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Tuning the size and composition of manganese oxide nanoparticles through varying temperature ramp and aging time

  • Celia Martinez de la Torre,

    Roles Conceptualization, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Chemical and Biomedical Engineering, West Virginia University, Morgantown, WV, United States of America

  • Jasmine H. Grossman,

    Roles Conceptualization, Writing – review & editing

    Affiliation Department of Chemical and Biomedical Engineering, West Virginia University, Morgantown, WV, United States of America

  • Andrey A. Bobko,

    Roles Conceptualization, Formal analysis, Investigation, Writing – review & editing

    Affiliation Department of Biochemistry and In Vivo Multifunctional Magnetic Resonance Center, West Virginia University, Morgantown, WV, United States of America

  • Margaret F. Bennewitz

    Roles Conceptualization, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Chemical and Biomedical Engineering, West Virginia University, Morgantown, WV, United States of America


Manganese oxide (MnO) nanoparticles (NPs) can serve as robust pH-sensitive contrast agents for magnetic resonance imaging (MRI) due to Mn2+ release at low pH, which generates a ~30 fold change in T1 relaxivity. Strategies to control NP size, composition, and Mn2+ dissolution rates are essential to improve diagnostic performance of pH-responsive MnO NPs. We are the first to demonstrate that MnO NP size and composition can be tuned by the temperature ramping rate and aging time used during thermal decomposition of manganese(II) acetylacetonate. Two different temperature ramping rates (10°C/min and 20°C/min) were applied to reach 300°C and NPs were aged at that temperature for 5, 15, or 30 min. A faster ramping rate and shorter aging time produced the smallest NPs of ~23 nm. Shorter aging times created a mixture of MnO and Mn3O4 NPs, whereas longer aging times formed MnO. Our results indicate that a 20°C/min ramp rate with an aging time of 30 min was the ideal temperature condition to form the smallest pure MnO NPs of ~32 nm. However, Mn2+ dissolution rates at low pH were unaffected by synthesis conditions. Although Mn2+ production was high at pH 5 mimicking endosomes inside cells, minimal Mn2+ was released at pH 6.5 and 7.4, which mimic the tumor extracellular space and blood, respectively. To further elucidate the effects of NP composition and size on Mn2+ release and MRI contrast, the ideal MnO NP formulation (~32 nm) was compared with smaller MnO and Mn3O4 NPs. Small MnO NPs produced the highest amount of Mn2+ at acidic pH with maximum T1 MRI signal; Mn3O4 NPs generated the lowest MRI signal. MnO NPs encapsulated within poly(lactide-co-glycolide) (PLGA) retained significantly higher Mn2+ release and MRI signal compared to PLGA Mn3O4 NPs. Therefore, MnO instead of Mn3O4 should be targeted intracellularly to maximize MRI contrast.


The use of metal oxide nanoparticles (NPs) has been increasing over the past decades due to their magnetic, electric, and catalytic properties. Of particular interest to biomedical applications is the ability of metal oxide NPs, such as iron oxide and manganese oxide (MnO), to serve as contrast agents for magnetic resonance imaging (MRI) [1]. Typically, the metal oxide crystals are encapsulated within a polymer to promote hydrophilicity and biocompatibility. Iron oxide NPs are superparamagnetic and cause dark contrast on T2 and T2* MRI. The negative contrast of iron oxide NPs is so robust that even single cells can be visualized on MRI if each cell accumulates at least 1 pg of iron [24]. However, iron oxide NPs elicit strong MRI signal in their intact form and therefore constantly generate contrast, or are always in the “ON” state. Furthermore, naturally occurring iron present inside the liver, spleen, bone marrow and blood leads to dark contrast that can be difficult to differentiate from applied iron oxide NPs. As an alternative, manganese oxide (MnO) NPs provide the advantage over iron oxide in that they can provide switchable, bright contrast on T1 MRI due to the paramagnetic properties of the Mn2+ ion. Our group and other studies have shown that intact MnO NPs are in an “OFF” state and create minimal T1 MRI signal due to the Mn2+ ions being tightly bound and inaccessible to the surrounding water molecules [510]. In acidic media, MnO dissolves to form Mn2+, which coordinates with water molecules to decrease T1 and produce a positive MRI signal, thus turning “ON” MRI contrast [510].

Compared to gold standard and pH-sensitive gadolinium T1 MRI contrast agents, MnO NPs have superior MRI properties. Clinically used gadolinium chelates are not pH-sensitive, and are always in an “ON” state, which highlights any well vascularized structure and can lead to false positive diagnoses in which a benign tumor can be mistaken for a malignant tumor [1113]. In addition, many standard gadolinium agents such as MultiHance have low relaxivities of ~4 mM-1s-1 at 1.5T-4.7T [14]; Mn2+ has a higher relaxivity of ~ 7–8 mM-1s-1 at the same field strengths [5, 15, 16]. Furthermore, when gadolinium agents are altered to be pH sensitive, T1 relaxivity changes only ~2–4 times [17, 18] over pH 5 to 7.4. Polymeric MnO NPs are more powerful smart contrast agents, producing a relaxivity change of ~30 times, as intact NPs have very low r1 (0.12–0.21 mM-1s-1) [5, 19] at pH 7.4 and release Mn2+ at pH 5 to increase relaxivity to 7 mM-1s-1. MnO NPs with targeting agents can be utilized for enhanced specificity for detection of cancerous tumors through NP dissolution inside tumor cells within low pH endosomes or lysosomes.

To enhance MRI signal generation, it is necessary to fine-tune synthesis strategies to control and reduce the size of MnO NPs. It was hypothesized that smaller NP diameters will increase the surface area to volume ratio to facilitate faster dissolution of MnO to Mn2+ to generate higher MRI signal under acidic conditions and allow for more efficient packing of MnO NPs into polymeric or liposomal delivery systems. MnO NPs are commonly synthesized by thermal decomposition of a manganese-based compound such as manganese acetylacetonate (Mn(II) ACAC) [2022], Mn oleate [23], Mn acetate [21, 2426], Mn carbonate [27] or Mn stearate [28]. Several different variables can be modified to optimize the physical and chemical properties of the synthesized MnO NPs including the type of inert gas [2022], peak reaction temperature [2123, 26], total reaction time [23, 24, 28], and types/ratios of initial chemical compounds [2022, 24, 25] utilized in the reaction. To date, the effects of temperature ramp rate and aging time on both size and composition have not been explored. Herein, we systematically evaluate how two temperature ramping rates (10°C/min and 20°C/min) combined with increasing aging times (5, 15 and 30 min) at 300°C can be utilized to synthesize smaller NPs of pure MnO composition. MRI was utilized to evaluate T1 signal enhancement of Mn2+ ions released from NPs with different sizes and compositions to determine which formulation maximized MRI contrast at pH 5, 6.5 and 7.4.

Materials and methods


Mn(II) ACAC, oleylamine (70%, technical grade), poly(vinyl alcohol) (PVA), and rhodamine 6G were obtained from Sigma-Aldrich. Dibenzyl ether (≥99%, Acros Organics), hexane (≥98.5%, Macron Fine Chemicals), dichloromethane (≥99.5% stabilized ACS, BDH Chemicals), Dulbecco's phosphate buffered saline (PBS), sodium citrate dihydrate (BDH Chemicals), citric acid (VWR Chemicals, LLC), and manganese(II) chloride tetrahydrate (98–101% ACS, VWR Chemicals, LLC) were purchased from VWR. Hydrochloric acid (HCl) TraceMetal™ Grade was acquired from Fisher Scientific. Ethanol (Decon Laboratories Inc.) was obtained internally from West Virginia University’s Environmental Health and Safety Services. Ester-terminated 50:50 poly(D,L-lactide-co-glycolide) (PLGA) (inherent viscosity: 0.55–0.75 dL/g) was obtained from Lactel Absorbable Polymers.

Synthesis of MnO NPs

All work for MnO NP synthesis should be performed under a chemical fume hood with proper PPE including safety glasses, nitrile gloves and a lab coat. MnO NPs were fabricated using a standard thermal decomposition reaction of Mn(II) ACAC dissolved in oleylamine and dibenzyl ether based on the synthesis of magnetite (Fe3O4) NPs by Xu et al. [29] Mn(II) ACAC (6 mmol) was dissolved in 40 mL of oleylamine and 20 mL of dibenzyl ether. The solution was heated with a heating mantle connected to a thermocouple probe immersed into the reaction mixture and a programmable temperature controller. According to the user defined temperature profile, the mixture was heated from room temperature to 60°C over 30 min under a constant flow of inert N2 gas. A constant N2 flow was needed to successfully remove all oxygen from the reaction and obtain the desired product, MnO NPs. Then, the temperature was quickly raised to 300°C under N2 gas using two different ramp rates of 20°C/min or 10°C/min and aged at 300°C for either 5, 15, or 30 min. To assess variability between synthesized NP batches, each of the 6 different temperature profiles were independently run 3 times, obtaining a total of 18 batches of MnO NPs. All of the 18 batches were utilized for further experiments. Upon completion of the reaction, the heating mantle was removed to allow the solution to cool down to room temperature. The MnO NPs were pelleted in Nalgene® Oak Ridge centrifuge tubes following centrifugation at 17,400 x g for 10 min and washed 4 to 5 times in hexane and ethanol using the same centrifugation procedure. Resulting MnO NPs were resuspended in hexane and left in a fume hood to dry overnight. After overnight drying, the MnO NPs were baked over 24 hr in an oven at 100°C. The resulting MnO NPs synthesized by thermal decomposition were hydrophobic and capped with oleylamine.

PLGA encapsulation of MnO NPs

For a separate set of experiments, MnO NPs were encapsulated with PLGA using an oil-in-water emulsion technique mediated by sonication [5]. Approximately 100 mg of PLGA was dissolved in 2 mL of dichloromethane (DCM) in a test tube. Once the polymer was fully dissolved, 50 mg of MnO NPs and 500 μL of a 2 mg/mL DCM solution of rhodamine 6G were added to the tube of the polymer/solvent mixture. The organic mixture was bath sonicated while being added dropwise to 4 mL of an aqueous 5% w/v solution of PVA while vortexing at high speed. The mixture was vortexed for an additional 10 s and then sonicated 3 X [15 s ON– 5 sec OFF] at 40% amplitude with a Qsonica Sonicator 125 Watts to create the single emulsion. Immediately after sonication, the emulsion was poured into 60 mL of an aqueous 0.3% w/v PVA solution, under rapid mixing on a stir plate. The PLGA MnO NPs were stirred for 3 hr to evaporate the DCM and were collected by centrifugation at 17,400 x g for 10 min. NPs were washed 3 times with deionized water, resuspended in deionized water, frozen overnight at -80°C, and dried on a lyophilizer for 3 days.

Physical and chemical characterization of MnO NPs

To prepare samples for transmission electron microscopy (TEM), dried MnO NPs and PLGA MnO NPs were suspended in ethanol and deionized water, respectively, using bath sonication. After NP resuspension, 15 μL of the MnO NP mixture was dropped and air dried on 300 mesh copper PELCO® TEM grid support films of carbon type-B (Ted Pella, Inc.). Images were taken using a JEOL JEM-2100 transmission electron microscope at 200 kV for the MnO NPs and 120 kV for the PLGA MnO NPs.

X-ray diffraction patterns (XRD) of MnO NPs were obtained using a Panalytical X’Pert Pro X-ray diffractometer equipped with a Cu K-Alpha X-ray source operating at 45 kV and 40 mA in the Bragg-Brentano geometry. The spectra were collected over a 2θ range of 5° to 110° at a step size of 0.017° with a 1D silicon strip X-ray detector. The obtained XRD patterns were analyzed using the X’Pert HighScore Plus program. By comparing the XRD spectra of our synthesized MnO NPs with known spectra for MnO and Mn3O4, the program obtained an estimated composition for our samples.

Scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDS) was performed to analyze the elemental composition of the MnO NP samples using a Hitachi Scanning Electron Microscope S4700 operated at 15 kV with the EDAX Team EDS System.

X-ray photoelectron spectroscopy (XPS) measurements were performed using a PHI VersaProbe 5000 Scanning X-Ray Photoelectron Spectrometer (ULVAC-PHI, Inc.) at room temperature and under vacuum greater than 1e-6 Pascal. All measurements were performed using a focused Al K-Alpha X-ray source at a photon energy of 1486 eV and power of 25 W with an X-ray spot size of 100 μm. The take-off angle of the photoelectron was set at 45o. Compositional survey scans were obtained using a pass energy of 117.4 eV and an energy step of 0.5 eV. High-resolution detailed scans of each element were acquired using a pass energy of 23.5 eV and an energy step of 0.1 eV. All XPS spectra were referenced to the C1s peak at a binding energy of 284.8 eV.

Fourier-transform infrared spectroscopy (FTIR) measurements on MnO NP samples, oleylamine, PLGA MnO NP samples, and PLGA were performed using a DIGILAB FTS 7000 FTIR spectrometer equipped with a GladiATR attenuated total reflectance (ATR) module from PIKE Technologies.

Size distribution of the PLGA MnO NPs was measured through dynamic light scattering (DLS) using a Nano Powder Sizer Malvern Instrument Zetasizer Nano ZS. Six milligrams of the PLGA MnO NPs were suspended in 10 mL of deionized water and bath sonicated prior to analysis.

Mn2+ controlled release experiments

To evaluate Mn2+ release under different pH conditions, 10 mg of each MnO NP batch was suspended in 1 mL of PBS pH 7.4, 20 mM citrate buffer pH 6.5, or 20 mM citrate buffer pH 5 to simulate the pH of the blood, the tumor microenvironment, and cellular endosomes/lysosomes, respectively. The same three pH conditions were used to evaluate Mn2+ release from unencapsulated smaller MnO NPs (19 ± 6 nm) and unencapsulated smaller Mn3O4 NPs (17 ± 5 nm) to further observe the effect of size and chemical composition on release rate. Similarly, PLGA MnO NPs were suspended under the same conditions to assess Mn2+ release from hydrophilic NPs. Citrate buffers were made by adding anhydrous citric acid and sodium citrate dihydrate to deionized water. The MnO NP or PLGA MnO NP solutions were incubated in Eppendorf tubes under continuous slow rotation (6 rpm) to ensure gentle mixing over 24 hr at 37°C to simulate body temperature. At 1, 2, 4, 8 and 24 hr, the Eppendorf tubes were centrifuged at 17,400 x g for 10 min and the supernatants were collected and analyzed for released Mn2+ content by inductively coupled plasma-optical emission spectrometry (ICP-OES). The remaining pelleted MnO NPs or PLGA MnO NPs were resuspended in 1 mL of fresh buffer and placed back into the rotating incubator until the next time point was collected. The maximum amount of Mn2+ contained within each NP batch was calculated through measuring the total Mn2+ content of unencapsulated MnO NPs (10 mg) or PLGA MnO NPs (10 mg) fully dissolved in 150 μL of HCl trace metal grade using bath sonication. Mn2+ amounts were analyzed using Agilent 720 ICP-OES (1400 watts) with a plasma flow of 15.0 L/min, auxiliary flow of 1.50 L/min, and nebulizer flow of 0.75 L/min. Each sample was evaluated 5 times with a replicate and stabilization time of 10 and 15 s, respectively, and results were averaged. The % Mn2+ released at each time point was calculated using Eq 1 below. The Mn2+ cumulative release graph was created by adding together the % Mn2+ released from each of the previous time points.


PLGA MnO NP % loading capacity was calculated using the total Mn2+ content of unencapsulated MnO NPs (10 mg) and PLGA MnO NPs (10 mg) with Eq 2 below.


MRI properties of NPs

Two different MRI experiments were performed. First, the r1 molar relaxivities of Mn2+ at pH 7.4 (PBS), pH 6.5 (20 mM citrate buffer), and pH 5 (20 mM citrate buffer) were determined. To measure r1, manganese(II) chloride tetrahydrate was dissolved in the three buffers to achieve Mn2+ concentrations of 0.182, 0.102, 0.058, 0.032, and 0.018 mM. MRI of the Mn2+ solutions was performed at 1.0 T on a Bruker ICON MRI. T1 measurements were generated by a RARE sequence using an echo time of 10.68 ms. A total of 10 repetition times (25.2, 50, 100, 200, 400, 800, 1,600, 3,200, 6,400, and 12,800 ms) were used to acquire images of the tubes. Using Matlab and the T1 longitudinal relaxation equation (Eq 3), T1 fitting was accomplished: (3) where Mz is the longitudinal magnetization aligned along the z-axis at some time, t, and Mo is the magnetization at equilibrium. The r1 relaxivity for Mn2+ at pH 7.4, pH 6.5 and pH 5 was calculated using Eq 4: (4) where 1/T1 is the measured relaxation rate, 1/T1,0 is the relaxation rate of the solvent only, and [Mn2+] is the concentration of Mn2+. The relaxivity is the slope of the linear fitted line when 1/T1−1/T1,0 is plotted versus [Mn2+].

Second, unencapsulated or PLGA encapsulated MnO NPs were suspended in pH 7.4, pH 6.5 and pH 5 as described for Mn2+ controlled release experiments. After 24 hours, the supernatant from 8 to 24 hr for the unencapsulated NPs was collected and diluted 100 fold. For PLGA encapsulated NPs, the supernatant from all time points were combined and diluted 100 fold. Longitudinal MRI properties of the collected supernatants containing released Mn2+ were measured at 1 T using the same MRI parameters as above. T1 values of the supernatants were measured using Eq 3. Additionally, Mn2+ concentrations were calculated from the measured T1 values and the r1 values for Mn2+ at each pH using Eq 4. Mn2+ concentrations measured by MRI were compared with Mn2+ concentrations measured by ICP-OES.


Approximately 25 to 35 TEM images and 800 to 900 MnO NPs were quantified per temperature condition using the line trace tool in ImageJ to measure the NP diameter. Each temperature condition contained 3 independent batches of synthesized MnO NPs. Statistical significance of mean NP diameters, Mn2+ controlled release, and MRI T1 values between groups were evaluated using the 2-tailed unpaired Student’s t-test with Bonferroni correction to account for multiple comparisons, where *p < 0.05 was defined as significant and **p < 0.01 was defined as highly significant. The polydispersity index (PDI) of MnO NPs for each temperature condition was calculated from the TEM images using Eq 5: (5) where σ is the standard deviation of the MnO NP diameters, and d is the mean diameter of MnO NPs.

Results and discussion

MnO NPs were fabricated using a standard thermal decomposition reaction of Mn(II) ACAC dissolved in oleylamine and dibenzyl ether (Fig 1). Precise control over the temperature rise was achieved through programming a temperature controller (S1 Fig), which received real-time feedback through a thermocouple probe placed inside the reaction mixture. Two different temperature variables were studied, specifically heating rates and aging times, in attempts to achieve pure MnO NPs of smaller sizes. For simplicity, in the rest of the manuscript, we will refer to all synthesized manganese oxide NPs as MnO NPs, unless otherwise specified.

Fig 1. Thermal decomposition of Mn(II) ACAC was performed in oleylamine and dibenzyl ether at two temperature ramp rates and increasing aging times at 300°C to form MnO NPs coated with oleylamine.

Smaller NP diameters result from faster temperature ramp rate and shorter aging time

TEM was used to assess MnO NP size and a representative image from each temperature condition is shown in Fig 2. Our MnO NPs generally displayed a rounded octagon morphology similar to MnO NPs obtained by Nolis et al. [21] from heating manganese acetate and oleylamine to 250 and 300°C; however, their MnO NP diameters were much larger (100 nm and 70 nm, respectively, at 250°C and 300°C).

Fig 2.

TEM images of MnO NPs generated from each of the 6 different temperature profiles: 20°C/min ramp with a) 5 min at 300°C, b) 15 min at 300°C, c) 30 min at 300°C, and 10°C/min ramp with d) 5 min at 300°C, e) 15 min at 300°C, and f) 30 min at 300°C. The MnO NPs have a rounded octagon shape, but some variation in size. Scale bars are 20 nm.

As the ramping rate decreased and the aging time at 300°C increased, the average MnO NP diameter grew by a maximum of nearly 54%. When the fastest ramp (20°C/min) and shortest aging time (5 min) was used, the MnO NPs were the smallest, with an average diameter of 23 ± 9 nm. As the aging time was increased to 15 and 30 min, the average MnO NP size increased to 32 ± 11 nm and 32 ± 12 nm, respectively. When the ramp rate was decreased to 10°C/min, the average size of MnO NPs increased to 27 ± 10 nm, 36 ± 12 nm, and 36 ± 13 nm at 5, 15, and 30 min at 300°C, respectively. As shown in Fig 3, the average NP diameter was significantly different between ramping rates at all aging times. Significance was also achieved within both ramping rates when comparing aging times of 5 min to 15 min and 5 min to 30 min. Despite the high standard deviation of NP size, significance was achieved due to the large sample size analyzed (800–900 NPs per temperature condition). PDI values for MnO NPs from each temperature condition were calculated and found to be ≤ 0.15 (S1 Table). It is important to note that after 15 min at 300°C, the MnO NP size stabilized in both ramping conditions.

Fig 3. Average MnO NP diameters achieved with different ramping rates and aging times at 300°C.

A faster ramping rate and a shorter aging time produced the smallest NPs. A total of 800–900 NPs were analyzed from TEM images per temperature condition. Error bars are average ± standard deviation. **p<0.01 was defined as highly significant.

The increase in MnO NP size with a slower temperature ramp and an increase in aging time at 300°C was likely due to a longer total reaction time, leading to more opportunity for NP growth and coalescence (S1 Table). Histograms comparing the size distributions at the two different temperature ramps are shown in S2 Fig. Chen et al. [28] also observed a rise in NP growth with increasing aging times at 310°C, which was associated with two distinct growth patterns: only minimal increases in NP size were observed from 3 to 30 min at 310°C, whereas a much larger increase in NP size was achieved from 100 to 285 min at 310°C.

Our MnO NPs tended to exhibit a variation in size, likely due to several factors. First, MnO NP growth could follow an Ostwald ripening process, whereby smaller NPs begin to dissolve and add onto larger ones to cause polydispersity [28]. Second, a subset of smaller MnO NPs could coalesce or join together into larger NPs as the reaction proceeds to also lead to size variation [3032]. Third, the concentration of oleylamine has been shown to contribute to NP size distribution. When a lower stabilizer concentration is used, the NPs do not have enough capping, which can allow for their aggregation [33].

Mn3O4 is incompletely reduced to MnO by faster temperature ramp rate and shorter aging time

Remarkably, the MnO NP size was not the only characteristic affected when the temperature profile was changed. XRD was used to evaluate MnO NP crystal structure and bulk composition. Fig 4A–4F shows the XRD spectra of each NP for all 6 temperature conditions, while Fig 4G and 4H show the characteristic XRD peaks of Mn3O4 and MnO, respectively. All synthesized NPs (Fig 4A–4F) clearly display the 5 highest characteristic peaks for MnO (Fig 4H), whereas the first three spectra (Fig 4A–4C) also contain the 3 highest characteristic peaks of Mn3O4 (Fig 4G). Therefore, the top 3 temperature profiles with the shortest total reaction times resulted in a mixture of Mn3O4 and MnO NPs and the bottom 3 temperature profiles with longer total reaction times led to a more pure MnO NP formulation. Table 1 displays the estimated percent NP composition that X’Pert HighScore Plus calculated for each temperature profile based on its database of known compounds. As the overall temperature reaction times were increased, the MnO percentage composition increased and Mn3O4 percentage composition decreased. We hypothesize that when less time was applied into the synthesis, the reaction did not have enough thermal energy to occur completely, obtaining a mixture of MnO and Mn3O4.

Fig 4.

XRD spectra of Mn3O4/MnO NP mixture or MnO NPs generated with the following temperature profiles: a) 5 min at 300°C with 20°C/min ramp, b) 5 min at 300°C with 10°C/min ramp, c) 15 min at 300°C with 20°C/min ramp, d) 15 min at 300°C with 10°C/min ramp, e) 30 min at 300°C with 20°C/min ramp, and f) 30 min at 300°C with 10°C/min ramp. The standard diffraction peaks for known g) Mn3O4 and h) MnO are shown from X’Pert HighScore. Upon comparison with the standard spectra, a-c) shows Mn3O4/MnO NP mixtures, whereas d-f) shows MnO NPs.

Table 1. MnO and Mn3O4 composition (%) for NP trials based on X’Pert HighScore database.

To our knowledge, our study is the first to show that the ramping rate and aging time at 300°C impact the composition of the synthesized NPs. Mn3O4 or MnO/Mn3O4 NP mixtures were previously observed by Nolis et al. [21] and Seo et al. [22], but the aging temperature used was much lower between 150–200°C. Our study reveals that MnO NPs still contain some Mn3O4 composition even at 300°C when applying faster ramp rates and shorter aging times. Based on our results and the literature, we hypothesize that the formation of MnO NPs is initiated by first forming Mn3O4 NPs at lower temperatures (150–200°C) during thermal decomposition of Mn(II) ACAC. As the reaction time and temperature is increased, the Mn3O4 NPs begin to be reduced to MnO NPs through an endothermic reaction: Mn3O4 → 3MnO + ½O2 [34]. Shorter aging times at 300°C do not allow for complete conversion of Mn3O4 to MnO, and lead to a mixed MnO/Mn3O4 composition. Longer aging times provide more thermal energy needed to obtain a full reduction to a pure MnO composition. Once again, for simplicity, in the rest of the manuscript, we will refer to all synthesized manganese oxide NPs as MnO NPs.

MnO NP surfaces are coated with Mn3O4 and oleylamine

To complement the bulk analysis of XRD, SEM/EDS, XPS and FTIR were used to assess the surface chemistry of MnO NPs formed with different temperature profiles. SEM/EDS and XPS confirmed the elemental composition of our NP samples to be mainly manganese and oxygen (S3 Fig and Fig 5A, respectively). The magnitude of the Mn3s peak splitting (Fig 5B) can be used to identify the oxidation state of surface bound manganese. A ΔE of 6.1eV indicates MnO (Mn2+), while a ΔE of ≥ 5.4 eV indicates Mn2O3 (Mn3+) [35]. Previous literature has shown that since Mn3O4 is a mixture of Mn2+ and Mn3+ oxidation states, the Mn3s peak splitting has an intermediate ΔE of 5.6 eV [36]. Fig 5B shows that all NP samples, regardless of the temperature profile, show the characteristic peak splitting of Mn3O4. XPS results demonstrate that the surface of the NPs oxidizes after synthesis in the presence of air to form a coating of Mn3O4, consistent with what others have found [36]. Together, XRD and XPS show that reaction conditions affect the overall bulk composition of the synthesized NPs (Mn3O4/MnO versus MnO), but that all NPs are oxidized to include a layer of Mn3O4 on the surface.

Fig 5.

XPS spectra of MnO NP samples for each temperature profile showing the a) whole spectral region and b) the Mn3s region. The whole spectral region indicates the presence of manganese, oxygen, and carbon in the NP samples, while the Mn3s region shows peak splitting characteristic of surface oxidation to Mn3O4 (Mn2+/Mn3+ oxidation states).

The NP surface chemistry was further studied with FTIR to corroborate hydrophobic capping with oleylamine. Fig 6 presents the FTIR spectra of each NP for all 6 temperature conditions. All NP samples show the characteristic modes of oleyl groups: peaks around 2850–2854 and 2918–2926 cm-1 (marked by asterisks) due to the symmetric and asymmetric CH2 stretching modes, respectively [37]. Additionally, the peaks around 1593 cm-1 and 3300 cm-1 (marked by squares) are attributed to the NH2 bending vibration, and the symmetric and asymmetric stretching vibration of the amine group (NH2), respectively [38]. MnO NP FTIR spectra had similar peaks to those present in the oleylamine only spectra (S4 Fig). These peaks consolidate NP capping formed by oleylamine, which is consistent with the literature when synthesizing metal oxide NPs [29, 39]. Lastly, peaks around 600 cm-1 (marked by a triangle) correspond to the vibration of Mn-O and Mn-O-Mn bonds, confirming the chemistry found through XRD, SEM/EDS and XPS [40].

Fig 6.

FTIR spectra of the following temperature profiles: 20°C/min ramp with a) 5 min at 300°C, b) 15 min at 300°C, c) 30 min at 300°C, and 10°C/min ramp with d) 5 min at 300°C, e) 15 min at 300°C, and f) 30 min at 300°C. Asterisks represent oleyl groups, squares correspond to amine groups, and triangles show the vibration of Mn-O and Mn-O-Mn bonds. The oleyl group spectral regions are enlarged in the boxed insets to resolve the two distinct peaks.

Mn2+ release rate from MnO NPs is maximum at pH 5 and unaffected by synthesis conditions

As mentioned before, size reduction of MnO NPs is important to increase the surface area to volume ratio to generate a higher dissolution rate of MnO to Mn2+ in acidic media to create a greater T1 MRI signal. The controlled release profile of Mn2+ from MnO NPs was tested over time in 3 different pH conditions: pH 7.4 to mimic the normal physiological pH of the blood, pH 6.5 to mimic the slightly acidic extracellular pH in cancerous tumors due to increased lactic acid production, and pH 5 to mimic the acidic pH of endosomes and lysosomes inside cells. It is well known that following cell uptake, metallic NPs are shuttled to endosomes inside cells [41]. Fig 7 shows a representative Mn2+ controlled release curve for MnO NPs formed from the 20°C/min ramp aged at 300°C for 30 min. Similar to what we have shown previously [5], MnO dissociation into Mn2+ at physiological pH 7.4 was extremely minimal (faint dotted line in Fig 7), meaning that no MRI signal enhancement would be produced in the blood. At pH 6.5, only ~9% (~0.5 mg) of the total manganese content was released as Mn2+ after 24 hr (dashed line in Fig 7), which will likely result in a very weak enhancement of MRI signal if the MnO NPs remain in the extracellular space of cancerous tumors. Consistent with our previous findings [5], pH 5 showed the most robust release of Mn2+ over 24 hr of ~46% (~2.9 mg) (solid black line in Fig 7). The other MnO NP formulations (S5 Fig) showed similar controlled release curves with no significant differences between Mn3O4/MnO NP mixtures and MnO only. The total Mn2+ amount released at each temperature condition and pH are shown in S2 Table.

Fig 7.

Cumulative release of Mn2+ from MnO NPs over 24 hr after incubation in PBS pH 7.4 (dotted line), 20 mM citrate buffer pH 6.5 (dashed line), and 20 mM citrate buffer pH 5 (solid line). Mn2+ release increased with a decrease in pH. The controlled release curve is shown for the MnO NPs generated with 30 min at 300°C and a 20°C/min ramp. Time points are shown for 1, 2, 4, 8 and 24 hr. Error bars show mean ± standard deviation.

The similarity of Mn2+ release from all NP formulations in acidic media was surprising, as Godunov et al. [42] have shown that Mn3O4 dissolves incompletely in dilute acidic conditions due to the formation of Mn2+ ions as well as MnO2, whereas MnO dissolves completely. Their study also demonstrated that MnO dissolves at a faster rate than Mn3O4 in concentrated acidic solutions, but both compounds completely dissociate [42]. The similarity of our Mn2+ controlled release curves between NP formulations could indicate that the slower anticipated Mn2+ release from MnO/Mn3O4 NP mixtures could be counteracted by their smaller NP diameter with increased surface area. Similarly, MnO NPs would be expected to exhibit enhanced Mn2+ production, but had larger diameters, which could slow their release.

Small diameter MnO NPs have maximum Mn2+ release and MRI signal enhancement

To further understand how NP size and chemical composition affected Mn2+ controlled release and T1 MRI properties, we compared the synthesized optimal MnO NPs above (32 nm ± 12 nm) with smaller MnO NPs (19 ± 6 nm) and smaller Mn3O4 NPs (17 ± 5 nm). TEM images and XRD spectra of the small MnO and small Mn3O4 NPs are shown in S6 Fig. When compared to large MnO NPs, small MnO NPs released ~7% more Mn2+ after 24 hours at pH 5 likely due to their increased surface area to volume ratio; however, the increased Mn2+ production from small MnO NPs was not statistically significant (Fig 8). Chemical composition of NPs had a much larger impact on Mn2+ release. Small Mn3O4 NPs released significantly less Mn2+ (~20% reduction) than small MnO NPs (Fig 8). Similar trends were observed at pH 6.5.

Fig 8. Average cumulative release of Mn2+ from 17 nm Mn3O4, 19 nm MnO, and 32 nm MnO NPs after 24 hr of incubation in PBS pH 7.4, 20 mM citrate buffer pH 6.5, and 20 mM citrate buffer pH 5.

Mn2+ release was highest from small MnO NPs and lowest from small Mn3O4 NPs at pH 5 and 6.5. Three different batches of each type of NPs were analyzed during controlled release. Error bars are average ± standard deviation.* p<0.05 and **p<0.01 were defined as significant and highly significant, respectively.

Next, the impact of NP size and chemical composition on T1 MRI signal enhancement was assessed to determine which formulation would be most favorable for MRI applications. The smallest MnO NPs were the most efficient MRI contrast agents by producing the lowest T1 value at all pH levels (Table 2). This is not surprising, as small MnO NPs released the greatest amount of Mn2+ after 24 hours at pH 5 and 6.5 (Fig 8). Large MnO NPs were slightly less efficient with a ~20% increase in T1 compared to small MnO NPs. Small Mn3O4 NPs were the least effective MRI contrast agents, with a ~42% increase in T1 compared to small MnO NPs that was statistically significant (Table 2). Similar to small MnO NPs, the obtained T1 values for large MnO NPs and small Mn3O4 NPs also mirrored the trends seen in the Mn2+ controlled release experiments at pH 6.5 and 5 (Fig 8). Therefore, for MRI applications, it would be favorable to utilize MnO NPs rather than Mn3O4 due to the generation of a greater concentration of Mn2+ ions which produces a larger signal enhancement on T1 MRI (S7 Fig). Furthermore, the MRI data confirmed the results from ICP-EOS when using the Mn2+ calibration curves to calculate the Mn2+ concentration in the solutions (S7 Fig). Since the calibration curves only contained diluted Mn2+ ions and the calculated Mn concentration from MRI and ICP-OES were similar at pH 6.5 and 5 (S3 Table), it is likely that Mn3+ ions minimally contributed to the MRI signal and Mn2+ ions were the dominant species responsible for the MRI signal increase. According to Gale et al. [43], chelated Mn2+ has a 6.6 fold higher r1 relaxivity compared to chelated Mn3+ at 1.4 T, which supports that our main MRI signal likely originates from Mn2+.

Table 2. MRI T1 values of supernatants collected between 8 and 24 hours after NP incubation at pH 7.4, 6.5 and 5.

MnO NPs encapsulated in PLGA retain maximum Mn2+ release and MRI signal enhancement compared to PLGA Mn3O4 NPs

The MnO NPs synthesized herein are hydrophobic initially and capped with oleylamine (Fig 6). The hydrophobicity of MnO NPs in our study may provide a limitation to the assessment of NP dissolution kinetics. Therefore, hydrophilic NPs were fabricated by encapsulating large MnO NPs (32 nm), small MnO NPs (19 nm) and small Mn3O4 NPs (17 nm) within PLGA, a clinically approved biocompatible and biodegradable polymer, to confirm the trend observed with hydrophobic NPs. TEM showed dark metal oxide nanocrystals trapped within the polymeric NPs (S8 Fig) while FTIR confirmed successful surface coating with PLGA through matching characteristic FTIR spectral peaks of PLGA MnO NPs with PLGA only (circles shown in S9 and S10 Figs). PLGA MnO NPs had ~30% loading capacity (S4 Table) and an average diameter between 220 and 255 nm based on DLS analysis (S11 Fig).

To test the effects of polymer coating on Mn2+ generation and MRI signal, the same controlled release experiment was performed on PLGA MnO NPs, and the supernatants were analyzed with ICP-OES and MRI. PLGA MnO (19 nm) NPs had the highest Mn2+ release after 24 hr at pH 5; approximately 90% of the encapsulated MnO NPs dissociated to Mn2+ compared to only ~45% of encapsulated Mn3O4 NPs (Fig 9). All three PLGA NPs had ~25% cumulative release rates at pH 6.5 after 24 hours, with negligible release at PBS pH 7.4 (Fig 9). As can be seen by comparing Figs 8 and 9, polymer encapsulation did not significantly impact the overall trends in Mn2+ controlled release. MnO retained its ability to produce Mn2+ at a much faster rate compared to Mn3O4 regardless of the surface groups.

Fig 9. Average cumulative release of Mn2+ from PLGA Mn3O4 (17 nm), PLGA MnO (19 nm), and PLGA MnO (32 nm) NPs after 24 hr of incubation at pH 7.4, pH 6.5, and pH 5.

Mn2+ release was significantly greater from PLGA MnO NPs compared to PLGA Mn3O4 NPs at pH 5. Three different batches of each type of NPs were analyzed during controlled release. Error bars are average ± standard deviation. **p<0.01 was defined as highly significant.

PLGA MnO (19 nm) NPs had a significantly lower T1 value compared to PLGA Mn3O4 (17 nm) NPs after 24 hours at pH 5 (Table 3), which is consistent with the enhanced Mn2+ generation of MnO shown by ICP-OES. Both PLGA MnO NP formulations had comparable T1 values at all pHs (Table 3 and S12 Fig), which shows that unencapsulated MnO NP size does not have a significant impact on Mn2+ release or MRI signal once NPs are encapsulated within a polymer. Since a different number of metal oxide NPs can be encapsulated within each individual PLGA NP, variability in loading within each sample could have contributed to a loss of MRI trends between PLGA MnO (19 nm) NPs and PLGA MnO (32 nm) NPs. Altogether, our results support the use of MnO over Mn3O4 due to higher Mn2+ generation and MRI signal with and without PLGA encapsulation.

Table 3. MRI T1 values of supernatants collected 24 hours after PLGA NP incubation at pH 7.4, 6.5 and 5.

In addition to utilizing MnO over Mn3O4, another strategy to maximize MRI signal from MnO NPs in vitro and in vivo would be to employ NP targeting to enhance uptake into tumor cells to take advantage of the low acidic conditions of endosomes and lysosomes to aid in increased Mn2+ generation. Several receptors are overexpressed on tumor cells depending on the cancer type such as the folate receptor, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), the transferrin receptor, and the mucin-1 (MUC-1) receptor [44], among others. NPs can be conjugated with either antibodies or peptides designed to bind specifically to these receptors to enable targeting. Small targeting peptides provide several advantages over antibody targeting including reduced production cost, low molecular weight and reduced immunogenicity [45].

Besides PLGA encapsulation, NPs can be made hydrophilic through ligand exchange [35] or lipid capping [46]. Further NP modifications to enhance functionality include adding stealth polymers to the surface such as polyethylene glycol (PEG) [47] to extend blood circulation times to promote NP accumulation in tumors as well as adding chemotherapeutic drugs or microRNA to develop theranostic systems to track drug delivery to tumors. If PEG is used, targeting moieties should be added to the end of the PEG chains, as targeting agents attached to the NP surface would experience steric hindrance by long PEG chains and be inaccessible to bind with tumor cell receptors [48]. Although PEG can greatly enhance blood circulation times and NP accumulation in tumors, it can decrease NP uptake into tumor cells [49]. As an alternative approach, cleavable PEG chains can be utilized, which can be designed for cleavage at low tumor pH or by enzymes overexpressed at the cancer site [50]. Phospholipid versus polymer encapsulation techniques have different advantages. Phospholipid coating will minimally add to the overall NP size and facilitate synthesis of small NPs, as the hydrophobic lipid tail will associate with the hydrophobic NP surface and the hydrophilic head will point out towards the aqueous media [46, 51, 52]. Phospholipids conjugated to fluorescent dyes, polymers, and different reactive functional groups (e.g. free acid, amine, alkene, azido, etc.) are readily available from commercial sources such as Avanti Polar Lipids. When utilizing phospholipids, it is important to use long chain saturated lipids with a phase transition temperature >37°C to assure better stability and to purchase reactive functional groups and polymers attached to the lipid head groups to ensure these moieties are facing out towards the aqueous media. Polymeric encapsulation typically produces larger NPs, but is very customizable; fluorescent dyes, metal oxide NPs, and drug can all be added during the synthesis phase for simultaneous encapsulation [5, 53, 54]. Release rate of the contents can be controlled through changing the polymer composition.

It will also be important to evaluate potential Mn toxicity in the consideration of adopting MnO NPs for MRI of tumors. Mn toxicity is thought to arise from the release of free Mn2+ ions, as Mn2+ mimics Ca2+ and can enter neurons and muscles. The ability of Mn2+ to travel down neurons has been used for manganese enhanced MRI (MEMRI) in animals to visualize neuronal activity. Bock et al. [55] have shown that MEMRI in rats had no adverse effects using 30 mg/kg of free Mn2+, injected every 2 days for 12 days, totaling 180 mg/kg Mn2+. MnO NPs should be better tolerated in vivo, as they carry MnO, not free Mn2+ directly. Through incorporating specific NP targeting to tumor cells and confining Mn2+ release to low pH tumor endosomes (Figs 79), the systemic dose of free Mn2+ should be minimized. Nonetheless, it will be necessary to thoroughly evaluate MnO NP hepatic, cardiac, and sensorimotor toxicity in vivo over time in tumor bearing animals to assess any off-target effects. The key results from our study are summarized below in Fig 10.

Fig 10. Schematic presentation illustrating how size and composition of MnO NPs can be fine-tuned by altering temperature ramping rate and aging time.

A shorter aging time and faster ramp rate produced smaller NPs with a mixed composition of MnO/Mn3O4. Larger NPs comprised of MnO only were synthesized by extending the aging time and using a slower temperature ramp. Mn2+ production was highest at pH 5, mimicking cell endosomes. Unencapsulated small MnO NPs released the greatest amount of Mn2+ and had the highest MRI signal enhancement (yellow) compared to unencapsulated large MnO NPs and small Mn3O4 NPs. Although there was no significant difference between large and small MnO NPs after PLGA encapsulation, both PLGA MnO NP formulations released significantly more Mn2+ compared to PLGA Mn3O4 NPs and generated much higher T1 signal enhancement (not shown).


In conclusion, we are the first to demonstrate that modification of the temperature ramp rate and aging time at 300°C can be used to fine-tune both the diameter and composition of MnO NPs (Fig 10). The fastest ramp and shortest aging time produced the smallest NP size through limiting the overall reaction time and NP growth; however, a mixture of Mn3O4 and MnO NPs was obtained with shorter aging times due to the incomplete reduction of Mn3O4 to MnO. To achieve pure MnO, which is most desirable for MRI applications, longer aging times at 300°C were needed, but MnO NP size increased as well. In our study, the 20°C/min temperature ramp with a 30 minute aging time at 300°C was the most ideal temperature condition to form the smallest pure MnO NPs. XPS and FTIR confirmed NP surface oxidation to Mn3O4 and oleylamine capping, respectively. Remarkably, ramping rate and aging time had a negligible effect on the Mn2+ release rate, indicating that NP size and composition characteristics could be counteracting each other, as MnO/Mn3O4 NPs tended to be smaller than MnO NPs. To further explore the impact of NP size and chemical composition on Mn2+ release rate and MRI signal, the ideal MnO NPs synthesized in this study (32 nm) were compared with smaller MnO NPs (19 nm) and smaller Mn3O4 NPs (17 nm), with and without PLGA encapsulation. As predicted, the smallest unencapsulated MnO NPs released the most Mn2+ ions at pH 5 and 6.5 and led to the greatest reduction in T1 longitudinal relaxation time, with the highest MRI signal. PLGA encapsulation of large and small MnO NPs reduced the trends observed with unencapsulated MnO NPs possibly due to variability of metal oxide loading within individual NPs. With and without PLGA coating, Mn3O4 NPs were consistently the least effective MRI contrast agents; therefore, it is recommended to utilize MnO over Mn3O4 for MRI applications to expedite Mn2+ release and the resulting MRI signal produced. Future studies will explore varying the chemical reactant ratios to further decrease NP size and polydispersity, and using novel surface functionalization to enhance MnO NP endocytosis into cancer cells to maximize MRI contrast through Mn2+ generation.

Supporting information

S1 Fig. Temperature profiles of MnO NP synthesis.

Reactant mixtures were heated from room temperature to 60°C over 30 minutes and then to 300°C using two different temperature ramps of a) 20°C/min or b) 10°C/min. Both temperature profiles show an aging temperature at 300°C for 30 minutes prior to cooling. Note how the temperatures measured during the experiments (red circles) closely match the theoretical programmed settings for the temperature controller (black lines), indicating precise control of MnO NP fabrication conditions.


S2 Fig.

Size distributions of the diameter of MnO NPs produced using the following temperature profiles: a) 5 min at 300°C with a 20°C/min vs. 10°C/min ramp, b) 15 min at 300°C with a 20°C/min vs. 10°C/min ramp, and c) 30 min at 300°C with a 20°C/min vs. 10°C/min ramp. MnO NP diameter increases as the ramping rate decreases and aging time at 300°C increases. The average size for each distribution is shown in S1 Table.


S3 Fig.

EDS spectra of the MnO NP samples with the following temperature profiles: a) 5 min at 300°C with 20°C/min ramp, b) 5 min at 300°C with 10°C/min ramp, c) 15 min at 300°C with 20°C/min ramp, d) 15 min at 300°C with 10°C/min ramp, e) 30 min at 300 oC with 20°C/min ramp, and f) 30 min at 300°C with 10°C/min ramp. EDS confirmed the presence of Mn and O elements in NP samples.


S4 Fig. FTIR spectrum of oleylamine.

Asterisks represent oleyl groups, while squares represent amine groups.


S5 Fig. Cumulative release of Mn2+ from MnO NPs over 24 hr after incubation in PBS pH 7.4 (dotted line), 20 mM citrate buffer pH 6.5 (dashed line), and 20 mM citrate buffer pH 5 (solid line).

Controlled release curves are shown for MnO NPs generated with the following temperature profiles: a) 5 min at 300°C with 20°C/min ramp, b) 5 min at 300°C with 10°C/min ramp, c) 15 min at 300°C with 20°C/min ramp, d) 15 min at 300°C with 10°C/min ramp, and e) 30 min at 300°C with 10°C/min ramp. Mn2+ release increased with a decrease in pH. Time points are shown for 1, 2, 4, 8 and 24 hr. Error bars show mean ± standard deviation.


S6 Fig. XRD and TEM of small Mn3O4 NPs and small MnO NPs.

XRD spectra of a) 17 nm Mn3O4 NPs and b) 19 nm MnO NPs. The standard diffraction peaks for known c) Mn3O4 and d) MnO are shown from X’Pert HighScore. Through comparing with the standard diffraction peaks, Mn3O4 NPs are 73–100% Mn3O4 composition and MnO NPs are 67–73% MnO composition. TEM images of e) 17 nm Mn3O4 and f) 19 nm MnO NPs. NPs are smaller in size compared to Fig 2 and have a lower size variation. Scale bar is 50 nm.


S7 Fig. MRI properties of Mn2+ standard curve solutions and Mn2+ supernatants collected from dissolving MnO and Mn3O4 NPs.

a) r1 values for free Mn2+ in 20 mM citrate buffer pH 5 (black), 20 mM citrate buffer pH 6.5 (blue), and PBS pH 7.4 (red). T1-weighted MRI images shown in b-e) were acquired at 1 T with a 400 ms repetition time. T1 MRI of increasing Mn2+ concentrations in b) 20 mM citrate buffer pH 5, c) 20 mM citrate buffer pH 6.5, d) PBS pH 7.4. e) shows T1 MRI images of supernatants collected from small Mn3O4 (17 nm), small MnO (19 nm) and large MnO (32 nm) NPs suspended in pH 5 citrate buffer for 24 hours. MRI signal enhancement is greatest from small MnO NPs and least from small Mn3O4 NPs.


S8 Fig. TEM images of PLGA encapsulated NPs formed by single emulsion.

Three different types of metal oxide NPs were coated with PLGA including a) 17 nm Mn3O4, b) 19 nm MnO, and c) 32 nm MnO. Metal oxide NPs can be visualized as dark circles inside of the PLGA. NP loading capacity was ~30%. Scale bars are 100 nm.


S9 Fig.

FTIR spectra of PLGA encapsulated NPs: a) PLGA Mn3O4 (17 nm), b) PLGA MnO (19 nm), and c) PLGA MnO (32 nm). All NPs possess the characteristic peaks of PLGA, represented by circles, as shown in S10 Fig.


S10 Fig. FTIR spectrum of PLGA.

Circles represent characteristic peaks of PLGA. The peaks at 2993 cm−1 and 2989 cm−1 show the C–H stretch of CH2, and C–H stretch of–C–H–, respectively. The peak at 1751 cm−1 is assigned to the C = O stretching vibration of the ester bond and 1165–1087 cm−1 corresponds to the C–O stretching.


S11 Fig.

Size distributions of PLGA NP diameters by DLS analysis: a) PLGA Mn3O4 (17 nm), b) PLGA MnO (19 nm), and c) PLGA MnO (32 nm). Highest peak for NP diameters is in the 220 to 255 nm bin size range.


S12 Fig. MRI properties of Mn2+ supernatants collected from dissolving PLGA MnO and PLGA Mn3O4 NPs.

T1 MRI images of supernatants collected from PLGA Mn3O4 (17 nm), PLGA MnO (19 nm) and PLGA MnO (32 nm) NPs suspended in pH 5 citrate buffer, pH 6.5 citrate buffer, and pH 7.4 PBS for 24 hours. MRI signal enhancement is significantly greater from PLGA MnO NPs compared to PLGA Mn3O4 NPs.


S1 Table. Total reaction time, average diameter and PDI of MnO NPs for each temperature condition.


S2 Table. Total amount of Mn2+ released (ave ± stdev) from MnO NPs over 24 hr at different pH.


S3 Table. Concentration of Mn2+ obtained from controlled release of NPs at 24 hr analyzed by ICP-OES and MRI.


S4 Table. Loading capacity of PLGA NPs (mg MnxOy/mg NP) by ICP-OES.



The authors would like to thank Dr. Marcela Redigolo for advice on TEM and SEM sample preparation and imaging, Dr. Qiang Wang for interpretation and analysis of the XRD, XPS, and FTIR spectra, Robert Vincent for performing ICP-OES measurements of Mn2+ controlled release samples, Domenic Cipollone for executing the DLS measurements of PLGA MnO NPs, Dr. John Zondlo and Hunter Snoderly for setting up and programming the temperature controller, James Hall for his assistance in assembling the experimental setup for MnO NP synthesis, Alexander Pueschel for helping to analyze MnO NP TEM images, Joy Wu for her involvement in optimizing the PLGA MnO NP encapsulation conditions, Jiahua (Cathy) Li and Huy Pham for their literature search contribution, the WVU Shared Research Facility for use of their equipment including the TEM, SEM/EDS, XRD, XPS, and FTIR and the In Vivo Multifunctional Magnetic Resonance center for the use of their 1 T MRI machine.


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