Materials
Tenofovir (≥ 98% HPLC) was procured from Clearsynth, Bangalore, India. Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Penicillin-Streptomycin (Pen-Strep), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS) and HEPES buffer from Gibco, Invitrogen, New York, USA. Cyanine5.5 NHS ester, the amine-reactive far-red emitting fluorescent dye, was purchased from Abcam, Waltham, USA. Dimethyl sulfoxide (DMSO), 3-(4,5dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and Cysteamine hydrochloride ≥ 98% (titration) from Sigma Aldrich, Burlington, USA. The other chemicals were reagent/HLPC grade and were employed with no additional refinement.
Gold nanoparticles synthesis
The citrate reduction process was used to synthesize AuNPs [92]. Before synthesis, all the glassware was rinsed with aqua regia and washed thoroughly with distilled water. Briefly, a 20 mL aqueous solution of 0.1 mg/mL Tetrachloroauric acid (HAuCl4; 99% pure, Sigma-Aldrich, Burlington, USA) was used as a metallic precursor and allowed to heat to the point of boiling. Then 2 mL of the reducing agent, 1% sodium citrate solution, was added instantly and stirred for 10 min. Infographic, Fig. 1A, illustrates the reaction scheme for preparing AuNPs.
Characterization of AuNPs and AuNP-TNF nanoconjugate
Particle size and surface charge analysis
Using DLS, the size and ζ potential were estimated. The hydrodynamic size and the surface charge of AuNPs and AuNP-TNF were measured by DLS and ζ potential, respectively. (Zeta sizer nano instrument, Malvern, Cambridge, UK), The nanoformulation was dispersed in water at a medium at 25 ℃. Clear disposable ζ cells were used for measurements were taken in triplicates, and mean particle size reported.
FE-SEM analysis
The size and morphology of the synthesized AuNPs were evaluated using FE-SEM. The FE-SEM images were captured at a 20 kV accelerating voltage to investigate the surface morphology of the synthesized AuNPs. (Nova NanoSEM™ 450; FEI, Hillsboro, USA). Briefly, a small amount of AuNPs was deposited over an aluminum substrate and pasted over double-sided carbon tape. Subsequently, the sputtered coated samples were analyzed. The EDS was carried out along with FE-SEM for qualitative and semiquantitative analysis of elements present in the materials under study (Bruker; XFlash® 6I30, Karlsruhe, Germany).
TEM analysis
TEM is extensively used to image the size of nanoparticles, disclose phase/crystallographic orientation information via a diffraction pattern, and determine chemical composition via the energy spectrum. In this study, the size and morphology of the synthesized AuNPs were also evaluated using TEM. A drop of AuNPs was deposited on carbon film-covered copper mesh TEM grids, air-dried for 60 min, and then imaged. (Tecnai G2; U-twin, Hillsboro, USA).
Drug conjugation to AuNPs
Amine coupling through reactive esters is the most frequent strategy for connecting ligands covalently to a drug's hydrophilic solid surface, such as TNF. In this study, TNF was conjugated to AuNPs surface by using EDC and NHS coupling. Briefly, 2 mg AuNPs were activated by adding 6.96 µmol of EDC and NHS in an aqueous environment. The reaction mixture was stirred at ambient temperature for 2 h in the dark. (Fig. 1A).
Subsequently, 6.96 µmole of TNF solubilized in water was introduced to the reaction mixture and, for 24 h, stirred in the dark. (Fig. 1B) The conjugate was then pelleted by centrifugation at 12,400 g for 20 min and washed thrice with water to eliminate any unreacted reagents and by-products. The supernatant drug concentration was calculated by measuring absorbance at 259 nm [31]. The following Eq. (1) was used to compute the conjugation capacity:-
$$Conjugation\,capacity (\%) = (Total\,drug - drug\,in\,supernatant)/(total\,drug)\times 100$$
(1)
DLS was used to determine the size and surface charge of the AuNP-TNF. Using a UV-Vis spectrophotometer, the spectrum was recorded in the 400-700 nm range. Additionally, as described below, FT-IR was carried out in the 400-4000 cm−1 range.
UV–-Vis spectroscopy
UV-Vis absorption spectra of AuNPs and AuNPs conjugated TNF were recorded to determine the successful conjugation of TNF on the AuNPs surface. The UV-Vis analysis was performed using a UV-VIS spectrophotometer, Shimadzu 1800, Kyoto, Japan, in double beam standard quartz cuvettes with the spectral range of 400-700 nm wavelength at Room Temperature (RT); the UV-Vis absorption spectra were obtained [93]. Briefly, 5 μL of AuNPs or AuNP-TFV were diluted with Millipore water and analyzed by the spectrophotometer.
TGA analysis
TGA was used to evaluate bare AuNPs and TNF-conjugated AuNPs (PerkinElmer-STA 6000 Simultaneous Thermal Analyzer, Waltham, USA). Under flowing nitrogen, individual compounds were heated to 800 ℃ at 55 ℃/min while retained on an aluminum pan. In the presence of air, under the same heating rate from 800 ℃ to 1000 ℃, the compounds were then decomposed.
FT-IR spectroscopy
Additionally, AuNPs were analyzed with FT-IR (IRAffinity−1, Shimadzu, Columbia, USA). The AuNPs were dispersed in KBr pellets, and the samples were examined in transmission mode in the spectral band 400-4000 cm−1. The scanning speed was 20 mm/sec at RT, and the spectral resolution was 4 cm−1.
Synthesis of Cy5.5-tagged AuNPs
The synthesis was carried out as per the method reported in Wai and New (2020) [94]. Briefly, to 20 mL of 0.1 mg/mL HAuCl4 solution, 200 µL of 213 mM cysteamine hydrochloride was poured. Under dark conditions at RT, the mixture was vigorously stirred. Next, the solution was agitated for 10 min after 5 µL of freshly prepared cold 10 mM NaBH4 was added and then mildly stirred for an extra 30 min. The solution was stored overnight in the dark. Subsequently, Cy5.5 NHS ester was added to the AuNPs, swirled for 4 h in the dark, and then washed with distilled water. The Cy5.5-tagged AuNPs were used for internalization, uptake, and in vivo studies.
In vitro experiments
Cell cultures
The genetically altered HeLa cell line, TZM-bl (JC53-bl), HEK 293 T, and Human Monocytic THP-1 cell lines were acquired from the National Institutes of Health (NIH), AIDS Research and Reference Reagent Program (ARRRP), Bethesda, USA. For primary screening of the synthesized nano-ART, the TZM-bl reporter cell line was employed. These cells express CD4, CXCR4, and CCR5 receptors, which are essential for HIV-infected cells. Additionally, Escherichia coli β-galactosidase enzyme and Tat-responsive reporter genes for firefly Luciferase (Luc) are inserted in these cells. The HIV-1 Long Terminal Repeat (LTR) promoter regulates the expression of these genes. This cell line was maintained in DMEM enriched with 25 mM HEPES buffer solution, penicillin (100 U/mL), streptomycin (100 mg/mL), and 10% FBS. Cell cultures were retained at 37 ℃ in a humidified atmosphere of 5% CO2. Adherent HEK 293 T cells were employed to propagate the infectious virus particles. HEK 293 T cells were also maintained under identical conditions as the TZM-bl cells. In addition, the THP-1 cells were also employed as an in vitro target cell system to simulate the efficiency of synthesized nano-ART during HIV infection. The cells were maintained in a complete RPMI 1640 growth medium fortified with 25 mM HEPES solution, 100 U/mL penicillin G, 100 mg/mL streptomycin, and 10% FBS. Further, naïve THP-1 cells were differentiated into MΦ by re-suspension in the complete growth medium, supplemented with 200 ng/mL (324 nM) phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Burlington, USA) for 2 or 3 days followed by 1 day of rest in PMA-free growth medium. Disseminated morphology, cell adhesion, increased granularity, and abnormal nucleus shape, which are hallmarks of THP-1 derived macrophages (MΦ), were promoted after the treatment, as observed by optical microscopy. Ficoll Histopaque® (1077; Sigma-Aldrich, Burlington, USA) was used to separate PBMCs from the whole blood of a healthy donor using a density gradient centrifugation technique. PBMCs were activated with 5 µg/mL Phytohaemagglutin (PHA-p; Sigma-Aldrich, Burlington, USA) and maintained in RPMI 1640 medium enriched with 10 U/mL Interleukin-2 (IL-2; Roche, Branchburg, USA), and 10% FBS.
Propagation of viruses
HIV-1 primary isolates
In the present study, the anti-HIV1 activity against HIV1VB28 (CCR5 tropic, Subtype C isolate, Virus repository, ICMR- NARI, Pune, India) and HIV1UG070 (CXCR4 tropic, Subtype D [Uganda], NIH ARRRP, Bethesda, USA) was assessed. The activated PBMC cells were infected with the viral strains to develop virus stocks. The ELISA was conducted to monitor the viral growth using the HIV-1 p24 antigen detection kit (Advanced Bioscience Laboratories; Inc, Rockville, USA). Virus cell-free culture supernatants were harvested, centrifuged, filtered, and preserved at -80 ℃ in aliquots. The TCID50 (50% tissue culture infective dose) of each virus stock was calculated after titration.
The pseudotyped
The HIV-1 NL4-3 molecular clone (pNL4-3) encoding full-length HIV-1 was received from the NIH-ARRRP, Bethesda, USA. TOP10 competent cells were used to propagate the plasmid. (E. coli DH5 alpha; Invitrogen, New York, USA). Briefly, TOP10 competent cells were grown overnight on an incubator shaker at 37 ℃ in Luria Bertani broth enriched with ampicillin (100 µg/mL). Using a Qiaquick™ spin miniprep kit (Qiagen, Germantown, USA), the plasmid was extracted and quantified on a Nanodrop UV spectrophotometer. The medium was replenished with a fresh medium prior to transfection. Then, as per the manufacturer's protocol, 1.5 g of plasmid DNA was transfected into HEK 293 T cells (4 × 105 /well) in a 6 well plate (Techno Plastic Products, Trasadingen, Switzerland) using X-tremeGene HP transfection reagent (Roche, Basal, Switzerland). The culture supernatant was collected, centrifuged, filtered (PALL® syringe filter [0.22 µm]; Pall Corporation, New York, USA), and retained at -80 ℃ until needed. The Multiplicity of Infection (MOI) of the virus stock was determined, after which it was titrated in the MΦ.
Cell proliferation assay
To measure cell viability, the tetrazolium-based MTT assay was executed. In TZM-bl, PBMCs, and MΦ, the CC50 values of free Tenofovir, Gold nanoparticles, and Gold nanoparticles conjugated with Tenofovir were determined. The TZM-bl cells (0.01e6 /well) were plated into flat-bottom 96 well microplates and stored at 37 ℃ in a humidified atmosphere of 5% CO2 overnight.
The naïve THP-1 cells were differentiated into MΦ in flat-bottom 96 well microplates as per the procedure described above. Subsequently, the medium was replenished with a serum-free medium. Following that, two-fold serial dilutions of free TNF, AuNPs, and AuNP-TNF were prepared and transferred into wells. The TZM-bl and MΦ cells plates were incubated for 48 h and 5 days at 37 ℃ in a humidified atmosphere of 5% CO2, respectively. The 0.2e6 /well of stimulated PBMCs (previously described) were seeded in U-bottom 96 well microplates and treated with various concentrations of compounds. The plates were incubated at 37 ℃ in a humidified atmosphere of 5% CO2 for 5 days. MTT solution (20 μL of 5 mg/mL strength) was added to each well after incubation. The plates were maintained for 4 h at 37 ℃ in similar conditions. Finally, the formed formazan crystals were dissolved with DMSO. At 550/630 nm, the Optical Density (OD) values were measured, and the results were represented in terms of CC50.
Genotoxicity
Single-Strand DNA Breaks (SSBs) and DNA Double-Strand Breaks (DSBs) along with Alkali Labile Sites (ALS) were detected using the alkaline comet assay. This assay is widely referred to as "Single-cell micro-gel electrophoresis." Here, stimulated PBMCs were incubated with TNF, AuNPs, and AuNP-TNF at 5 and 150 μg/mL concentrations for 3 and 24 h, respectively, at 37 ℃ in a humidified atmosphere of 5% CO2. The stimulated PBMCs without treatment was considered a negative control, and cells exposed to 40 µg/mL Cyclophosphamide were regarded as a positive control. The medium containing treatment compound was replenished, and 50 µL of the mincing solution was added to each sample (10% DMSO and 20 mM EDTA in Hank's Balanced Salt Solution (HBSS, devoid of Ca2+, Mg2+, and phenol red, pH 7.5). The comet assay was carried out in accordance with the previously reported procedure [95, 96]. In short, 20 µL of cell lysate (0.2e6 cells) were combined with 150 μL of 0.5% Low Melting Point Agarose (LPMA; Sigma-Aldrich, Burlington, USA). Then, onto chilled microscope slides pre-coated with 1.0% Normal Melting Agarose (NMA), 20 μL aliquots of each sample were dropped. After adding newly prepared 1% Triton X-100 lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris Base, pH 10.0), the cells were lysed on the ice at 4 ℃ for 1 h. They were then immersed in an alkaline buffer (0.3 M NaOH, 1 mM EDTA, pH > 13.0) for 20 min to unwind. Subsequently, for 20 min, the electrophoresis was carried out in the same buffer at a power supply of 25 V and a current of 300 mA. The slides were then neutralized for 2 × 5 min in 0.4 M Tris buffer and maintained for 5 min in Milli Q water. The slides were fixed by dipping them for 5 min in chilled methanol and then dehydrated overnight. Eventually, by immersing air-dried slides in 1X EtBr (GeNei™, Bengaluru, India) for 15 min, the DNA was stained. A fluorescent microscope (AxioCam MRmImager 2®; Carl Zeiss, Oberkochen, Germany) was used to obtain the single-cell image. The results were then scored using the Comet Imager software (version 2.2) and analyzed with FIJI software. (Image J; Version 2.0.0, NIH, Bethesda, USA).
Fifty cells per sample were scored (fifty comets on each duplicate gel). The findings were represented as the average of the following parameters: % DNA damage and Olive Tail Moment (OTM). The OTM was calculated using the Eq. (2):-
$$Olive\,Tail\,Moment= (tail\,mean-head\,mean) \times \%\,of\,DNA\,in\,the\,tail$$
(2)
For each compound and concentration, three different experiments were carried out.
In vitro hemolysis assay
AuNP-TNF must be distributed systemically to be effective HIV therapeutic, which is accomplished chiefly by blood circulation throughout the body. As a result, the nanoparticles' blood compatibility assessment is an effort to examine the nanoparticles' toxicity profile for Intravenous (i.v.) administration. A colorimetric hemolysis assay was used to assess the quantity of liberated red-colored hemoglobin, representing the RBCs disintegration level. The analysis was carried out according to the previously published protocol [63, 97, 98]. Thus, the in vivo scenario may be mimicked with this experiment.
Under medical supervision, the blood collected from a healthy donor is stabilized with EDTA and processed promptly. First, using centrifugation (800 g, 5 min), the RBCs were separated. Isolated RBCs were washed five times, pelleted, and resuspended in PBS. Subsequently, to acquire a 4% RBC suspension, the RBCs were diluted in a 5% w/v glucose solution. Finally, RBCs suspended in PBS and Triton-X 100 (1% v/v) were deployed as negative and positive controls, respectively.
TNF, AuNPs, and AuNP-TNF were incubated with RBC suspensions at concentrations of 5, 10, and 20 μg/mL. For 2 h, the suspensions were kept at 37 ℃ in a humidified atmosphere of 5% CO2.
Using a microplate reader, the OD of released hemoglobin was measured at 540 nm. The proportion of hemolysis was determined using the Eq. (3):-
$$\% Hemolysis = \left[ {\left( {A{-}B} \right)/\left( {C - B} \right)} \right] \times 100$$
(3)
where; A represents test OD, B represents negative control OD, and C represents positive control OD. For each term of the equation, the background interference was subtracted from each OD value. Three independent replicates of the experiment were carried out.
Annexin V‑FITC/Propidium Iodide (PI) apoptosis assay
TZM-bl cells (1 × 106/well) were treated with TNF, AuNPs, and AuNP-TNF at 80, 420, and 500 µg/mL concentrations, respectively. According to the manufacturer's procedure (Annexin V-FITC/PI Apoptosis Assay kit; Invitrogen, New York, USA), staining of the treated cells was done for 10 min at RT in the dark with 5 µL PI for 5 min and 5 µL Annexin V-FITC. Cells without treatment served as a negative control, and cells treated with 10 µM Camtothectin (CPT), an inhibitor of DNA topoisomerase that induces Programmed Cell Death (PCD), served as a positive control. Using a FACSAria™ Fusion flow cytometer (Becton Dickenson, Franklin Lakes, USA), the amount of apoptosis induced by compounds in TZM-bl cells was quantified. In addition, apoptosis was evaluated using FlowJo software (version 10.0). The results were presented as the overall apoptosis rate (the early and late apoptotic cell population).
Virucidal assay
The efficacy of free TNF, AuNPs, and AuNP-TNF to inhibit HIV-1 replication was assessed in different HIV-1 infected cells. The TZM-bl cells (0.01e6 /well) plated on the previous day were infected with the pre-titrated viral stocks of HIV1VB28 or HIV1UG070. The plates were incubated for 2 h. This was followed by adding two-fold serial dilutions of the sub-toxic concentrations of free TNF, AuNPs, and AuNP-TNF. The plates were then incubated at 37 ℃ in a humidified atmosphere of 5% CO2 for 48 h. The cells grown without virus (100% inhibition) were considered the negative control, whereas the cells inoculated with the virus without compounds (100% infection) were considered the positive control. Post incubation, the Britelite plus reagent (Perkin Elmer, Waltham, USA) was added, the Relative Luminescence Units (RLUs) were quantified by a Luminometer (Victor 3; Perkin Elmer, Waltham, USA), and the percent inhibition was measured by the LUC software (version 04.4). The results were analyzed in terms of IC50. The TIs (\(TI= CC{50}/IC{50}\)) were calculated and compared with the free TNF.
For confirmation of the anti-HIV activity, the activated PBMCs (0.02e6/well) were inoculated with HIV-1 primary isolates (R5, HIV1VB28, and X4, HIV1UG070), and the experiments were conducted using similar procedures and conditions as described above. The supernatant was collected after 5 days of incubation and analyzed for HIV-1 capsid p24 antigen. The findings were represented as IC50 and TI, and the percent inhibition was measured.
The anti-HIV1 efficacy of free TNF, AuNPs, and AuNP-TNF has also been assessed in MΦ cells. The cells (0.02e6/well) were infected with a pre-titrated pNL4-3 stock (MOI of 0.01) and incubated at 37 ℃ in a humidified atmosphere of 5% CO2 for 4 h. Each well's media was aspirated, and the cells were rinsed at least thrice with fresh serum-free RPMI 1640 medium. Then, different concentrations of free TNF, AuNPs, and AuNP-TNF were added to the cells. The supernatant from respective wells was collected on days 1, 3, and 5 post-infection. The collected supernatants were analyzed for the p24 Gag protein using an HIV-1 p24 antigen detection kit to determine the antiretroviral efficacy of compounds at each time point. Each experiment included untreated and untreated HIV-1 infected cells as negative and positive controls. Three independent replicates of the investigation were carried out, and the results were expressed as IC50 for the luminescent cell-based assay and HIV-p24 antigen levels (pg/mL).
In vitro off-target activity evaluation to elucidate the mechanism of action
Enzymatic assays were carried out to understand the mode of action of free TNF, AuNPs, and AuNP-TNF in the HIV-1 triggered cells.
HIV-1 RT-ase assay
In accordance with the manufacturer's instructions, a colorimetric HIV-1 Reverse Transcriptase Assay (Roche, Mannheim, Germany) was employed to validate the activity. The potential of each compound to hinder the HIV-1 RT-ase enzyme was tested at various concentrations [99]. In a nutshell, for 60 min, the HIV-1 RT-ase and template nucleotide cocktail were incubated with different concentrations of compounds. Then, the mixtures were shifted to streptavidin-coated microtiter plates for an additional hour of incubation. As a result, the biotin and DIG-labelled template primer complex adhered to the streptavidin plates. The HRP enzyme conjugate was then added and incubated for an hour after rigorously rinsing to ensure that no unbound template remained. Absorbance at 405 nm and reference at 490 nm was measured after the substrate was added. The IC50 values were calculated. The IC50 values were calculated.
HIV-1 protease assay
The HIV Protease Activity Detection Kit (Sigma-Aldrich, Burlington, USA) was used to detect the inhibitory potential of AuNPs against HIV-1 protease. This kit is specific for the HIV Protease, a retroviral aspartyl protease critical for the HIV life-cycle. Briefly, a 1:1 master mix of the constrained substrate and Tris-EDTA (TE) buffer (pH 2.0) was prepared. Then, 20 µL of the master mix was pipetted to each microplate well. Later, 0.5 µL of His tagged HIV protease, recombinant from HIV-1 (Sigma-Aldrich, Burlington, USA) added to the positive control and experimental reaction wells. The microplate was incubated overnight at 37 ℃ in a humidified atmosphere of 5% CO2. Finally, 200 µL of the detector was added to each well, and the plate was incubated for 4-24 h at 37 ℃ in a humidified atmosphere of 5% CO2. At the excitation/ emission of 448/525 nm, the released GFP was measured. The blank fluorescence values were subtracted from the final fluorescence values of the sample(s) and the positive control. The data were obtained from three separate experiments, and the mean ± SD of RFU was determined.
Investigation of cellular uptake and internalization
Confocal laser scanning microscopy (CLSM)
In 6-well plates, TZM-bl cells (0.1e6/well) were plated on Poly-L-Lysine (Sigma-Aldrich, Burlington, USA) pre-coated coverslips and were incubated overnight at 37 ℃ in a humidified atmosphere of 5% CO2. The medium was replenished to a serum-free medium with 0.2 mg/mL of Cy5.5-AuNPs. The medium was aspirated after 2 h of treatment, and the treated cells were rinsed three times with PBS. Subsequently, the cells were fixed in 4% Paraformaldehyde (PFA; Sigma-Aldrich, Burlington, USA) in PBS (pH 7.4). For 3 min, the cells were permeabilized with 0.1% Triton™ X-100 (Sigma-Aldrich, Burlington, USA) in PBS. The washed cells were then stained with 1 ng/mL DAPI (for nuclei; Sigma-Aldrich, Burlington, USA). After staining, the coverslips were rinsed, dried, and mounted on slides using ProLong™ Gold Antifade Mountant (Invitrogen, New York, USA). Cells were imaged with oil using CLSM (Olympus FLUOVIEW FV3000, Tokyo, Japan) at 60X magnification. The images were analyzed using cellSens Dimension with multichannel 5D software.
Flow cytometry analysis (FCM)
Cy5.5-AuNPs were deployed as a tracer, and the internalization of AuNPs in cells was measured by flow cytometry. TZM-bl cells (0.1e6 /well) were plated in 6-well plates and incubated for 24 h at 37 ℃ in a humidified atmosphere of 5% CO2 to form a confluent monolayer. The culture medium was swapped with serum-free DMEM the following day, and the cells were allowed to rest for 30 min. Next, to induce cellular internalization of the nanoparticles, 0.2 mg/mL of the Cy5.5-AuNPs were dispersed in serum-free DMEM and introduced to the cells. The plates were then incubated for 0, 15, 30, 45, 60, and 120 min at 37 ℃ in a humidified atmosphere of 5% CO2. Finally, the cells were rinsed three times with PBS to eliminate free Cy5.5-AuNPs before being detached using Trypsin-EDTA 0.05% (Gibco-Invitrogen, New York, USA). For acquisition, the collected cells were resuspended in 500 μL PBS in a FACS tube and analyzed using a FACSAria™ Fusion flow cytometer (Becton Dickinson, Franklin Lakes, USA) to quantify cellular uptake. The data analysis was conducted using FACSDiva™ software. Three independent replicates of the experiment were carried out.
In vivo and ex vivo biodistribution
All studies were conducted following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Department of Animal Husbandry and Dairying (DAHD), Ministry of Fisheries, India. Ethical approval was obtained from the Institutional ethics committee (IEC) of the ICMR-NARI, Pune, India, and the Institutional Animal Ethics Committee (IAEC) of the Indian Institute of Science Education and Research (IISER), Pune, India. All mice utilized in this investigation were retained in standard dwelling conditions. They have been kept in Individually Ventilated Cages (IVC) with a 12 h light/dark cycle. The water and food were provided ad libitum.
In healthy mice, in vivo fluorescence imaging was employed to observe the real-time distribution of Cy5.5-AuNPs. This pre-clinical study is important to evaluate the efficacy of the synthesized AuNPs in reaching the tissues/organs. A single dose of 0.2 mL of Cy5.5 NHS ester-AuNPs (5 mg/kg, n = 3 for each timepoint) or PBS (control, n = 1 for each timepoint) was administrated intravenously to seven weeks old male BALB/c mice (≈ 25 g of weights). Using an in vivo imaging system (IVIS Spectrum; PerkinElmer, USA), the fluorescence distribution of whole-body (mice were under isoflurane) was captured at various time intervals post i.v. administration (0.25 h, 1 h, 2 h, 24 h, 48 h, and 7 days). The IVIS® imaging system's scanning parameters were configured to emission/excitation at 673/707 nm, having a field of view of 13.5 cm and a fluency rate of 2 mW/cm2. Mice were sacrificed humanely at each time point following the scan. NIR fluorescence pictures of ex vivo organs were acquired after the main organs, the liver, heart, kidney, lung, and brain, were excised (673em/707ex filters). Using Living Image® 4.72 software (64-Bit), the acquired pictures were processed, and Regions of Interest (ROI) were generated. After subtracting the background signal, the average radiant efficiency [p/s/cm2/sr]/ [W/cm2] was determined. The harvested organs were preserved in 10% neutral buffered formalin (Sigma-Aldrich, Burlington, USA) for histopathology analysis. FLIT was also recorded, which leverages the signal's geometry, depth, and intensity to create a 3D reconstruction of the mouse and provides anatomical localization of the fluorescence signal [100, 101].
Histopathological staining and analysis
The organs of mice collected at each time point were fixed with 4% PFA, embedded in paraffin, sliced into thin sections, and stained with Hematoxylin/Eosin (H&E). Using a digital microscope, the slides were examined. For histological examination, each tissue (liver, heart, kidney, lung, and brain) was immersed in a 10% buffered neutral formaldehyde solution for 48 h. The tissues were dehydrated and embedded in paraffin once the chemical fixation was accomplished. A microtome was used to cut thin tissue sections with a thickness of 4-5 μm (Leica RM 2025; Wetzlar, Germany). Subsequently, the paraffin coat was dissolved in a water bath and closed on slides coated with Poly-L-Lysine. After being placed in an oven at 37 ℃, the sections were deparaffinized in xylol, and dehydrated by lowering the percentage of ethyl alcohol in the alcohol series. Finally, they were stained with H&E staining. The optical microscope (Olympus CH40; Tokyo, Japan) and a digital camera were used to inspect the slides.
Statistical analyses
All experiments were performed in triplicate, and means with standard errors were computed. The statistical significance of differences in values between the study groups and controls was determined using one-way ANOVA. Differences with p ≤ 0.05 were regarded as statistically significant. GraphPad Prism 5 for Windows (GraphPad Software, USA) was used to conduct all statistical analyses.