Chemicals
All organic solvents utilized in experiments were of high-performance liquid chromatography (HPLC) grade and purchased form Fisher scientific UK. Ferric Sulphatehexahydrate Fe2(SO4)3·6H2O, Ferrous sulphateheptahydrate (FeSO4·7H2O), 3-(Trimethoxysilyl)propyl methacrylate (MPTES), 4-dimethyl amino pyridine (DMAP), azobisisobutyronitrile (AIBN), ammonium hydroxide, dicyclohexylcarbodiimide (DCC), metronidazole and amphotericin B were obtained from Sigma-Aldrich, Merck Darmstadt (Germany) through a local supplier.
Synthesis of Met-MNPs and Met-MNPs-Amp
The synthesis of Met-MNPs-Ampwas carried out in multiple steps described in Fig. 1. First, compound 1 was synthesized by the following procedure. Methacrylic acid (0.77 g, 9.0 mmol), 4-Dimethyl amino pyridine (0.061 g, 0.5 mmol) and dicyclohexylcarbodiimide (DCC, 1.85 g, 9.0 mmol) were taken in a round bottom flask containing tetrahydrofuran (THF) (20 mL) connected with a condenser. The reaction was stirred for 10 min at 0 °C under Ar atmosphere. Metronidazole (0.51 g, 3.0 mmol) was added later and stirred at 0 °C for 6 h. The resulting mixture was concentrated in vacuo and then subjected to column chromatography using flash silica as a stationary phase. Compound 1 was obtained using hexane and ethyl acetate (6:4 v/v) as mobile phases. Rf: 0.60 (DCM:MeOH, 9: 1, v/v). Yield 35%; M.P.: 160–170 °C, EI-MS m/z 239.1. 1H NMR (300 MHz MeOD) δ ppm: 7.9 (s 1H imidazole), 2.4 (s 3H CH3), 5.9 (s 1H C=C), 5.5 (s 1H C=C), 3.02 (s 3H CH3) 4.7 (t 2H CH2), 4.5 (t 2H CH2).
For the second step, a co-precipitation method was adopted for the preparation of narrow range MNPs first by using the previously described protocol (Petcharoen and Sirivat 2012). Here, MNPs was further etched with 3-(Trimethoxysilyl)propyl methacrylate (MPTES) through silanization reaction with slight modification (Saif et al. 2015). Briefly, a MNPs (10 mg/mL) suspension was prepared in ethanol and then MPTES was added in such a manner that the ratio between NPs to MPTES remains 1:6 and left with constant stirring at 60 °C for 4 h. The resultant brownish suspension was washed several times with ethanol and freeze dried. Finally, polymerization was adopted with the aim to functionalize compound 1 on MPTES coated MNPs. Typically, MPTES-MNPs (10 mg/mL) dispersion was prepared in anhydrous ACN under Ar atmosphere. Then, compound 1 (1.20 g, 5 mmol) was added to the above dispersion and after being stirred to 10 min, AIBN (1.32 g, 8 mmol) was added to the resulting mixture and refluxed for 15 h under Ar atmosphere at 60 °C. The prepared Met-MNPs underwent successive washing with ACN and were freeze dried. Met-MNPs were further exploited for their drug entrapment potential using a passive drug loading technique. Briefly, Met-MNPs were incubated with various equivalents of AmpB in methanol for 24 h on a shaker at 200 rpm under ambient conditions. The resulting Met-MNPs-Amp was removed by means of a permanent magnet and washed sequentially with water to remove the unloaded drug and stored at 4 °C for further analysis.
Characterization of Met-MNPs-Amp
Size, size distribution and morphology
The average hydrodynamic diameter and polydispersity index (PDI) of vacant and Met-MNPs-Amp were investigated via Zetasizer (Zetasizer Nano ZS90 Malvern Instruments, Malvern, UK). Concisely diluted nanoparticles were cautiously transferred to a transparent plastic cuvette to avoid any bubble formation. The cuvette was then placed in the cell holder of the instrument and analysis was taken at 90° scattering at 25 °C. The medium viscosity and refractive index were constant and kept at 1.0, 1.33 and 80.4 mPa, respectively. Nanoparticles were also characterized for morphology using atomic force microscopy (AFM, Agilent 5500). A drop of the formulation was placed on a mica slide and air dried at ambient temperature and placed under a microscope. The morphology was investigated at non-contact model.
Drug entrapment efficiency determination
Entrapment efficiency is described as the amount of drug entrapped into a carrier with respect to the initial amount of drug added (Manatunga et al. 2017). Therefore, the entrapment efficiency was determined by measuring the amount of unloaded drug at 405 nm by UV spectroscopy (Shimadzu 1800 series, Shimadzu Japan). Drug entrapment was investigated inthe following relation.
$$\% {\text{EE}} = {{\left( {{\text{Q}}_{\text{t}} - {\text{Q}}_{\text{p}} } \right)} \mathord{\left/ {\vphantom {{\left( {{\text{Q}}_{\text{t}} - {\text{Q}}_{\text{p}} } \right)} {{\text{Q}}_{\text{t}} }}} \right. \kern-0pt} {{\text{Q}}_{\text{t}} }} \times 100$$
Qp: Quantity of free drug, Qt: Quantity of drug added, % EE: Entrapment efficiency of loaded drug in percent.
Fourier Transformed Infrared (FT-IR) Spectroscopy
Fourier transformed infrared (FT-IR, IR-470 spectrometer (Shimadzu, Kyoto, Japan)) analysis was performed in order to elucidate the possible drug entrapment and surface functionalization. Small amounts of powdered nanoparticles were mixed with KBr and subjected to a high pressure of 200 Psi to obtain self-supporting disks.
Biocompatibility studies
Hemocompatibility study
Human blood was obtained from healthy individuals in the University of Karachi, Pakistan following relevant guidelines and regulations (Ethics committee approval ICCBS/IEC/LET/015/2018). Ethylenediaminetetraacetic acid (EDTA) stabilized fresh human blood samples (5.0 mL) were added to 10 mL of phosphate-buffered saline (PBS). Then, red blood cells (RBCs) were isolated via centrifugation at 6000 rpm and washed several times with PBS solution. The purified RBCs were further diluted in 50 mL PBS and Triton X was used as the positive control, respectively. Then, 0.2 mL of diluted RBC suspension and 0.8 mL of Met-MNPs solutions in a range of 200–1000 µg/mL were mixed by vortexing. All sample tubes were kept in static condition at room temperature for 3 h. Finally, the mixtures were centrifuged at 12,000 rpm for 10 min, and 1.5 mL of the supernatant of each sample was transferred to a cuvette. The absorbance values of the supernatants at 540 nm were determined by UV–Vis spectrophotometry. The percent hemolytic activity of RBCs was calculated using the following relation.
$$\% {\text{H}}.{\text{A}} = {{{\text{R}}_{\text{s}} } \mathord{\left/ {\vphantom {{{\text{R}}_{\text{s}} } {{\text{R}}_{\text{c}} }}} \right. \kern-0pt} {{\text{R}}_{\text{c}} }} \times 100$$
Rs: Absorbance of sample, Rc: Absorbance of positive control, % H.A: Hemolytic activity in percent.
In vitro cytotoxicity
The synthesized nano carrier was screened for its cytotoxicity using MTT assay. Human cervical adenocarcinoma cells (HeLa) (ATCC® CCL-2™), were obtained from American Type Culture Collection (ATCC),and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% foetal bovine serum, 1% minimum essential medium amino acids, 1% l-glutamine and 1% antibiotics at 37 °C with 5% CO2 (Rajendran et al. 2019). NIH 3T3 cells (ATCC CRL-1658) were purchased from the ATCC and cultured in Dulbecco`s modified eagle`s medium (DMEM) having foetal bovine serum (10%) (Invitrogen, USA) and antibiotics (streptomycin and penicillin—about 50 U/mL).Both cell lines were incubated into well plates with 96 wells and 8 × 103 and 6 × 104 cells/well thickness, individually, in a (200 µL) refined media. After incubation for about 24 h, fresh media was introduced (200 µL) consisting of Met-MNPs at various concentrations from 25 to 100 μg/mL. Cells incubated in media without NPs were used as the negative control and developed for 48 h. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide solution (MTT) in PBS was introduced into each well (20 µL; 5 mg/mL). The unreacted solution was expelled after 4 h incubation. The resulting formazan crystals were dissolved and introduced to 200 μL DMSO per well before being analysed at 570 nm in a microplate reader. For the positive control and reference, standard Doxorubicin and Cyclohexylamine were used. The following relation was utilized to calculate the % cell viability.
$${\text{Cell}}\;{\text{viability}} = {{{\text{A}}_{\text{t}} } \mathord{\left/ {\vphantom {{{\text{A}}_{\text{t}} } {{\text{A}}_{\text{c}} }}} \right. \kern-0pt} {{\text{A}}_{\text{c}} }} \times 100$$
At: Mean of Absorbance value of Test Sample, Ac: Mean of Absorbance value of Control.
Culture of amoebae
Acanthamoeba castellanii of T4 genotype (ATCC 50492) was purchased from American Tissue Culture Collection (ATCC). A. castellanii was cultured in growth medium consisting of Proteose peptone (0.75% w/v), yeast extract (0.75% w/v), and d-glucose (1.5% w/v) (PYG) (Anwar et al. 2019b).
Amoebicidal assays
Amoebicidal assays were performed as previously described (Rajendran et al. 2019). Briefly, 5 × 105 amoebae were incubated with compounds at various concentrations for 24 h at 30 °C. The positive control used was chlorhexidine, respective solvents were used as solvent controls and RPMI-1640 alone was used as the negative control. A haemocytometer and 0.1% trypan blue solution were used to distinguish between live (unstained) and dead (stained) cells, in trypan blue exclusion assays. The number of viable amoebae determined were represented as % cell death for graphical illustration.
Cysticidal assays
Acanthamoeba castellanii cysts were prepared by methods described previously (Anwar et al. 2019c). After the formation of mature cysts, 5 × 105 cysts were treated with various concentrations of Met-MNPs-Amp in 24-well plates in the presence of RPMI-1640 and incubated for 72 h at 30 °C. Chlorhexidine was used against A. castellanii as the positive control. RPMI-1640 alone was used as the negative control. After the incubation, the remaining viable cysts were enumerated using a hemocytometer by trypan blue exclusion method.