodium chloride, 100 mM EDTA disodium salt, 0.1% SDS, 0.26 M NaOH [pH. 13]) for 2 h at 4 °C in the dark. After lysis, the slides were washed with dH 2 O, transferred to an electrophoresis unit, covered with freshly prepared electrophoresis buffer (0.03 M NaOH, 2 mM EDTA disodium salt, pH 12.3), left for unwinding of DNA for 30 minutes, and the cells were electrophoresed for 22 minutes at 22 V. The cells were neutralized with neutralization buffer (500 mMTrisHCl, pH 8.0) for 15 minutes, washed 3-4 times with dH 2 O, and stained with PI (4 μg/mL) for 1 hour. The slides were dried and then observed using the fluorescence microscope.