Materials
All the chemical precursors used for the synthesis of SPIONs and G4 PAMAM Dendrimer were of analytical grade and were used without any further purifications. Wherever necessary cell culture grade Milli-Q water was used. Ethylene diamine (EDA), Methyl Acrylate (MA), Iron (III) nitrate nonahydrate (Fe(NO3)3. 9H2O), Citric acid (C₆H₈O₇), Sodium Hydroxide (NaOH), APTES, EDC, NHS, MES Buffer were procured from Sigma-Aldrich. Solvents such as methanol, ethanol, dimethyl sulfoxide (DMSO), toluene, and methanol were procured from SRL Chemicals India. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin–EDTA, phosphate-buffered saline solution (PBS), antibiotics (penicillin-streptomycin solution), trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), paraformaldehyde, hydrochloric acid (HCl) and aq. Potassium ferrocyanide (C₆FeK₄N₆) were procured from HiMedia Laboratories. Test drug Ivermectin (API grade) was procured from Ami Chemicals, Mumbai. 3T3 cell line was procured from Dr. Caroline’s Lab, Navi Mumbai.
Synthesis Of Dendrimer Coated Spions:
The synthesis of SPIONs was carried out using microwave assisted processes standardized in our lab which has been reported earlier [3] with slight modifications. Briefly, 30mM of aqueous Iron (III) nitrate nonahydrate [Fe(NO3)3. 9H2O] is kept under constant stirring while adding 0.025 mM ethanolic ascorbic acid. This solution is further stirred for about 5 more minutes and then irradiated in the Microwave Synthesizer (CEM Discover mono mode) for 3 minutes at 1300C and 65 W. The black precipitate of Fe3O4 is collect with the help of N52 neodymium magnet and washed 3–5 times with water and dried at 60°C. The particles are then calcinated at in a muffle furnace for about an hour at 500°C.
Further, the SPIONs were dispersed in D.I. water by sonicating for 45 minutes. The resultant colloidal solution was subjected to addition of 2% (V/V) APTES solution and was stirred for about 15 hours at R.T. [4–6]. The APTES functionalised SPIONs (SPION@APTES) were separated using N54 magnets, dried and stored in cool, dark conditions until further use.
Dendrimers were synthesized by Michael addition as described in literature [6–8] with slight modifications. Briefly, EDA in methanol solvent was allowed to [6, 7] react with MA for 24 hours at 45 °C giving − 0.5 G (half generation core) under nitrogen atmosphere. The − 0.5 G core is then reacted with EDA to give G0 (Core) of PAMAM dendrimer. This process is repeated iteratively till G4 PAMAM dendrimers have been synthesised. The G4 PAMAM dendrimers are then coated onto SPION@APTES by carbodiimide chemistry using EDC & NHS. The resultant final product of interest is G4 PAMAM Dendrimer Coated SPIONs (DC-SPIONs) which is used subjected to further experimental studies.
Characterization
SPIONs and DC-SPIONs both characterized using FTIR spectroscopy (IR affinity- 1S, Shimadzu), atomic force microscopy (AFM) (JPK, Germany); XRD (Bruker Discover D08, Germany), vibrating sample magnetometer (VSM, Oxford Instruments, UK) and MRI (GE Healthcare 3.0T MRI System, USA). SPIONs alone were characterized by HR-TEM (Tecnai G2, F30 300 kV) while dendrimers alone were characterized by boiling point and NMR analysis (Bruker AVANCE NEO NanoBay 400MHz).
MTT Assay
MTT assay was used to evaluate the metabolically active and viable cells. 3T3 cells were seeded in a a 96-well plate at in complete DMEM with 10% FBS and allowed to reach about 70–80% confluency. Various concentrations of SPIONs, Dendrimers and DC-SPIONs were added to the cells and were allowed to be incubated for 24 hours at 37°C under 5% CO2. After 24 hours, the media was changed and MTT reagent was added to each well, and the plate was incubated for 4 h at 37°C. Formation for formazan crystals was observed after which, the media was gently removed and the crystals were dissolved in 10% DMSO. The readings were immediately recorded using a Multiskan Sky High UV-Vis spectrophotometric reader between 550–600 nm.
Prussian Blue staining
Prussian blue staining is used to detect the amount of ferric iron (SPIONs) post cellular internalization. Primarily, the 70–80% confluent 3T3 cells are exposed to various concentrations of SPIONs, Dendrimers and DC-SPIONs and incubated for 24 hours at 37°C under 5% CO2. Post incubation, the cells are gently washed and fixed using 4% paraformaldehyde. The fixed cells are flooded with Pearl’s solutions (aq. Hydrochloric acid and aq. Potassium ferrocyanide) for about 20 mins, followed by a counter stain with nuclear fast red dye. The cells are observed for bright blue specks indicative of presence of ferric ions [9–11].
In Vitro MRI Contrast studies
The study was carried out using a 3T clinical MRI machine and the method followed was similar to the one described in one of our previous publications with slight modifications [3]. Briefly, 3T3 cell line was utilized for this study. About 1 x 106 cells per well were seeded in a 24 well plate and allowed to establish about 70–80% confluency. Various concentrations of SPIONs, Dendrimers and DC-SPIONs were added to the wells and then incubated for 24 hours at 37°C under 5% CO2. Post incubation, the media was gently aspirated and cells were washed using PBS. The cells were harvested using trypsin and collected as a pellet which was again redispersed in PBS. For preparation of cell phantoms, the suspension was mixed with equal volume of 2% agarose solution and allowed to settle in a labelled 24-well plate. The sample plate was now used to generate the phantom images of T2 contrast [12–17]. The data acquisition parameters in this case were: Echo time (TE) = 10 ms, Repetition time (TR) = 7000 ms. The relaxivity (r2) values were calculated using the following equation: r2 = (1/T2) sec− 1.