Lyophilized Iron Oxide Nanoparticles Encapsulated in Amphotericin B: A Novel Targeted Nano Drug Delivery System for the Treatment of Systemic Fungal Infections

We formulated and tested a targeted nanodrug delivery system to help treat life-threatening invasive fungal infections, such as cryptococcal meningitis. Various designs of iron oxide nanoparticles (IONP) (34–40 nm) coated with bovine serum albumin and coated and targeted with amphotericin B (AMB-IONP), were formulated by applying a layer-by-layer approach. The nanoparticles were monodispersed and spherical in shape, and the lead formulation was found to be in an optimum range for nanomedicine with size (≤36 nm), zeta potential (−20 mV), and poly dispersity index (≤0.2), and the drug loading was 13.6 ± 6.9 µg of AMB/mg of IONP. The drug release profile indicated a burst release of up to 3 h, followed by a sustained drug release of up to 72 h. The lead showed a time-dependent cellular uptake in C. albicans and C. glabrata clinical isolates, and exhibited an improved efficacy (16–25-fold) over a marketed conventional AMB-deoxycholate product in susceptibility testing. Intracellular trafficking of AMB-IONP by TEM and confocal laser scanning microscopy confirmed the successful delivery of the AMB payload at and/or inside the fungal cells leading to potential therapeutic advantages over the AMB-deoxycholate product. A short-term stability study at 5 °C and 25 °C for up to two months showed that the lyophilized form was stable.

bound albumin solution. Immediately, this 0.5 mL of AMB-bound BSA mixture was added to the surface-activated IONP mixture. The mixture was reacted for two hours with continuous mixing at ambient temperature. Then 10 µL of the quenching buffer was added, mixed well and incubated at ambient temperature for at least 10 minutes. Then the nanoparticles were washed as per procedure above and stored.
Design E: The order of the layers from inside to outside are IONP and AMB (Figure 1 design E main article). 0.5 mL of AMB solution (50-100 µg/mL) was added to the activated IONP mixture. The mixture was reacted for two hours with continuous mixing at ambient temperature. Then 10 µL of the quenching buffer was added, mixed well and incubated at ambient temperature for at least 10 minutes. Then the nanoparticles were washed as per procedure above and stored.

S2. Drug loading (Refereed in Section 2.2 of Main Article)
Loaded AMB amounts in the AMB-IONP were determined by a validated HPLC method developed previously [1]. All chromatographic studies were conducted on validated and qualified equipment. The HPLC system consists of a Waters HPLC Alliance system on an e2695 separations module with a Waters 2998 PDA detector (Milford, MA, USA). Samples were injected using a Waters auto-injector and the instrument was controlled by use of Empower2® software (Milford, MA, USA). Components were separated on a Waters XBridgeTM C18 reversed-phase column (Milford, MA, USA) with 150 mm × 4.6 mm dimensions and 3.5 µm particle size. The column was kept thermostatic at 30°C in a Waters column oven (Milford, MA, USA). The detection and quantification of AMB was performed using an isocratic method with a mobile phase of acetic acid (0.73%)-acetonitrile (59:41, v/v). The flow rate of the solvent was 1.0 mL/min with a runtime of 6 minutes. The drug was extracted from the formulation by dispersing the AMB-IONP in DMSO at a dilution factor of 25 and then subjecting to sonication for 30 minutes. The dispersion was then subjected to magnetic separation for 10-12 h at 4 °C. An aliquot was taken for HPLC analysis. Samples were injected into the column at a constant volume of 20 µL and a PDA detector at 408 nm was employed to obtain the response for AMB.
The AMB peak is well resolved from the adjacent peaks with a retention time of 2.7 min. The drug loaded into the formulations was directly determined from each formulation by extracting the drug into DMSO. The drug loading was determined by amount of drug in nanoparticles over amount of nanoparticles. The amount of drug loaded in the formulations was calculated from a standard curve which was linear between 0.050 to 1 µg/mL with a coefficient of determination (r 2 ) value of 0.9987.

S3. Particle size and Zeta potential (Refereed in Section 2.2 of Main Article)
The mean hydrodynamic size (Z-average) of IONP and AMB-IONP were determined by a dynamic light scattering (also known as photon correlation spectroscopy) technique using Malvern Zetasizer Nano ZS90 (Westborough, MA, USA) equipped with 50 mW diode laser as the light source, operating at 532/633 nm. Particle-scattered photons were detected at an angle of 90°. Each sample was determined in duplicate. ζ-potential was estimated with an electrophoretic light scattering (also known as laser Doppler microelectrophoresis) technique with the Malvern Zetasizer Nano ZS90. Figure S1. Schematic diagram of the in-house experimental setup for in vitro drug release study.

S5. Physicochemical Characterization of Lyophilized AMB-IONP (Refereed in Section 2.16 of Main Article)
The lyophilized product of the AMB-IONP was subjected to visual inspection to determine height of the cake, any cake separation, and product on rubber closures, meltback, and presence of any surface abnormalities such as air bubbles or puffing.
The lyophilized cake was then subjected to moisture content analysis using a Mettler Toledo DL31 Karl Fischer Titrator (Mettler Toledo Inc., Boston, MA, USA). The equipment was calibrated before use on the day of analysis. The sample of about 5 mg of the lyophilized product was weighed accurately and added to the titration mixture. The measurements were performed in triplicate and the moisture content in the lyophilized cake was calculated as % w/w.
The lyophilized product was reconstituted with water and the time for re-dispersibility after reconstitution was recorded. The mean hydrodynamic size (Z-average) and ζ-potential of the reconstituted AMB-IONP were determined.   Figure S4. DSC thermograms of different sucrose weight ratios to IONP. (Notes: The data indicates that the Tgʹ values increased with an increase of sucrose weight ratio in the mixture. The Tgʹ value was found to be about -25 °C. Thus, primary drying temperature of 2-5 °C < Tgʹ was used to inhibit collapse.). Figure S5. Physical appearance of lyophilized product of AMB-IONP with different weight ratios of sucrose. (Sucrose weight ratio of 16 and higher found to be optimum without any visible abnormalities of the cake).