Tuneable manganese oxide nanoparticle based theranostic agents for potential diagnosis and drug delivery

Among various magnetic nanoparticles, manganese oxide nanoparticles are considered as established T1 magnetic resonance imaging (MRI) contrast agents for preclinical research. The implications of their degradation properties and use as therapeutic carriers in drug delivery systems have not been explored. In addition, how the chemical composition and size of manganese oxide nanoparticles, as well as the surrounding environment, influence their degradation and MRI contrast properties (T1vs. T2) have not been studied in great detail. A fundamental understanding of their characteristic properties, such as degradation, is highly desirable for developing simultaneous diagnosis and therapeutic solutions. Here, we demonstrate how the precursor type and reaction environment affect the size and chemical composition of manganese oxide nanoparticles and evaluate their influence on the nanoparticle degradability and release of the drug l-3,4-dihydroxyphenylalanine (l-dopa). The results show that the degradation rate (and the associated release of drug l-dopa molecules) of manganese oxide nanoparticles depends on their size, composition and the surrounding environment (aqueous or biometric fluid). The dependence of MRI relaxivities of manganese oxide nanoparticles on the size, chemical composition and nanoparticle degradation in water is also established. A preliminary cell viability study reveals the cytocompatible properties of l-dopa functionalized manganese oxide nanoparticles. Overall, this work provides new insights into smartly designed manganese oxide nanoparticles with multitasking capabilities to target bioimaging and therapeutic applications.


Synthesis of MONP-8 and MONP-6:
These NPs were synthesized by thermal decomposition of manganese oleate. Firstly, the manganese oleate precursor was synthesized by mixing manganese (II) chloride hexahydrate (20 mmol), sodium oleate (60 mmol), deionized water (30 mL), hexane (70 mL) and ethanol (40 mL) in a 250 mL round bottom flask. The reaction mixture was vigorously stirred at 70 °C for 4 h under argon atmosphere. After the reaction, the organic component was separated from the aqueous phase and washed three times with deionized water to remove reaction byproducts. The product was further purified in a rotary evaporator to remove residual hexane, ethanol and water, before being transferred to a glass vial and stored in a fridge (4 °C). For the synthesis of MONP-8, the reaction mixture containing 3.4 g of manganese oleate, 1.2 mL oleic acid, and 50 mL of 1-octadecene (1-hexadecene for MONP-6) was heated to an end temperature of 320°C (300°C for MONP-6) at the rate of 3°C min -1 , and kept for 45 minutes at the end temperature. After the reaction, MONPs were obtained by precipitating the reaction mixture with 40 mL toluene and 120 mL acetone.
Functionalization of MONPs with L-dopa: 5 mg of dried MONPs was dispersed in 5 mL chloroform and mixed with 20 mg of L-dopa and 3 mL DMSO. The mixture was heated to 70°C and left to react for 1 hour under constant stirring. The surface-coated nanoparticles were separated by centrifugation (4400 rpm, 10 minutes). Following the removal of the supernatant, the precipitate was dispersed in acetone and centrifuged again to remove the unbound L-dopa molecules. The functional NPs were dispersed in deionized water prior to use.
Characterization: Transmission electron microscopy (TEM) images were taken with a TEM (Philips CM30), operating at 120 kV and JEOL 2100 operating at 200 kV. X-ray diffraction (XRD) analysis was performed with an X-ray diffractometer (Malvern PANanalytical X'pert Pro) using Cu Kα (λ = 0.154nm) in the 2θ range of 15° -80°. Spectral data was analysed using ICDD PDF-4+ 2019 software. X-ray photoelectron spectroscopy (XPS) measurements on MONPs were performed using XPS K-Alpha spectrometer (Thermo Fischer Scientific) with a monochromatic AL K-Alpha (1486.6eV) X-ray source. Survey spectral peaks were obtained using 1eV energy step and pass energy of 200eV. The high resolution XPS spectra were collected using 0.1eV energy steps and pass energy of 50 eV. XPS data was analysed using S3 Thermo Avantage V5.9902 whilst elemental spectral data was analysed using CasaXPS. Background subtraction of the elemental spectra was performed using the Shirley method and peak deconvolution with a Gaussian-Lorentzian function (70% Gaussian, 30% Lorentz).

Time dependent degradation study:
To measure the degradation rate of MONPs in water, 5 mg functionalised nanoparticles were dispersed in 5 mL of MilliQ water and sonicated to ensure proper mixing. The hydrodynamic radius measurements, performed using a Malvern Zetasizer Nano ZS instrument at 25°, were taken at 0, 0.5, 1, 2, 4, and 24 hours after dispersion. Prior to each measurement, the solution was sonicated for 2 minutes to ensure proper dispersion. To measure the degradation under physiological conditions, the protocol above was modified. 5 mg of functionalised nanoparticles was dispersed in 5 mL of physiological fluid (25 v/v% FBS in MilliQ water). Prior to each measurement, the nanoparticles were diluted with MilliQ water (6.25 v/v% nanoparticle solution). All results were analysed using the Zetasizer software.
Relaxivity measurement: A 0.5 T time domain NMR relaxometry (Minispec Analyzer MQ20, Bruker) was used for T1 and T2 measurements. A total of 800 μL of each sample was poured in a 10 mm sample tube. All the measurements were made at 40°C using a thermostatic circulator water bath. T2 decay curves were acquired using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with a repetition time (TR) of 1s and 1000 spin-echoes with an echo time spacing of 0.25 ms. T1 values were obtained using an inversion-recovery pulse sequence with a repetition time (TR) of 2 seconds and 20 logarithmically spaced inversion times ranging from 0.5 ms to 1 s. Relaxivities (mM -1 s--1 ) were calculated by taking the inverse of T1 and T2 times (seconds) and dividing these values by the total metal ion (Mn) concentration (mM) for each sample as determined by inductively coupled plasma mass spectroscopy (ICP-MS).

Magnetic properties:
Magnetic properties of MONPs were measured with a quantum design physical properties measurement system (PPMS) with vibrating sample magnetometer (VSM). ZFC/FC curves. M(H) were measured by cooling the sample at zero field from room temperature to 2K and warming up from 2K to 380K at the rate of 1K/min with small field 500 Oe. M(H) curves at 5K were measured from sweeping the field from -60kOe to +60kOe.
Cytotoxicity: Human foreskin fibroblasts were cultured in complete media containing DMEM, supplemented with 10% (v/v) fetal calf serum, 30 mg/ml penicillin, and 100 mg/mL streptomycin. Cells were cultured at 37°C with 5% CO2, and culture medium changed every 3 days until cells were passaged at 80-90% confluence. All fibroblasts were at passage 12. Cells were then seeded at a density of 10x10 3 cells/well before incubation with MONPs at various concentrations. The cell viability was assessed after 1 day, 3 days and 7 days with an MTS assay (using Cell Titre 96 Aqueous non radiologic cell proliferation assay kit (Promega, USA)) following the manufacturer's instructions. The absorbance was recorded using a Multiskan Go Spectrophotometer at a wavelength of 490nm. ICP-MS was used to quantify the concentration of MONPs present in each group.  Table S1. Elemental (at%) of MONPs determined from XPS survey spectra before and after the surface functionalization of MONP with L-dopa. Oleylamine capped MONP-N 2 and MONP-Air were synthesized in the presence of oleylamine solvent. Oleic acid capped MONP-8 and MONP-6 were synthesized in the presence of octadecene and oleic acid.