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

Abstract

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.

1. 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 been supplied as shaped (rod or ring) systems and amorphous or crystalline dispersion for ophthalmic inserts, implants, devices, and oral formulations [2]. HME-processed products may have the following outcomes: solubility/bioavailability improvement, taste masking, and controlled/targeted drug release [2]. Various types of pharmaceutical dosage forms, such as films, granules, multicomponent systems (e.g., salts, co-amorphous systems, and co-crystal systems), nanoparticles, pellet, self-microemulsifying drug delivery systems, semi-solid products (e.g., creams, gels, and ointments), and solid implants, can be prepared by the HME technique [3]. These days, an HME system has been coupled with other downstream equipment (e.g., high-pressure homogenizer, pelletizer, and 3D printer) for producing advanced formulations [3].

In cases of organic small chemicals, HME has been selected for improving aqueous solubility, dissolution, and absorption, controlling drug release, and masking bitter tastes [4,5,6,7]. However, there were few reports regarding the fabrication of delivery systems for inorganic substances by HME technique [8,9,10]. In our previous studies [8,9,10], hydrophilic polymer and surfactants have been introduced to make colloidal systems of mineral salts (e.g., ZnSO₄, FeSO₄, and CuSO₄) after dispersing in aqueous media. Herein, polyethylene glycol (PEG) 6000 was used as a hydrophilic polymer and Span 80 and Tween 80 were added as non-ionic surfactants for the homogeneous dispersion of MnSO₄. PEG 6000 can act as a polymer matrix for the distribution of MnSO₄ and both Span 80 and Tween 80 can reduce the surface tension during the particle formation process in the aqueous media. The combination of PEG 6000, Span 80, and Tween 80 has been successfully applied to make colloidal systems of ZnSO₄, FeSO₄, and CuSO₄ [8,9,10]. Although the same feeding ratio of PEG 6000, Span 80, and Tween 80 has been used for HME processing, those metal salts (e.g., ZnSO₄, FeSO₄, and CuSO₄) in our previous studies have different physicochemical properties (i.e., solubility and dispersibility) compared with MnSO₄. HME processing and formulation design were successfully applied to a MnSO₄ molecule in this study. Those HME-processed mineral salts-based particles exhibited nano-sized structures following dispersion in aqueous media. Nano-sized particulate systems may generally possess several advantages such as specific delivery to the target region, the improvement of drug safety and efficacy, and controlled drug cargo release, as reported [11]. This study may have critical implications in the fabrication of MnSO₄-based nanoparticles (NPs) with polymer and surfactants by HME processing.

Moreover, HME-processed MnSO₄ NPs were applied to the chemodynamic therapy of colorectal cancer in this investigation. Cancer cells may have higher H₂O₂ levels compared to normal cells; therefore, a H₂O₂-responsive system may possess cancer cell targeting capability [12]. Mn²⁺ ion, usually provided by MnO₂, can provide a Fenton-like reaction; therefore, it can generate hydroxyl radical from cellular H₂O₂ [13,14,15]. In the current study, MnSO₄ has been used as the source of Mn²⁺ ion, and the HME process was introduced to make nano-size structures for enhanced entry to cancer cells. The generation of toxic hydroxyl radicals may inhibit the proliferation of cancer cells through the elevation of the cellular reactive oxygen species (ROS) level and the induction of other cell death mechanisms (e.g., apoptosis). It is expected that the developed MnSO₄ NPs may be efficient chemodynamic therapeutic agents for colorectal cancer.

2. Materials and Methods

2.1. Materials

MnSO₄ (MnSO₄·H₂O) was acquired from TMC Co., Ltd. (Anyang, Republic of Korea). PEG 6000 was provided by Samchun Pure Chemical Co., Ltd. (Pyeongtaek, Republic of Korea). Span 80 and Tween 80 were purchased from Daejung Chemical & Metals Co., Ltd. (Siheung, Republic of Korea). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate (TMB DH), and methylene blue (MB) were purchased from Sigma–Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Gibco Life Technologies, Inc. (Grand Island, NY, USA).

2.2. Production and Particle Characterization of MnSO₄ Extrudate-Based Nanocomposites

2.2.1. Production of MnSO₄ Formulations by HME

MnSO₄, PEG 6000, Span 80, and Tween 80 were blended at a 20:64:12:4 ratio (w/w/w/w) prior to putting those materials into the extruder [8,9,10,16]. Those blended materials were translocated to the hopper part of HME (45 g/min rate). A hot-melt extruder installed with twin-screw (STS-25HS, Hankook E.M. Ltd., Pyeongtaek, Republic of Korea) and a round-shaped die (1 mm diameter) was utilized for the fabrication of MnSO₄-based extrudates. Temperatures in the barrel and die parts were set as 45 °C and 40 °C, respectively. The screw speed was maintained at 150 rpm in this HME step. After moving through the conveying and kneading parts in the barrel, specimens were extruded from the die part. Extruded materials were hardened and pulverized by the grinder (HBL-3500S, Samyang Electronics Co., Gunpo, Republic of Korea).

2.2.2. Particle Characterization of MnSO₄ NPs

The mean diameter of the MnSO₄ powder was determined by laser diffraction particle size analysis. MnSO₄ 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 particle properties of MnSO₄ NPs in distilled water (DW) were investigated. Hydrodynamic diameter, polydispersity index, and zeta potential values of MnSO₄ NPs dispersed in DW (at 10 mg/mL) were measured by dynamic light scattering and laser Doppler methods (ELS-Z1000; Otsuka Electronics, Tokyo, Japan) [8,9,10].

The morphology of MnSO₄ NPs (in DW) was observed by transmission electron microscopy (TEM). MnSO₄ 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 MnSO₄ NPs was determined by inductively coupled plasma-optical 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 MnSO₄ NPs were determined by a dynamic light scattering method (ELS-Z1000, Otsuka Electronics). MnSO₄ 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.

2.3. Solid-State Studies

2.3.1. Fourier-Transform Infrared (FT-IR) Spectroscopy Analysis

Alteration in the chemical functions of MnSO₄, freeze-dried mixture (MnSO₄, PEG 6000, Span 80, and Tween 80), and MnSO₄ NPs was studied by FT-IR analysis. Transmittance (%) values of MnSO₄, the freeze-dried mixture, and MnSO₄ NPs were scanned using a Frontier FT-IR spectrometer (PerkinElmer Inc., Buckinghamshire, UK). FT-IR spectra were obtained in the attenuated total reflectance mode. Transmittance (%) values of MnSO₄, the freeze-dried mixture, and MnSO₄ NPs were monitored in a 400–4000 cm⁻¹ wavenumber range.

2.3.2. X-ray Diffractometry (XRD) Assay

The crystal properties of MnSO₄, the freeze-dried mixture, and MnSO₄ 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.

2.3.3. X-ray Photoelectron Spectroscopy (XPS) Study

The elemental composition of MnSO₄ and MnSO₄ NPs was determined by an XPS (K-Alpha+, Thermo Fisher Scientific, East Grinstead, UK) system. The atomic contents (%) in MnSO₄ (Mn 2p, O 1s, Ca 2p, C 1s, and S 2p) and MnSO₄ NPs (Mn 2p, O 1s, C 1s, and S 2p) were measured. Al K_α X-ray (as a source gun) was used in this test. Peak patterns of C 1s, O 1s, Mn 2p, and S 2p were analyzed in a narrow binding energy range.

2.3.4. Field Emission Scanning Electron Microscopy (FE-SEM)/Energy Dispersive Spectrometry (EDS) Study

FE-SEM (JSM-7900F, JEOL, Tokyo, Japan) combined with EDS was applied to identify the distribution of elements on MnSO₄ and MnSO₄ NPs. Atomic contents of C, O, S, and Mn in MnSO₄ and MnSO₄ NPs were quantitatively determined.

2.4. Catalytic Assays

2.4.1. 3,3′,5,5′- Tetramethylbenzidine (TMB) Assay

The hydroxyl radical generation activity was examined by a TMB-based assay [18]. Each specimen (0.2 mL) of MnSO₄ and MnSO₄ NPs (at 1000 μg/mL MnSO₄ concentration) was put into the microtube and 0.03% H₂O₂ 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).

2.4.2. MB Assay

The hydroxyl radical scavenging effect was studied by an MB-based assay [19]. Each specimen (0.5 mL) of MnSO₄ and MnSO₄ NPs (at 1000 μg/mL MnSO₄ concentration) was put into a dialysis tube (molecular weight cut-off: 12–14 kDa). An H₂O₂ 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.

2.5. In Vitro Anticancer Activity Tests

2.5.1. Antiproliferation Assay

The antiproliferation potential of MnSO₄ and MnSO₄ 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 × 10³ cells per well) were seeded in 96-well plate and they were incubated at 37 °C for 24 h. MnSO₄ and MnSO₄ NPs (at 10, 25, 50, 100, and 200 μg/mL MnSO₄ 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.

2.5.2. Cellular ROS Assay

CT-26 cells were added to a 6-well plate at 1.0 × 10⁵ cells/well density. The next day, each specimen of MnSO₄ and MnSO₄ NPs (at 200 μg/mL MnSO₄ 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).

2.5.3. Apoptosis Assay

CT-26 cells were seeded in a 6-well plate at a density of 1.0 × 10⁵ 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 × 10⁶ cells/mL. The cells were stained with FITC Annexin V and propidium iodide (PI) in the binding buffer and analyzed by flow cytometry.

2.6. 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).

3. Results and Discussion

3.1. 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-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 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 Mn²⁺ ion.

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 D_v (10), D_v (50), and D_v (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 MnSO₄ composites (MnSO₄ NPs) were explored by size, distribution, zeta potential, encapsulation efficiency, and morphology analyses (Figure 2B–F). The hydrodynamic diameter of MnSO₄ NPs in DW was 169 nm, and that group exhibited unimodal size distribution (Figure 2B,C). MnSO₄ 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, MnSO₄ 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 (MnSO₄ powder) to nano (MnSO₄ NPs) size was accomplished with the HME process with the hydrophilic polymer and surfactants. The zeta potential value of MnSO₄ 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 MnSO₄ in MnSO₄ NPs was around 100%, indicating the successful encapsulation of MnSO₄ 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 MnSO₄ NPs were higher than that in DW (Figure 2B,E). This may be due to the interactions between MnSO₄ NPs and buffer salts in PBS or proteins in serum media. Nevertheless, the hydrodynamic diameters of MnSO₄ 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 MnSO₄ 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 MnSO₄ 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 MnSO₄ NPs for cancer treatment.

3.2. Solid-State Features of MnSO₄ NPs

The solid-state features of fabricated MnSO₄ NPs by HME process were explored by FT-IR, XRD, XPS, and SEM-EDS tests (Figure 3, Figure 4, Figure 5 and Figure 6 and Figures S1–S4, Tables S1 and S2). The existence of MnSO₄ and its interactions with pharmaceutical additives were elucidated by FT-IR analysis (Figure 3 and Table S1). MnSO₄ was characterized by several shifts, such as v₁SO₄²⁻ (1017 cm⁻¹), v₃SO₄²⁻ (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). MnSO₄ in MnSO₄ NPs was also featured by v₁SO₄²⁻ (960 cm⁻¹), v₃SO₄²⁻ (1096 cm⁻¹), and the O–H stretching band (3411 cm⁻¹) (Figure 3C). The FT-IR data of the freeze-dried mixture and MnSO₄ NPs groups were almost similar, indicating similar interactions between MnSO₄ and pharmaceutical excipients during freeze-drying and the HME process (Table S1). The unaltered doublet Mn–O peaks of MnSO₄ (529/508 cm⁻¹) in MnSO₄ NPs (529/509 cm⁻¹) envisaged the incorporation of MnSO₄ in MnSO₄ 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 –CH₂– of Span 80, Tween 80, and PEG 6000, respectively [27]. The appearance of alkane- and ester-specific peaks in the FT-IR spectra of MnSO₄ NPs indicated the successful formation of MnSO₄-based nanovesicles by the HME process.

Figure 3. FT-IR spectra of (A) MnSO₄, (B) freeze-dried mixture, and (C) MnSO₄ NPs. Wavenumber-dependent transmittance value (%T) is plotted.

Figure 3. FT-IR spectra of (A) MnSO₄, (B) freeze-dried mixture, and (C) MnSO₄ NPs. Wavenumber-dependent transmittance value (%T) is plotted.

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.

Figure 4. XRD patterns of (A) MnSO₄, (B) freeze-dried mixture, and (C) MnSO₄ NPs.

Figure 4. XRD patterns of (A) MnSO₄, (B) freeze-dried mixture, and (C) MnSO₄ NPs.

The atomic distribution in the outer surface of prepared specimens was investigated with an XPS assay (Figure 5 and Figure 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.

Figure 5. XPS data of (A) MnSO₄ and (B) MnSO₄ NPs.

Figure 5. XPS data of (A) MnSO₄ and (B) MnSO₄ NPs.

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].

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 MnSO₄ [31] shifted to 654.29 and 641.59 eV, respectively, in the Mn 2p spectrum of MnSO₄ NPs (Figure S3). Such a relatively lesser negative shift (i.e., –0.26 and –0.42 eV, respectively) of Mn 2p peaks implied the prevalence of weak ionic interaction between Mn(II) of MnSO₄ and –(C=O)–O–/>CH–OH/–CH₂–OH of PEG 6000, Span 80, and Tween 80 [32]. It was further confirmed by the shift of O 1s and S 2p peaks of SO₄²⁻ from 531.68 and 168.29/169.33 eV ) of MnSO₄ to 531.57 and 164.65/168.75 eV (Figures S2B and S4B) in MnSO₄ 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 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.

Figure 6. SEM images and EDS analysis data of MnSO₄ and MnSO₄ NPs. Weight portion (Wt%) and atomic contents (Atomic %) are included in the inset.

Figure 6. SEM images and EDS analysis data of MnSO₄ and MnSO₄ NPs. Weight portion (Wt%) and atomic contents (Atomic %) are included in the inset.

3.3. Catalytic Features

The catalytic activity of developed MnSO₄ NPs was investigated with TMB and MB assays (Figure 7). Mn²⁺ 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.

3.4. 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 concentration-dependent cytotoxicity profiles. The IC₅₀ values (presented as MnSO₄ concentration in both groups) of MnSO₄ and MnSO₄ NPs were 100.5 and 86.0 μg/mL, respectively. In particular, the MnSO₄ NPs group exhibited higher cytotoxicity than MnSO₄ group at 200 μg/mL MnSO₄ concentration (p < 0.05). Compared to the micron size of MnSO₄, MnSO₄ NPs had colloidal size range in the aqueous media (Figure 2). A smaller particle size may have higher surface area; therefore, MnSO₄ NPs will produce a higher OH radical amount in cancer cells rather than MnSO₄ 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 MnSO₄ NPs compared to MnSO₄. This 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), Mn²⁺ 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 Mn²⁺ 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.

4. 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 HME-processed 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, Mn²⁺ 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.