Manganese Sulfate Nanocomposites Fabricated by Hot-Melt Extrusion for Chemodynamic Therapy of Colorectal Cancer

The development of metal salts-based nanocomposites is highly desired for the Fenton or Fenton-like reaction-based chemodynamic therapy of cancer. Manganese sulfate (MnSO4)-dispersed nanoparticles (NPs) were fabricated with a hot-melt extrusion (HME) system for the chemodynamic therapy of colorectal cancer in this study. MnSO4 was homogeneously distributed in polyethylene glycol (PEG) 6000 (as a hydrophilic polymer) with the aid of surfactants (Span 80 and Tween 80) by HME processing. Nano-size distribution was achieved after dispersing the pulverized extrudate of MnSO4-based composite in the aqueous media. The distribution of MnSO4 in HME extrudate and the interactions between MnSO4 and pharmaceutical additives were elucidated by Fourier-transform infrared, X-ray diffractometry, X-ray photoelectron spectroscopy, and scanning electron microscopy analyses. Hydroxyl radical generation efficiency by the Fenton-like chemistry capability of Mn2+ ion was also confirmed by catalytic assays. By using the intrinsic H2O2 in cancer cells, MnSO4 NPs provided an elevated cellular reactive oxygen species level, apoptosis induction capability, and antiproliferation efficiency. The designed HME-processed MnSO4 formulation can be efficiently used for the chemodynamic therapy of colorectal cancer.


Introduction
Hot-melt extrusion (HME) has been widely engaged for preparing drug formulations due to its continuous process, cost-effectiveness, large scale-up production, no downstream process, high throughput characteristic, solvent-free property, and unit operation [1][2][3]. Several machine and operation factors, such as screw type (e.g., single, twin, and multi screws), screw design, screw speed, feed rate, barrel temperature, and die shape, may govern the physicochemical properties of extrudates [1]. Additionally, physical and chemical features of active pharmaceutical ingredients, carriers, and plasticizers should be considered for the optimization of HME process [1]. In the commercial market, several products have

Particle Characterization of MnSO 4 NPs
The mean diameter of the MnSO 4 powder was determined by laser diffraction particle size analysis. MnSO 4 was dispersed in ethanol and the size-dependent volume density profile was obtained by a laser diffraction particle size analyzer (Mastersizer 3000, Malvern Instruments Ltd., Malvern, UK) [17].
The morphology of MnSO 4 NPs (in DW) was observed by transmission electron microscopy (TEM). MnSO 4 NPs (10 mg/mL in DW) were put onto the copper grid with film, the sample was stained with uranyl acetate (1%), and the liquid content was dried in an air stream for 10 min. The particle shape was observed by TEM (CM120; Philips, Amsterdam, The Netherlands).
The content of Mn in MnSO 4 NPs was determined by inductively coupled plasmaoptical emission spectrometry (ICP-OES; Optima 7300 DV, PerkinElmer, Inc., Waltham, MA, USA). Before ICP-OES analysis, the specimen was dissolved in nitric acid.
Incubation time-dependent mean diameter and size distribution features of MnSO 4 NPs were determined by a dynamic light scattering method (ELS-Z1000, Otsuka Electronics). MnSO 4 NPs (at 10 mg/mL) were dispersed in phosphate buffered saline (PBS, pH 7.4) or FBS solution (50%, v/v) and they were incubated for 24 h. After incubating for 0.5, 1, 3, 6, and 24 h, hydrodynamic diameter and polydispersity index values were measured.

X-ray Diffractometry (XRD) Assay
The crystal properties of MnSO 4 , the freeze-dried mixture, and MnSO 4 NPs were explored by a Philips X Pert PRO MPD diffractometer (PANalytical Corp., Almero, The Netherlands). The intensity values according to angle (2θ) at the 5-80 • range were measured. The generator voltage and tube current were set as 40 kV and 30 mA, respectively. The scan time per step and scan step size were 8.67 s and 0.013 • , respectively. The hydroxyl radical generation activity was examined by a TMB-based assay [18]. Each specimen (0.2 mL) of MnSO 4 and MnSO 4 NPs (at 1000 µg/mL MnSO 4 concentration) was put into the microtube and 0.03% H 2 O 2 solution (0.2 mL) was added. Then, 10 mM TMB DH (0.4 mL) was added to each sample and they were incubated at 37 • C for reaction. The reactant (0.2 mL) was acquired at 0, 10, 20, and 30 min and the corresponding absorbance was detected at 650 nm by a microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA, USA).

MB Assay
The hydroxyl radical scavenging effect was studied by an MB-based assay [19]. Each specimen (0.5 mL) of MnSO 4 and MnSO 4 NPs (at 1000 µg/mL MnSO 4 concentration) was put into a dialysis tube (molecular weight cut-off: 12-14 kDa). An H 2 O 2 solution (0.03%, 2.5 mL) was added to that sample and they were incubated at 37 • C for 30 min. Subsequently, an MB solution (20 µg/mL) was added to that mixture and the reactant (0.2 mL) was collected at 0, 15, 30, 60, and 120 min. The absorbance at 650 nm was measured by a microplate reader.

Antiproliferation Assay
The antiproliferation potential of MnSO 4 and MnSO 4 NPs was assessed in CT-26 (colon carcinoma) cells with a colorimetric assay. CT-26 cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Those cells were cultured in DMEM containing FBS (10%, v/v) and penicillin-streptomycin (1%, v/v) at 37 • C. CT-26 cells (at 5.0 × 10 3 cells per well) were seeded in 96-well plate and they were incubated at 37 • C for 24 h. MnSO 4 and MnSO 4 NPs (at 10, 25, 50, 100, and 200 µg/mL MnSO 4 concentrations) were applied to the cells and they were incubated for 72 h. After eliminating each sample, CellTiter 96 ® AQueous One Solution Cell Proliferation Assay Reagent (Promega Corp., Fitchburg, WI, USA) was applied to the cells and incubated at 37 • C. The absorbance at 490 nm was detected by a microplate reader.

Cellular ROS Assay
CT-26 cells were added to a 6-well plate at 1.0 × 10 5 cells/well density. The next day, each specimen of MnSO 4 and MnSO 4 NPs (at 200 µg/mL MnSO 4 concentration) was treated to the cells and they were incubated for 24 h. The cells were washed with cold PBS two times and stained by DCFH-DA (10 µM) for 20 min at 37 • C. Then, the cells were resuspended with FBS solution (2%, v/v). The fluorescence intensity of cellular ROS was measured by flow cytometry (BD Bioscience, San Diego, CA, USA).

Apoptosis Assay
CT-26 cells were seeded in a 6-well plate at a density of 1.0 × 10 5 cells per well. Each specimen of MnSO 4 and MnSO 4 NPs (at 200 µg/mL MnSO 4 concentration) was treated to the cells and incubated for 24 h. The cells were washed with cold PBS two times and resuspended in the binding buffer at a concentration of 1.0 × 10 6 cells/mL. The cells were stained with FITC Annexin V and propidium iodide (PI) in the binding buffer and analyzed by flow cytometry.

Data Analysis
Statistical analyses of data were performed with a two-tailed t-test and analysis of variance. Each experiment was repeated at least three times. Data are provided as the mean ± standard deviation (SD).

Fabrication and Particle Property Tests of MnSO 4 NPs
MnSO 4 NPs were fabricated by the HME process for the chemodynamic therapy of cancer in this study ( Figure 1). MnSO 4 was homogeneously dispersed in hydrophilic polymer (PEG 6000) and non-ionic surfactants (Span 80 and Tween 80) by a twin-screw-based HME system. As observed in our previous reports [8,9], the combination of PEG 6000, Span 80, and Tween 80 was successfully engaged to prepare nano-sized particles of FeSO 4 or CuSO 4 following their dispersion in the aqueous environment. MnSO 4 /PEG 6000/Span 80/Tween 80 extrudate was acquired by the HME process and was pulverized for the convenient preparation of nano-size vesicles after dispersing in the aqueous media. Nano-sized particles of MnSO 4 may be easily introduced to cancer cells [20] and they may attribute to the conversion of intrinsic H 2 O 2 to hydroxyl radical by a Fenton-like reaction of Mn 2+ ion. treated to the cells and they were incubated for 24 h. The cells were washed with cold PBS two times and stained by DCFH-DA (10 μM) for 20 min at 37 °C. Then, the cells were resuspended with FBS solution (2%, v/v). The fluorescence intensity of cellular ROS was measured by flow cytometry (BD Bioscience, San Diego, CA, USA).

Apoptosis Assay
CT-26 cells were seeded in a 6-well plate at a density of 1.0 × 10 5 cells per well. Each specimen of MnSO4 and MnSO4 NPs (at 200 μg/mL MnSO4 concentration) was treated to the cells and incubated for 24 h. The cells were washed with cold PBS two times and resuspended in the binding buffer at a concentration of 1.0 × 10 6 cells/mL. The cells were stained with FITC Annexin V and propidium iodide (PI) in the binding buffer and analyzed by flow cytometry.

Data Analysis
Statistical analyses of data were performed with a two-tailed t-test and analysis of variance. Each experiment was repeated at least three times. Data are provided as the mean ± standard deviation (SD).

Fabrication and Particle Property Tests of MnSO4 NPs
MnSO4 NPs were fabricated by the HME process for the chemodynamic therapy of cancer in this study ( Figure 1). MnSO4 was homogeneously dispersed in hydrophilic polymer (PEG 6000) and non-ionic surfactants (Span 80 and Tween 80) by a twin-screwbased HME system. As observed in our previous reports [8,9], the combination of PEG 6000, Span 80, and Tween 80 was successfully engaged to prepare nano-sized particles of FeSO4 or CuSO4 following their dispersion in the aqueous environment. MnSO4/PEG 6000/Span 80/Tween 80 extrudate was acquired by the HME process and was pulverized for the convenient preparation of nano-size vesicles after dispersing in the aqueous media. Nano-sized particles of MnSO4 may be easily introduced to cancer cells [20] and they may attribute to the conversion of intrinsic H2O2 to hydroxyl radical by a Fenton-like reaction of Mn 2+ ion. NPs production by HME process. Figure 1. Schematic illustration of MnSO 4 NPs production by HME process.
The particle features of MnSO 4 NPs produced by the HME technique were explored as shown in Figure 2. The diameter of MnSO 4 dispersed in ethanol was determined by a particle size analyzer ( Figure 2A). The D v (10), D v (50), and D v (90) values of MnSO 4 were 78, 116, and 169 µm, respectively. The micron size of the MnSO 4 powder was confirmed by particle size analysis.
Pharmaceutics 2023, 15, 1831 6 of 13 The particle features of MnSO4 NPs produced by the HME technique were explored as shown in Figure 2. The diameter of MnSO4 dispersed in ethanol was determined by a particle size analyzer ( Figure 2A). The Dv (10), Dv (50), and Dv (90) values of MnSO4 were 78, 116, and 169 μm, respectively. The micron size of the MnSO4 powder was confirmed by particle size analysis.  The particle properties of HME-processed MnSO 4 composites (MnSO 4 NPs) were explored by size, distribution, zeta potential, encapsulation efficiency, and morphology analyses ( Figure 2B-F). The hydrodynamic diameter of MnSO 4 NPs in DW was 169 nm, and that group exhibited unimodal size distribution ( Figure 2B,C). MnSO 4 seems to be successfully dispersed in the hydrophilic polymer (PEG 6000) and surfactants (Span 80 and Tween 80), reducing the surface tension of particles. By the aid of twin-screw-enabled HME processing, MnSO 4 seems to be homogeneously dispersed in the polymer matrix and it produced nano-sized particles in the aqueous media. Therefore, a significant size reduction effect from micron (MnSO 4 powder) to nano (MnSO 4 NPs) size was accomplished with the HME process with the hydrophilic polymer and surfactants. The zeta potential value of MnSO 4 NPs was −10.7 mV and spherical in shape, with the corresponding diameter, was also shown in the TEM image ( Figure 2B,D). The encapsulation efficiency of MnSO 4 in MnSO 4 NPs was around 100%, indicating the successful encapsulation of MnSO 4 in polymer/surfactant-based particles.
The particle stability in the biological matrices was evaluated by particle size analysis in aqueous buffer and serum media ( Figure 2E,F). In both PBS (pH 7.4) and serum solution (50% FBS), the initial hydrodynamic diameters of MnSO 4 NPs were higher than that in DW ( Figure 2B,E). This may be due to the interactions between MnSO 4 NPs and buffer salts in PBS or proteins in serum media. Nevertheless, the hydrodynamic diameters of MnSO 4 NPs in PBS (pH 7.4) and serum media (50% FBS) after 24 h incubation were 381 and 392 nm, respectively. The polydispersity index values of MnSO 4 NPs in PBS (pH 7.4) and serum media (50% FBS) after 24 h incubation were 0.28 and 0.25, respectively. In particular, the existence of the PEG layer in MnSO 4 NPs may inhibit the opsonization (which can lead to early elimination by mononuclear phagocyte system) and attribute to the maintenance of individual particles without the formation of aggregation [21,22]. Those results mean the maintenance of the nano-size even after incubation in the aqueous buffer or serum conditions, simulating the biological fluids. This may imply the safe application of developed MnSO 4 NPs for cancer treatment.

Solid-State Features of MnSO 4 NPs
The solid-state features of fabricated MnSO 4 NPs by HME process were explored by FT-IR, XRD, XPS, and SEM-EDS tests (Figures 3-6 and S1-S4, Tables S1 and S2). The existence of MnSO 4 and its interactions with pharmaceutical additives were elucidated by FT-IR analysis ( Figure 3 and Table S1). MnSO 4 was characterized by several shifts, such as v 1 SO 4 2− (1017 cm −1 ), v 3 SO 4 2− (1090 cm −1 ), and the O-H stretching band (3140 cm −1 ) ( Figure 3A) [23]. Those peaks were also observed at 960 cm −1 , 1094 cm −1 , and 3433 cm −1 , respectively, in the freeze-dried mixture group ( Figure 3B). MnSO 4 in MnSO 4 NPs was also featured by v 1 SO 4 2− (960 cm −1 ), v 3 SO 4 2− (1096 cm −1 ), and the O-H stretching band (3411 cm −1 ) ( Figure 3C). The FT-IR data of the freeze-dried mixture and MnSO 4 NPs groups were almost similar, indicating similar interactions between MnSO 4 and pharmaceutical excipients during freeze-drying and the HME process (Table S1). The unaltered doublet Mn-O peaks of MnSO 4 (529/508 cm −1 ) in MnSO 4 NPs (529/509 cm −1 ) envisaged the incorporation of MnSO 4 in MnSO 4 NPs [24][25][26]. In particular, peaks at 1740 cm −1 and 2884/1467 cm −1 were attributed to the C=O stretching of ester functionality of Span 80 and Tween 80 and the C-H stretching/deformation bands of -CH 2 -of Span 80, Tween 80, and PEG 6000, respectively [27]. The appearance of alkane-and ester-specific peaks in the FT-IR spectra of MnSO 4 NPs indicated the successful formation of MnSO 4 -based nanovesicles by the HME process. The crystalline feature of MnSO4 included in NPs was explored by XRD assay ( Figure  4). Representative peaks were shown at 18.04°, 25.38°, 28.37°, and 34.81° in the spectrum of MnSO4 [28]. Characteristic shifts were observed at 19.23°, 23.34°, and 25.42° in the profile of the MnSO4 NPs group. Those peaks in MnSO4 NPs group were very similar to The crystalline feature of MnSO 4 included in NPs was explored by XRD assay (Figure 4). Representative peaks were shown at 18.04 • , 25.38 • , 28.37 • , and 34.81 • in the spectrum of MnSO 4 [28]. Characteristic shifts were observed at 19.23 • , 23.34 • , and 25.42 • in the profile of the MnSO 4 NPs group. Those peaks in MnSO 4 NPs group were very similar to those in freeze-dried mixture group with attenuated intensity. The HME process can likely be attributed to less crystallization of the ingredients compared to freeze-drying. The existence of PEG 6000, Span 80, and Tween 80 and their interactions with MnSO 4 were observed in the XRD profile of the MnSO 4 NPs group. The crystalline feature of MnSO4 included in NPs was explored by XRD assay ( Figure  4). Representative peaks were shown at 18.04°, 25.38°, 28.37°, and 34.81° in the spectrum of MnSO4 [28]. Characteristic shifts were observed at 19.23°, 23.34°, and 25.42° in the profile of the MnSO4 NPs group. Those peaks in MnSO4 NPs group were very similar to those in freeze-dried mixture group with attenuated intensity. The HME process can likely be attributed to less crystallization of the ingredients compared to freeze-drying. The existence of PEG 6000, Span 80, and Tween 80 and their interactions with MnSO4 were observed in the XRD profile of the MnSO4 NPs group. The atomic distribution in the outer surface of prepared specimens was investigated with an XPS assay (Figures 5 and S1-S4, Table S2). The contents of Mn 2p, S 2p, and O 1s in the MnSO4 group were 8.48%, 13.28%, and 57.63%, respectively. However, in the MnSO4 NPs group, the values of Mn 2p, S 2p, and O 1s were changed to 0.61%, 0.54%, and 19.56%, respectively. HME processing with pharmaceutical excipients seems to alter the atomic distribution in fabricated MnSO4 NPs. The MnSO4 molecule might move to the inner part of the NP sample according to the data of XPS analysis. A dramatic change in C 1s and O 1s values between MnSO4 and MnSO4 NPs groups may be due to the existence of PEG 6000, Span 80, and Tween 80 in the tested region. The atomic distribution in the outer surface of prepared specimens was investigated with an XPS assay (Figures 5 and S1-S4, Table S2). The contents of Mn 2p, S 2p, and O 1s in the MnSO 4 group were 8.48%, 13.28%, and 57.63%, respectively. However, in the MnSO 4 NPs group, the values of Mn 2p, S 2p, and O 1s were changed to 0.61%, 0.54%, and 19.56%, respectively. HME processing with pharmaceutical excipients seems to alter the atomic distribution in fabricated MnSO 4 NPs. The MnSO 4 molecule might move to the inner part of the NP sample according to the data of XPS analysis. A dramatic change in C 1s and O 1s values between MnSO 4 and MnSO 4 NPs groups may be due to the existence of PEG 6000, Span 80, and Tween 80 in the tested region. The incorporation of PEG 6000, Span 80, and Tween 80 could also be inferred from the deconvoluted C 1s and O 1s spectrum of MnSO4 NPs. The deconvoluted C 1s peaks were observed at 284.53, 286.05, and 288.70 eV, attributed to -CH2-/-CH=CH-, >CH-OH/-CH2-OH, and -(C=O)-O-, respectively ( Figure S1 and Table S2) [29]. Moreover, two distinct peaks were detected at 532.35 and 533.22 eV in the deconvoluted O 1s spectrum of MnSO4 NPs assigned to -(C=O)-O-and >CH-OH/-CH2-OH ( Figure S2B) [30].
Atomic localization was also investigated with SEM equipped with an EDS technique ( Figure 6). The atomic percentage values of Mn and S in the MnSO4 group were 16.79% The incorporation of PEG 6000, Span 80, and Tween 80 could also be inferred from the deconvoluted C 1s and O 1s spectrum of MnSO 4 NPs. The deconvoluted C 1s peaks were observed at 284.53, 286.05, and 288.70 eV, attributed to -CH 2 -/-CH=CH-, >CH-OH/-CH 2 -OH, and -(C=O)-O-, respectively ( Figure S1 and Table S2) [29]. Moreover, two distinct peaks were detected at 532.35 and 533.22 eV in the deconvoluted O 1s spectrum of MnSO 4 NPs assigned to -(C=O)-O-and >CH-OH/-CH 2 -OH ( Figure S2B) [30].
The peaks at 654.55 and 642.01 eV attributed to Mn 2p 1/2 and Mn 2p 3/2 in Mn 2p spectrum of MnSO 4 [31] shifted to 654. 29 (Figures S2B and S4B) in MnSO 4 NPs, respectively. Atomic localization was also investigated with SEM equipped with an EDS technique ( Figure 6). The atomic percentage values of Mn and S in the MnSO 4 group were 16.79% and 12.70%, respectively. They were clearly changed to 11.74% (Mn) and 10.98% (S) in the MnSO 4 NPs group. The alteration of tested atoms in the MnSO 4 NPs group compared to the MnSO 4 group indicates the formation of nano-sized particles composed of MnSO 4 , PEG 6000, Span 80, and Tween 80 by the HME process. All of these findings revealed in the solid-state studies suggest the successful fabrication of MnSO 4 -based nanovesicles following dispersion in the aqueous media.
Atomic localization was also investigated with SEM equipped with an EDS technique ( Figure 6). The atomic percentage values of Mn and S in the MnSO4 group were 16.79% and 12.70%, respectively. They were clearly changed to 11.74% (Mn) and 10.98% (S) in the MnSO4 NPs group. The alteration of tested atoms in the MnSO4 NPs group compared to the MnSO4 group indicates the formation of nano-sized particles composed of MnSO4, PEG 6000, Span 80, and Tween 80 by the HME process. All of these findings revealed in the solid-state studies suggest the successful fabrication of MnSO4-based nanovesicles following dispersion in the aqueous media.

Catalytic Features
The catalytic activity of developed MnSO 4 NPs was investigated with TMB and MB assays (Figure 7). Mn 2+ ion (from MnSO 4 ) can degrade cellular H 2 O 2 to OH radical via a Fenton-like reaction [33]. The H 2 O 2 level in the tumor cell is clearly higher than that in normal cells; therefore, this H 2 O 2 -responsive catalytic vehicle may possess tumor-selective therapeutic effects. OH radical may oxidize TMB (colorless) to its blue product; thus, its absorbance at the 650 nm wavelength can be used for the assessment of OH radical generation [34]. In this study ( Figure 7A), there was an obvious difference between MnSO 4 and MnSO 4 NPs groups (p < 0.05). Of note, an increasing pattern of the absorbance value was observed in the MnSO 4 NPs group rather than the MnSO 4 group. Compared to micronsized MnSO 4 powder, MnSO 4 NPs may possess large surface area in the aqueous media. This may result in elevated Fenton-like reaction-based conversion of H 2 O 2 to OH radical.

Catalytic Features
The catalytic activity of developed MnSO4 NPs was investigated with TMB and MB assays (Figure 7). Mn 2+ ion (from MnSO4) can degrade cellular H2O2 to OH radical via a Fenton-like reaction [33]. The H2O2 level in the tumor cell is clearly higher than that in normal cells; therefore, this H2O2-responsive catalytic vehicle may possess tumor-selective therapeutic effects. OH radical may oxidize TMB (colorless) to its blue product; thus, its absorbance at the 650 nm wavelength can be used for the assessment of OH radical generation [34]. In this study ( Figure 7A), there was an obvious difference between MnSO4 and MnSO4 NPs groups (p < 0.05). Of note, an increasing pattern of the absorbance value was observed in the MnSO4 NPs group rather than the MnSO4 group. Compared to micron-sized MnSO4 powder, MnSO4 NPs may possess large surface area in the aqueous media. This may result in elevated Fenton-like reaction-based conversion of H2O2 to OH radical.
The OH radical generation efficiency of designed MnSO4 NPs was also assessed by MB-based assay ( Figure 7B). It is known that OH radical may attack the C-S + =C group as the first step of MB degradation and the degradation of the aromatic ring will make the MB solution transparent [35]. The MB degradation rate can be used for the evaluation of OH radical production [36]. As shown in Figure 7B, both MnSO4 and MnSO4 NPs groups had decreasing profiles of MB for 120 min incubation. Interestingly, there was a significant difference between MnSO4 and MnSO4 NPs groups at 120 min (p < 0.05). An approximately 43% reduction in the absorbance value of MB in the MnSO4 NPs group may imply the continuous production of OH radical during the tested reaction process. All of these data from catalytic assays suggest the successful generation of hydroxyl radicals aiming at the chemodynamic therapy of cancer.

In Vitro Anticancer Activities
The anticancer potential of developed MnSO4 NPs was explored in CT-26 cells (Figure 8). The antiproliferation activity of designed MnSO4 formulations was evaluated by an MTS-based assay ( Figure 8A). Both MnSO4 and MnSO4 NPs groups had The OH radical generation efficiency of designed MnSO 4 NPs was also assessed by MB-based assay ( Figure 7B). It is known that OH radical may attack the C-S + =C group as the first step of MB degradation and the degradation of the aromatic ring will make the MB solution transparent [35]. The MB degradation rate can be used for the evaluation of OH radical production [36]. As shown in Figure 7B, both MnSO 4 and MnSO 4 NPs groups had decreasing profiles of MB for 120 min incubation. Interestingly, there was a significant difference between MnSO 4 and MnSO 4 NPs groups at 120 min (p < 0.05). An approximately 43% reduction in the absorbance value of MB in the MnSO 4 NPs group may imply the continuous production of OH radical during the tested reaction process. All of these data from catalytic assays suggest the successful generation of hydroxyl radicals aiming at the chemodynamic therapy of cancer.

In Vitro Anticancer Activities
The anticancer potential of developed MnSO 4 NPs was explored in CT-26 cells (Figure 8 Figure 2). A smaller particle size may have higher surface area; therefore, MnSO 4 NPs will produce a higher OH radical amount in cancer cells rather than MnSO 4 based on the Fenton-like reaction. Particle size reduction with HME processing and the aid of hydrophilic polymer and surfactants may support the higher cytotoxicity of MnSO 4 NPs compared to MnSO 4 . This finding suggests the efficient antiproliferation potential of colorectal cancer cells following MnSO 4 NPs treatment. finding suggests the efficient antiproliferation potential of colorectal cancer cells following MnSO4 NPs treatment. Cellular ROS level was investigated after applying MnSO4 and MnSO4 NPs to CT-26 cells ( Figure 8B). At current tested conditions, the cellular ROS level of the MnSO4 NPs group was 42% higher than the MnSO4 group (p < 0.05). As revealed in the results of catalytic assays (Figure 7), Mn 2+ ion seems to provide Fenton-like chemistry-related OH radical generation from intrinsic H2O2, which is highly accumulated in cancer cells [13][14][15]. The smaller particle size of MnSO4 NPs, compared to MnSO4, may be the reason for the enhanced ROS level in cancer cells, principally due to the improved OH radical conversion.
The apoptosis induction capability of developed NPs was also assessed by annexin V-FITC and PI staining assay ( Figure 8C). The sum of percentage in the upper right (UR) and lower right (LR) panels implies the population of apoptosis after applying each treatment. In observed data ( Figure 8C), both MnSO4 and MnSO4 NPs induced a higher apoptosis rate than the control (no treatment) group, meaning the apoptosis induction capacity of the Mn 2+ ion. Notably, the population percentage of (UR + LR) panel in the MnSO4 NPs group was significantly higher than those of the control and MnSO4 groups (p < 0.05). An elevated cellular ROS level may lead to improved apoptosis induction efficiency, ultimately explaining the higher antiproliferation potential. All of these experimental data in CT-26 cells may indicate the application of MnSO4 NPs to its chemodynamic therapy.

Conclusions
Micron-sized MnSO4 powder was dispersed in the hydrophilic polymer (PEG 6000) and non-ionic surfactants (Span 80 and Tween 80) to make nano-sized particles after dispersing in aqueous media with the aid of HME processing. A twin-screw-installed HME system with temperature control successfully produced a homogeneous extrudate composed of MnSO4 and pharmaceutical excipients. Following pulverization, those HMEprocessed MnSO4 particles exhibited a nano-size diameter, spherical shape, and negative zeta potential. The proper distribution of MnSO4 in the hydrophilic polymer with surfactants was demonstrated by several solid-state studies. The hydroxyl radical generation activity of MnSO4 NPs was elucidated by catalytic assays. In colorectal cancer (CT-26) cells, Mn 2+ ion-related Fenton-like chemistry converted intrinsic H2O2 to OH radical and elevated the cellular ROS level, apoptosis induction efficiency, and antiproliferation potential. Developed MnSO4 NPs may be one of the promising candidates for the chemodynamic therapy of colorectal cancer.
Supplementary Materials: The following supporting information can be downloaded at:  Figure 8B). At current tested conditions, the cellular ROS level of the MnSO 4 NPs group was 42% higher than the MnSO 4 group (p < 0.05). As revealed in the results of catalytic assays (Figure 7), Mn 2+ ion seems to provide Fenton-like chemistry-related OH radical generation from intrinsic H 2 O 2 , which is highly accumulated in cancer cells [13][14][15]. The smaller particle size of MnSO 4 NPs, compared to MnSO 4 , may be the reason for the enhanced ROS level in cancer cells, principally due to the improved OH radical conversion.
The apoptosis induction capability of developed NPs was also assessed by annexin V-FITC and PI staining assay ( Figure 8C). The sum of percentage in the upper right (UR) and lower right (LR) panels implies the population of apoptosis after applying each treatment. In observed data ( Figure 8C), both MnSO 4 and MnSO 4 NPs induced a higher apoptosis rate than the control (no treatment) group, meaning the apoptosis induction capacity of the Mn 2+ ion. Notably, the population percentage of (UR + LR) panel in the MnSO 4 NPs group was significantly higher than those of the control and MnSO 4 groups (p < 0.05). An elevated cellular ROS level may lead to improved apoptosis induction efficiency, ultimately explaining the higher antiproliferation potential. All of these experimental data in CT-26 cells may indicate the application of MnSO 4 NPs to its chemodynamic therapy.

Conclusions
Micron-sized MnSO 4 powder was dispersed in the hydrophilic polymer (PEG 6000) and non-ionic surfactants (Span 80 and Tween 80) to make nano-sized particles after dispersing in aqueous media with the aid of HME processing. A twin-screw-installed HME system with temperature control successfully produced a homogeneous extrudate composed of MnSO 4 and pharmaceutical excipients. Following pulverization, those HME-processed MnSO 4 particles exhibited a nano-size diameter, spherical shape, and negative zeta potential. The proper distribution of MnSO 4 in the hydrophilic polymer with surfactants was demonstrated by several solid-state studies. The hydroxyl radical generation activity of MnSO 4 NPs was elucidated by catalytic assays. In colorectal cancer (CT-26) cells, Mn 2+ ion-related Fenton-like chemistry converted intrinsic H 2 O 2 to OH radical and elevated the cellular ROS level, apoptosis induction efficiency, and antiproliferation potential. Developed MnSO 4 NPs may be one of the promising candidates for the chemodynamic therapy of colorectal cancer.