# Induction of Heat Shock Protein-72 by Magnetic Nanofluid Hyperthermia in Cultured Retinal Ganglion Cells for Neuroprotective Treatment in Glaucoma

Authors: Jin Wook Jeoung; Minhong Jeun; Joo Hyun Park; Yu Jeong Kim; Seongtae Bae; Ki Ho Park
Journal: Journal of Nanomaterials
 (2015)

---

## Abstract

Background. Magnetic hyperthermia using superparamagnetic nanoparticle (SPNP) agents is considered a promising biotechnological approach to induce heat shock proteins (HSPs) in a target tissue because it can generate accurately controllable localized heating. Objectives. The main objective of this study is to demonstrate induction of HSPs in cultured retinal ganglion cells (RGCs) by using engineered Mn0.5Zn0.5Fe2O4 SPNP agents coated with polyethylene glycol (PEG) 500. Methods. The Mn0.5Zn0.5Fe2O4 nanoparticles were synthesized using a high temperature thermal decomposition method. The AC heating characteristics of PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles were investigated using an AC solenoid coil-capacitor system. Results. PEG 500-coated SPNPs efficiently penetrated into the cytoplasm of RGCs without causing obvious cytological changes and showed stable and well-saturated self-heating temperature rise characteristics. Immunofluorescent staining images showed that AC magnetic hyperthermia successfully induced HSP72 in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles. In Western blot analysis, a significant increase in immunoreactivity was observed for RGCs incubated with SPNPs in a fixed AC magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe). Conclusion. Our results demonstrate that the induction of HSP72 with a magnetic nanofluid hyperthermia could potentially be used as a neuroprotective treatment modality by way of enhancing a natural cytoprotective response.


---

## Body

## 1. Introduction

Glaucoma is characterized by progressive optic nerve damage with selective loss of retinal ganglion cells (RGCs) [1–3]. Recent clinical trials confirmed that the major modifiable risk factor for the development and progression of glaucoma is the intraocular pressure (IOP). The current treatment of glaucoma mainly involves reducing IOP and diurnal fluctuation. However, signs of glaucomatous progression can be seen in many patients with well-controlled IOP. In addition, disease onset and progression can occur in patients whose untreated IOPs are within the normal range. From this background, the importance of neuroprotection in the treatment of glaucoma has become a worldwide issue. During the past decades, many studies have focused on discovering neuroprotective agents along with IOP lowering medications in order to prevent retinal ganglion cell (RGC) death or even reverse the process of cell death [4].Heat shock proteins (HSPs), also known as stress proteins, are a group of proteins found in all living cells. It is rapidly induced by a variety of environmental stresses, such as hyperthermia, hypoxia, or ischemia, and its role is to protect cells against stress [5, 6]. This protection mainly attributes to members of the HSP70 family, especially 72 kDa heat shock protein (HSP72). HSP72, the major inducible form of the HSP70 family, has been found protecting cells from certain apoptotic stimuli such as oxidative stress, hypoxia, and inflammation [7–9].Magnetic hyperthermia using superparamagnetic nanoparticle (SPNP) agents is considered a promising biotechnological approach to induce HSPs in a target tissue because it can generate accurately controllable localized heating [10, 11]. Recently, our group reported on the feasibility of achieving neuroprotection with magnetically engineered superparamagnetic Mn0.5Zn0.5Fe2O4 nanoparticles for localized HSP induction [12]. However, no empirical study on HSP expression in neuronal cells induced by magnetically engineered SPNP agents has been presented. The purpose of this study is to demonstrate induction of HSPs in cultured RGCs by local magnetic hyperthermia using engineered superparamagnetic Mn0.5Zn0.5Fe2O4 nanoparticle agents.


## 2. Materials and Methods

### 2.1. Materials

Fe (III) acetylacetonate (precursor, >99.9%), Mn (III) acetate tetrahydrate (99.99%), Zn acetate dehydrate (99.999%), oleic acid (surfactant, 90%), oleylamine (surfactant, 70%), 1,2-hexadecanediol (reductant, 90%), and benzyl ether (solvent, 99%) were purchased from Sigma-Aldrich to synthesize Mn0.5Zn0.5Fe2O4 SPNPs. In order to coat the SPNPs with polyethylene glycol (PEG, 500 Da), PEG and triethylamine were purchased from Gelest Inc. and Sigma-Aldrich, respectively.


### 2.2. Synthesis of Mn0.5Zn0.5Fe2O4 and Coating with PEG 500

#### 2.2.1. Synthesis of Mn0.5Zn0.5Fe2O4

Mn0.5Zn0.5Fe2O4 was synthesized using a high temperature thermal decomposition method. Mn (0.5 mmol), Zn (0.5 mmol), Fe (III) (2 mmol), oleic acid (6 mmol), oleylamine (6 mmol), 1.2 hexadecanediol (10 mmol), and benzyl ether (20 mL) were mixed and gently stirred at room temperature. The ramping rate and heat treatment time were 13.3°C/min and 45 min, respectively. After heat treatment, the mixed solution was cooled to room temperature. Ethanol (40 mL) was added to the mixed solution to rinse the synthesized SPNPs, which were collected by centrifugation and then dried at room temperature.


#### 2.2.2. Coating of Mn0.5Zn0.5Fe2O4 with PEG 500

The synthesized Mn0.5Zn0.5Fe2O4 SPNAs were coated with PEG 500 Da to form a nanofluid. The SPNPs were dispersed in toluene (solvent, 7.5 mL), and then PEG (0.75 mL) and triethylamine (catalyst, 3.75 mL) were added. The mixed suspension was shaken for 24 hours at room temperature. The PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs were then collected by centrifugation and dispersed in distilled water.


### 2.3. Cell Culture

Transformed RGC-5 retinal ganglion cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin/streptomycin (Invitrogen), and 2 mM glutamine (Invitrogen) in a humidified incubator with 5% CO2 at 37°C.


### 2.4.In Vitro Cytotoxicity Assay: Analysis by Transmission Electron Microscopy (TEM)

TEM analysis was conducted for the RGC-5 cells treated with Mn0.5Zn0.5Fe2O4 particles to investigate the degree of SPNP penetration into the cells, cell apoptosis, and cell deformation, including nuclear fragmentation. RGC-5 cells were differentiated with 1 μM staurosporine for 1 hour and incubated with 300 μg/mL SPNP for 24 hours. Cells were fixed with 2.5% glutaraldehyde and washed with PBS followed by 0.1 M cacodylate buffer (pH 7.2) and postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for 1.5 hours at room temperature. The samples were then washed briefly with distilled water, dehydrated in ethanol, infiltrated with propylene oxide (Acros Organics) and EPON mixed with epoxy resin (Embed 812, nadic methyl anhydride, Poly Med@ 812, dodecenyl succinic anhydride, dimethylaminomethyl phenol, Electron Microscopy Polysciences), and finally embedded with epoxy resin alone. The epoxy resin mixed samples were loaded into capsules and polymerized at 38°C for 12 hours and 60°C for 48 hours. Thin sections were made using an RMC MT-XL ultramicrotome, collected on a copper grid, and viewed with a JEOL (KEM-1400) at 80 kV in the TEM mode.


### 2.5. Physical and Chemical Characterization of Mn0.5Zn0.5Fe2O4 SPNP Hyperthermia Agents

The shape, size, coating thickness, and size distribution of SPNPs were analyzed by high-resolution transmission electron microscopy (HR-TEM). TheT
AC
⁡
,
mag of PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs was characterized using an AC solenoid coil-capacitor system that we developed. The AC magnetically induced heating temperature of RGCs treated with the SPNPs (500 μg/mL) was measured at a fixed AC magnetic field of f
=
140 kHz and H
=
140 Oe. Heating stress was applied for times ranging from 600 to 1200 s. In addition, measurements were taken of the heating characteristics of RGCs treated with SPNPs at concentrations from 300 to 700 μg/mL under the same AC magnetic heating conditions.


### 2.6. Induction of Heat Shock Protein by the Magnetic Nanofluid Hyperthermia System

The induction of HSPs by AC magnetic nanofluid hyperthermia was conducted using the high performance PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs that we developed. In order to investigate the AC magnetically induced heating characteristics in RGCs, we developed an AC magnetic field generation system, which consists of AC coils, capacitors, DC power supplies, and wave generators (see Figure 1). This system was designed for bothin vitro andin vivo pilot studies. The applied frequency of this system can be automatically controlled from 30 to 370 kHz, and the applied AC magnetic field can be simultaneously changed from 0 to 200 Oe at each applied frequency.Figure 1
(a) An AC magnetic field generation system designed for bothin vitro andin vivo hyperthermia studies. This system consists of AC coils, capacitors, DC power supplies, and wave generators. (b) Microcentrifuge tube containing RGCs with inserted optical thermometer.
(a)
(b)Before magnetic hyperthermia, the RGC-5 cells were differentiated with 1μM staurosporine for 1 hour and incubated with Mn0.5Zn0.5Fe2O4 SPNPs for 24 hours. The cells were then washed with PBS (phosphate buffered saline) and treated with trypsin-EDTA (Life Technologies, USA) followed by addition of an equal volume of culture medium. Cells were centrifuged for 5 min; the supernatant was discarded and resuspended in culture medium. After a second 5-minute centrifugation, the supernatant was again discarded and the cells were harvested into 0.1 mL of the medium in a 0.6 mL Eppendorf (E) tube.After maintaining the temperature of the cells in the E-tube at 37°C in a temperature-controlled bath, we placed the E-tube and cells in the center of the magnetic coil of an induction generator, monitoring the temperature while the generator operated. At the fixed applied frequency of 140 kHz, we controlled the temperature by increasing or decreasing the strength of the AC magnetic field. We maintained the target temperature of 41.0°C for the desired period before stopping the generator.The RGCs incubated with Mn0.5Zn0.5Fe2O4 SPNPs were centrifuged to form a cell pellet. The tube and pelleted RGCs were immersed in a water bath to keep the temperature in the 36–37°C range. As shown in Figure 1, the temperature of the RGC-5 cells during hyperthermia was measured using an optical thermosensor (Fluoroptic temperature probe, Lumasense technologies, USA). The microcentrifuge tube with an optical thermometer inserted in the RGCs was placed at the center of the coil supplying the AC magnetic field. The warming rate of the SPNPs in the RGCs was controlled by changing the strength of H
appl within the biologically and physiologically safe range of 120 Oe (H
appl
·
f
appl
=
1.34
×
10
9 A m−1 s−1)–160 Oe (H
appl
·
f
appl
=
1.78
×
10
9 A m−1 s−1) at the fixed frequency of 140 kHz. The T
AC
⁡
,
mag of RGCs with SPNPs during HSPs induction was kept at 40.5°C ± 0.5°C for 900 sec.After 24 hours, the RGCs were counted and seeded onto a culture plate followed by fixation with 10% formaldehyde. The RGCs were washed twice with PBST (phosphate buffered saline tween-20) and then blocked. Anti-HSP72 (Enzo Life Sciences, USA) was added as primary antibody, followed by two washes with PBST. Alexa Fluor 488 goat anti-mouse IgG (FITC, fluorescein isothiocyanate) (Life Technologies, USA) was then added as secondary antibody, followed by two washes with PBST. To stain the nuclei, DAPI (4′,6-diamino-2-phenylindole) (Sigma, USA) was added, followed by two washes with PBST.


### 2.7. Western Blot Analysis

To quantify the induction of HSPs by hyperthermia using SPNPs, we performed Western blot analyses for four samples: three types of control group and an experimental sample with SPNPs (500μg/mL) and a magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe) applied for 900 sec. For the analysis, harvested cells were lysed in a lysis buffer (Intron Biotechnology, Korea), loaded onto a 10% SDS-polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with dried nonfat milk and incubated with the primary antibody: Hsp72 antibody (1 : 1000, Enzo Life Sciences, USA) and β-actin antibody (1 : 1000, Sigma, USA). The membrane was washed with Tris-buffered saline containing 0.1% tween-20 and incubated with goat anti-mouse IgG (1 : 5000, Santa Cruz, USA). The membrane was then washed and immunoblotting bands were detected using the WEST-ZOL detection system (iNtRON Biotechnology, Korea).


## 2.1. Materials

Fe (III) acetylacetonate (precursor, >99.9%), Mn (III) acetate tetrahydrate (99.99%), Zn acetate dehydrate (99.999%), oleic acid (surfactant, 90%), oleylamine (surfactant, 70%), 1,2-hexadecanediol (reductant, 90%), and benzyl ether (solvent, 99%) were purchased from Sigma-Aldrich to synthesize Mn0.5Zn0.5Fe2O4 SPNPs. In order to coat the SPNPs with polyethylene glycol (PEG, 500 Da), PEG and triethylamine were purchased from Gelest Inc. and Sigma-Aldrich, respectively.


## 2.2. Synthesis of Mn0.5Zn0.5Fe2O4 and Coating with PEG 500

### 2.2.1. Synthesis of Mn0.5Zn0.5Fe2O4

Mn0.5Zn0.5Fe2O4 was synthesized using a high temperature thermal decomposition method. Mn (0.5 mmol), Zn (0.5 mmol), Fe (III) (2 mmol), oleic acid (6 mmol), oleylamine (6 mmol), 1.2 hexadecanediol (10 mmol), and benzyl ether (20 mL) were mixed and gently stirred at room temperature. The ramping rate and heat treatment time were 13.3°C/min and 45 min, respectively. After heat treatment, the mixed solution was cooled to room temperature. Ethanol (40 mL) was added to the mixed solution to rinse the synthesized SPNPs, which were collected by centrifugation and then dried at room temperature.


### 2.2.2. Coating of Mn0.5Zn0.5Fe2O4 with PEG 500

The synthesized Mn0.5Zn0.5Fe2O4 SPNAs were coated with PEG 500 Da to form a nanofluid. The SPNPs were dispersed in toluene (solvent, 7.5 mL), and then PEG (0.75 mL) and triethylamine (catalyst, 3.75 mL) were added. The mixed suspension was shaken for 24 hours at room temperature. The PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs were then collected by centrifugation and dispersed in distilled water.


## 2.2.1. Synthesis of Mn0.5Zn0.5Fe2O4

Mn0.5Zn0.5Fe2O4 was synthesized using a high temperature thermal decomposition method. Mn (0.5 mmol), Zn (0.5 mmol), Fe (III) (2 mmol), oleic acid (6 mmol), oleylamine (6 mmol), 1.2 hexadecanediol (10 mmol), and benzyl ether (20 mL) were mixed and gently stirred at room temperature. The ramping rate and heat treatment time were 13.3°C/min and 45 min, respectively. After heat treatment, the mixed solution was cooled to room temperature. Ethanol (40 mL) was added to the mixed solution to rinse the synthesized SPNPs, which were collected by centrifugation and then dried at room temperature.


## 2.2.2. Coating of Mn0.5Zn0.5Fe2O4 with PEG 500

The synthesized Mn0.5Zn0.5Fe2O4 SPNAs were coated with PEG 500 Da to form a nanofluid. The SPNPs were dispersed in toluene (solvent, 7.5 mL), and then PEG (0.75 mL) and triethylamine (catalyst, 3.75 mL) were added. The mixed suspension was shaken for 24 hours at room temperature. The PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs were then collected by centrifugation and dispersed in distilled water.


## 2.3. Cell Culture

Transformed RGC-5 retinal ganglion cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin/streptomycin (Invitrogen), and 2 mM glutamine (Invitrogen) in a humidified incubator with 5% CO2 at 37°C.


## 2.4.In Vitro Cytotoxicity Assay: Analysis by Transmission Electron Microscopy (TEM)

TEM analysis was conducted for the RGC-5 cells treated with Mn0.5Zn0.5Fe2O4 particles to investigate the degree of SPNP penetration into the cells, cell apoptosis, and cell deformation, including nuclear fragmentation. RGC-5 cells were differentiated with 1 μM staurosporine for 1 hour and incubated with 300 μg/mL SPNP for 24 hours. Cells were fixed with 2.5% glutaraldehyde and washed with PBS followed by 0.1 M cacodylate buffer (pH 7.2) and postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for 1.5 hours at room temperature. The samples were then washed briefly with distilled water, dehydrated in ethanol, infiltrated with propylene oxide (Acros Organics) and EPON mixed with epoxy resin (Embed 812, nadic methyl anhydride, Poly Med@ 812, dodecenyl succinic anhydride, dimethylaminomethyl phenol, Electron Microscopy Polysciences), and finally embedded with epoxy resin alone. The epoxy resin mixed samples were loaded into capsules and polymerized at 38°C for 12 hours and 60°C for 48 hours. Thin sections were made using an RMC MT-XL ultramicrotome, collected on a copper grid, and viewed with a JEOL (KEM-1400) at 80 kV in the TEM mode.


## 2.5. Physical and Chemical Characterization of Mn0.5Zn0.5Fe2O4 SPNP Hyperthermia Agents

The shape, size, coating thickness, and size distribution of SPNPs were analyzed by high-resolution transmission electron microscopy (HR-TEM). TheT
AC
⁡
,
mag of PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs was characterized using an AC solenoid coil-capacitor system that we developed. The AC magnetically induced heating temperature of RGCs treated with the SPNPs (500 μg/mL) was measured at a fixed AC magnetic field of f
=
140 kHz and H
=
140 Oe. Heating stress was applied for times ranging from 600 to 1200 s. In addition, measurements were taken of the heating characteristics of RGCs treated with SPNPs at concentrations from 300 to 700 μg/mL under the same AC magnetic heating conditions.


## 2.6. Induction of Heat Shock Protein by the Magnetic Nanofluid Hyperthermia System

The induction of HSPs by AC magnetic nanofluid hyperthermia was conducted using the high performance PEG 500-coated Mn0.5Zn0.5Fe2O4 SPNPs that we developed. In order to investigate the AC magnetically induced heating characteristics in RGCs, we developed an AC magnetic field generation system, which consists of AC coils, capacitors, DC power supplies, and wave generators (see Figure 1). This system was designed for bothin vitro andin vivo pilot studies. The applied frequency of this system can be automatically controlled from 30 to 370 kHz, and the applied AC magnetic field can be simultaneously changed from 0 to 200 Oe at each applied frequency.Figure 1
(a) An AC magnetic field generation system designed for bothin vitro andin vivo hyperthermia studies. This system consists of AC coils, capacitors, DC power supplies, and wave generators. (b) Microcentrifuge tube containing RGCs with inserted optical thermometer.
(a)
(b)Before magnetic hyperthermia, the RGC-5 cells were differentiated with 1μM staurosporine for 1 hour and incubated with Mn0.5Zn0.5Fe2O4 SPNPs for 24 hours. The cells were then washed with PBS (phosphate buffered saline) and treated with trypsin-EDTA (Life Technologies, USA) followed by addition of an equal volume of culture medium. Cells were centrifuged for 5 min; the supernatant was discarded and resuspended in culture medium. After a second 5-minute centrifugation, the supernatant was again discarded and the cells were harvested into 0.1 mL of the medium in a 0.6 mL Eppendorf (E) tube.After maintaining the temperature of the cells in the E-tube at 37°C in a temperature-controlled bath, we placed the E-tube and cells in the center of the magnetic coil of an induction generator, monitoring the temperature while the generator operated. At the fixed applied frequency of 140 kHz, we controlled the temperature by increasing or decreasing the strength of the AC magnetic field. We maintained the target temperature of 41.0°C for the desired period before stopping the generator.The RGCs incubated with Mn0.5Zn0.5Fe2O4 SPNPs were centrifuged to form a cell pellet. The tube and pelleted RGCs were immersed in a water bath to keep the temperature in the 36–37°C range. As shown in Figure 1, the temperature of the RGC-5 cells during hyperthermia was measured using an optical thermosensor (Fluoroptic temperature probe, Lumasense technologies, USA). The microcentrifuge tube with an optical thermometer inserted in the RGCs was placed at the center of the coil supplying the AC magnetic field. The warming rate of the SPNPs in the RGCs was controlled by changing the strength of H
appl within the biologically and physiologically safe range of 120 Oe (H
appl
·
f
appl
=
1.34
×
10
9 A m−1 s−1)–160 Oe (H
appl
·
f
appl
=
1.78
×
10
9 A m−1 s−1) at the fixed frequency of 140 kHz. The T
AC
⁡
,
mag of RGCs with SPNPs during HSPs induction was kept at 40.5°C ± 0.5°C for 900 sec.After 24 hours, the RGCs were counted and seeded onto a culture plate followed by fixation with 10% formaldehyde. The RGCs were washed twice with PBST (phosphate buffered saline tween-20) and then blocked. Anti-HSP72 (Enzo Life Sciences, USA) was added as primary antibody, followed by two washes with PBST. Alexa Fluor 488 goat anti-mouse IgG (FITC, fluorescein isothiocyanate) (Life Technologies, USA) was then added as secondary antibody, followed by two washes with PBST. To stain the nuclei, DAPI (4′,6-diamino-2-phenylindole) (Sigma, USA) was added, followed by two washes with PBST.


## 2.7. Western Blot Analysis

To quantify the induction of HSPs by hyperthermia using SPNPs, we performed Western blot analyses for four samples: three types of control group and an experimental sample with SPNPs (500μg/mL) and a magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe) applied for 900 sec. For the analysis, harvested cells were lysed in a lysis buffer (Intron Biotechnology, Korea), loaded onto a 10% SDS-polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with dried nonfat milk and incubated with the primary antibody: Hsp72 antibody (1 : 1000, Enzo Life Sciences, USA) and β-actin antibody (1 : 1000, Sigma, USA). The membrane was washed with Tris-buffered saline containing 0.1% tween-20 and incubated with goat anti-mouse IgG (1 : 5000, Santa Cruz, USA). The membrane was then washed and immunoblotting bands were detected using the WEST-ZOL detection system (iNtRON Biotechnology, Korea).


## 3. Results and Discussion

This study was designed to demonstrate induction of HSPs in cultured RGCs by using engineered superparamagnetic Mn0.5Zn0.5Fe2O4 nanoparticle agents. We employed a localized magnetic hyperthermia system by using the SPNP agents as a new nanobiotechnological approach to effectively control the local induction of HSP72. Magnetic hyperthermia using SPNP agents is considered a promising biotechnological approach to the induction of HSPs in target tissues because it can generate localized heating with accurate temperature control. Evidence suggests that manipulation of the cellular stress response may be a strategy to protect neurons from damage following cerebral ischemia [13–15] or during the progression of neurodegenerative conditions such as Alzheimer’s disease [16, 17], Parkinson’s disease [18], and glaucoma [19–21]. In this study, we demonstrate that the induction of HSPs by localized magnetic hyperthermia using engineered SPNPs is promisingly feasible for neuroprotection modality.


### 3.1. Characterization of PEG 500-Coated Mn0.5Zn0.5Fe2O4

Figure2 shows the HR-TEM images of PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. The PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles have a round shape with a mean size of 7.20
±
0.85 nm and a narrow size distribution, and they are well segregated.Figure 2
High-resolution transmission electron microscopy image of superparamagnetic PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles dispersed in distilled water.


### 3.2.In Vitro Biocompatibility of PEG 500-Coated Mn0.5Zn0.5Fe2O4 Particles

In vitro biocompatibility was evaluated using TEM to study the degree of particle penetration into RGCs and any cytological changes caused by this penetration. Figure 3 illustrates that the PEG 500-coated SPNPs efficiently penetrated into the cytoplasm of RGCs without causing obvious cytological changes such as cell deformation or apoptosis. In addition, we found that the SPNPs did not penetrate the nuclei of RGCs, and so did not cause nuclear fragmentation.Figure 3
Transmission electron microscopy image of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. Nanoparticles (red circles) were found in the cytoplasm, but not in the nucleus (N), and they caused no morphological deformation. (a) Control, (b) 30 μg/mL Mn0.5Zn0.5Fe2O4.
(a)
(b)Our results suggest that these SPNPs can be located inside cells with no significant structural or morphological adverse effects. Furthermore, the PEG 500-coated nanoparticles inside the cells showed less agglomeration than those in our previous report [12], which may be due to the PEG coating and its weakly positively charged or uncharged surface. The results of TEM analyses of cultured RGCs suggest the potential feasibility of the coated nanoparticles as a localized hyperthermic agent for neuroprotection.


### 3.3. Characteristics of AC Magnetically Induced Heating by Mn0.5Zn0.5Fe2O4 Nanoparticles

Figure4 shows the temperatures of RGC treated with SPNPs (500 μg/mL) during AC magnetically induced heating. AC heating stress times varied from 600 sec to 1200 sec. The applied frequency and magnetic field were fixed at f
=
110 kHz and H
=
140 Oe, respectively, which are in the biologically safe and physiologically tolerable range. The PEG 500-coated nanoparticles produced stable and well-saturated heating characteristics. The temperature was controlled at a typical HSPs induction temperature of 41.0
±
0.5°C.Figure 4
AC magnetically induced heating temperatures of RGCs treated with SPNPs (500μg/mL) with different holding time of AC heating from 600 sec to 1200 sec. Applied frequency and magnetic field strength were constant at 140 kHz and 140 Oe, respectively. The temperature was controlled at a typical HSPs induction temperature of 41.0
±
0.5°C.Figure5 shows the AC magnetically induced heating temperature rise characteristics of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. The applied magnetic field and AC frequency were fixed at H
appl
=
140 Oe and f
appl
=
140 kHz, respectively, and the concentration of the nanofluid varied from 300 μg/mL to 700 μg/mL to explore the effects on HSP72 induction efficiency. As can be seen in Figure 5, the T
AC
⁡
,
max
⁡ characteristics and the corresponding AC heating response were strongly dependent on the concentration of the SPNPs (nanofluid solutions).Figure 5
AC magnetically induced temperature rise characteristics of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. Measurements were taken at the fixed applied frequency of 140 kHz and magnetic field of 140 Oe, with nanoparticle concentrations ranging from 300 μg/mL to 700 μg/mL. This figure describes the dependence of T
AC
⁡
,
max
⁡ characteristics on SPNP concentration.


### 3.4. Induction of Heat Shock Protein by Magnetic HyperthermiaIn Vitro

In this study, 3 types of control groups were used: (1) no SPNPs and no AC magnetic field, (2) no SPNPs but AC magnetic field applied (f
appl
=
140 kHz and H
appl
=
140 Oe), and (3) RGCs incubated with SPNPs (500 μg/mL) but no applied AC magnetic field. Figure 6 shows immunofluorescent staining images of HSP72 in RGCs treated with Mn0.5Zn0.5Fe2O4 nanoparticles after magnetic hyperthermia for 900 s at 41.0
±
0.5°C. In this study, the RGCs were stained using FITC and DAPI with antibodies to HSP72. As can be seen in Figure 6, HSP72 was not induced in the control groups 1, 2, and 3. However, HSP72 was successfully induced in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles and an adequate AC magnetic field. In addition, we found that the induction efficiency of HSP72 and cell death rate have a strong dependence on the temperature ramp rate, which was controlled by the externally applied AC magnetic field: cells experiencing a slower temperature elevation rate had more HSP72 induction and minimal cell loss.Figure 6
Immunofluorescent staining of HSP72. (a) Control group 1, without SPNPs and without magnetic field application. (b) Control group 2, without SPNPs but with magnetic field application (f
appl
=
140 kHz and H
appl
=
140 Oe). (c) Control group 3, without magnetic field application but incubated with SPNPs (500 μg/mL). (d) RGCs incubated with SPNPs (500 μg/mL) and with the AC magnetic field applied (f
appl
=
140 kHz and H
appl
=
140 Oe). HSP72 was not induced in the control groups 1, 2, and 3 but was successfully induced in RGCs incubated with SPNPs when an adequate magnetic field was applied.
(a)
(b)
(c)
(d)Specific biotechnical requirements [12] for any SPNP considered to be an agent for HSP induction are as follows: (1) it should have a high specific absorption rate, which enables it to rapidly reach the induction temperature (39–41°C) while the concentration of SPNPs is kept as low as possible; (2) it must reach the induction temperature with an applied magnetic field and frequency that fall within the ranges to ensure avoidance of bioelectrically induced damage [10] (i.e., H
<
190 Oe and f
<
120 kHz); and (3) it must be highly biocompatible and should easily penetrate the target cells. Our results demonstrate that PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles exhibited heating temperature characteristics that were stable and controllable at a typical HSP induction temperature of 41.0
±
0.5°C. Additionally, in this study, the temperature ramp rate was controlled by changing H
appl within the range from 120 Oe to 160 Oe at a fixed frequency of 140 kHz. The experimental results suggest that HSPs can be successfully induced in cultured RGCs while the applied magnetic field is maintained within a biologically and physiologically safe range.


### 3.5. Quantitative Analysis of HSP72 Expression by Western Blot Analysis

HSP72 expression in cultured RGCs was quantified by Western blot analysis (Figure7). There were weak immunoreactivities for HSP72 in the control groups 1, 2, and 3. However, a strong increase of immunoreactivity was observed in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles under the fixed AC magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe).Figure 7
Western blot analysis of HSP72. (1) Control group 1, without SPNPs and without magnetic field application. (2) Control group 2, without SPNPs but with magnetic field application (f
appl
=
140 kHz and H
appl
=
140 Oe). (3) Control group 3, without magnetic field application but incubated with SPNPs (500 μg/mL). (4) NP(+)/MF(+) group, RGCs incubated with SPNPs (500 μg/mL) and with the application of AC magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe).
(a)
(b)As can be seen in Figure6, we confirmed that HSP72 was successfully induced in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles when an appropriate magnetic field was applied. The results of Western blot analyses also showed a strong increase in the immunoreactivity of similarly treated RGCs. However, induction of HSP72 was not observed in the control group 3, indicating that mechanical stress due to incubation with Mn0.5Zn0.5Fe2O4 nanoparticles does not contribute to the induction of HSP72. In addition, the application of only the external magnetic field without SPNP agents (control group 2) does not induce HSP72 in cultured RGCs. Furthermore, it was found that the induction efficiency of HSP72 and the cell death rate are highly dependent on the temperature ramp rate. We speculate that a too rapid increase in temperature by using SPNP agents may inhibit RGCs from keeping enough thermal energy to induce stress proteins such as HSP72. Moreover, the more rapid temperature rise might lead to higher RGC cell death during HSP induction.Based on these results, the application of SPNP agents could be a promising biotechnical method for inducing localized HSP72. Because of specific limitations to intravenous injection of nanoparticles into the choroid, the SPNP agents can be injected into the intravitreal space of the eye. In a study performed by our group, the injected nanoparticles successfully diffused into the retina, with most found in the inner plexiform layer [12]. This is the layer most adjacent to the RGCs; therefore, highly effective heat transfer to the RGCs can be expected. These findings suggest that SPNP agents could potentially be used as neuroprotective mediators in glaucoma treatment.


## 3.1. Characterization of PEG 500-Coated Mn0.5Zn0.5Fe2O4

Figure2 shows the HR-TEM images of PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. The PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles have a round shape with a mean size of 7.20
±
0.85 nm and a narrow size distribution, and they are well segregated.Figure 2
High-resolution transmission electron microscopy image of superparamagnetic PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles dispersed in distilled water.


## 3.2.In Vitro Biocompatibility of PEG 500-Coated Mn0.5Zn0.5Fe2O4 Particles

In vitro biocompatibility was evaluated using TEM to study the degree of particle penetration into RGCs and any cytological changes caused by this penetration. Figure 3 illustrates that the PEG 500-coated SPNPs efficiently penetrated into the cytoplasm of RGCs without causing obvious cytological changes such as cell deformation or apoptosis. In addition, we found that the SPNPs did not penetrate the nuclei of RGCs, and so did not cause nuclear fragmentation.Figure 3
Transmission electron microscopy image of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. Nanoparticles (red circles) were found in the cytoplasm, but not in the nucleus (N), and they caused no morphological deformation. (a) Control, (b) 30 μg/mL Mn0.5Zn0.5Fe2O4.
(a)
(b)Our results suggest that these SPNPs can be located inside cells with no significant structural or morphological adverse effects. Furthermore, the PEG 500-coated nanoparticles inside the cells showed less agglomeration than those in our previous report [12], which may be due to the PEG coating and its weakly positively charged or uncharged surface. The results of TEM analyses of cultured RGCs suggest the potential feasibility of the coated nanoparticles as a localized hyperthermic agent for neuroprotection.


## 3.3. Characteristics of AC Magnetically Induced Heating by Mn0.5Zn0.5Fe2O4 Nanoparticles

Figure4 shows the temperatures of RGC treated with SPNPs (500 μg/mL) during AC magnetically induced heating. AC heating stress times varied from 600 sec to 1200 sec. The applied frequency and magnetic field were fixed at f
=
110 kHz and H
=
140 Oe, respectively, which are in the biologically safe and physiologically tolerable range. The PEG 500-coated nanoparticles produced stable and well-saturated heating characteristics. The temperature was controlled at a typical HSPs induction temperature of 41.0
±
0.5°C.Figure 4
AC magnetically induced heating temperatures of RGCs treated with SPNPs (500μg/mL) with different holding time of AC heating from 600 sec to 1200 sec. Applied frequency and magnetic field strength were constant at 140 kHz and 140 Oe, respectively. The temperature was controlled at a typical HSPs induction temperature of 41.0
±
0.5°C.Figure5 shows the AC magnetically induced heating temperature rise characteristics of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. The applied magnetic field and AC frequency were fixed at H
appl
=
140 Oe and f
appl
=
140 kHz, respectively, and the concentration of the nanofluid varied from 300 μg/mL to 700 μg/mL to explore the effects on HSP72 induction efficiency. As can be seen in Figure 5, the T
AC
⁡
,
max
⁡ characteristics and the corresponding AC heating response were strongly dependent on the concentration of the SPNPs (nanofluid solutions).Figure 5
AC magnetically induced temperature rise characteristics of RGCs treated with PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles. Measurements were taken at the fixed applied frequency of 140 kHz and magnetic field of 140 Oe, with nanoparticle concentrations ranging from 300 μg/mL to 700 μg/mL. This figure describes the dependence of T
AC
⁡
,
max
⁡ characteristics on SPNP concentration.


## 3.4. Induction of Heat Shock Protein by Magnetic HyperthermiaIn Vitro

In this study, 3 types of control groups were used: (1) no SPNPs and no AC magnetic field, (2) no SPNPs but AC magnetic field applied (f
appl
=
140 kHz and H
appl
=
140 Oe), and (3) RGCs incubated with SPNPs (500 μg/mL) but no applied AC magnetic field. Figure 6 shows immunofluorescent staining images of HSP72 in RGCs treated with Mn0.5Zn0.5Fe2O4 nanoparticles after magnetic hyperthermia for 900 s at 41.0
±
0.5°C. In this study, the RGCs were stained using FITC and DAPI with antibodies to HSP72. As can be seen in Figure 6, HSP72 was not induced in the control groups 1, 2, and 3. However, HSP72 was successfully induced in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles and an adequate AC magnetic field. In addition, we found that the induction efficiency of HSP72 and cell death rate have a strong dependence on the temperature ramp rate, which was controlled by the externally applied AC magnetic field: cells experiencing a slower temperature elevation rate had more HSP72 induction and minimal cell loss.Figure 6
Immunofluorescent staining of HSP72. (a) Control group 1, without SPNPs and without magnetic field application. (b) Control group 2, without SPNPs but with magnetic field application (f
appl
=
140 kHz and H
appl
=
140 Oe). (c) Control group 3, without magnetic field application but incubated with SPNPs (500 μg/mL). (d) RGCs incubated with SPNPs (500 μg/mL) and with the AC magnetic field applied (f
appl
=
140 kHz and H
appl
=
140 Oe). HSP72 was not induced in the control groups 1, 2, and 3 but was successfully induced in RGCs incubated with SPNPs when an adequate magnetic field was applied.
(a)
(b)
(c)
(d)Specific biotechnical requirements [12] for any SPNP considered to be an agent for HSP induction are as follows: (1) it should have a high specific absorption rate, which enables it to rapidly reach the induction temperature (39–41°C) while the concentration of SPNPs is kept as low as possible; (2) it must reach the induction temperature with an applied magnetic field and frequency that fall within the ranges to ensure avoidance of bioelectrically induced damage [10] (i.e., H
<
190 Oe and f
<
120 kHz); and (3) it must be highly biocompatible and should easily penetrate the target cells. Our results demonstrate that PEG 500-coated Mn0.5Zn0.5Fe2O4 nanoparticles exhibited heating temperature characteristics that were stable and controllable at a typical HSP induction temperature of 41.0
±
0.5°C. Additionally, in this study, the temperature ramp rate was controlled by changing H
appl within the range from 120 Oe to 160 Oe at a fixed frequency of 140 kHz. The experimental results suggest that HSPs can be successfully induced in cultured RGCs while the applied magnetic field is maintained within a biologically and physiologically safe range.


## 3.5. Quantitative Analysis of HSP72 Expression by Western Blot Analysis

HSP72 expression in cultured RGCs was quantified by Western blot analysis (Figure7). There were weak immunoreactivities for HSP72 in the control groups 1, 2, and 3. However, a strong increase of immunoreactivity was observed in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles under the fixed AC magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe).Figure 7
Western blot analysis of HSP72. (1) Control group 1, without SPNPs and without magnetic field application. (2) Control group 2, without SPNPs but with magnetic field application (f
appl
=
140 kHz and H
appl
=
140 Oe). (3) Control group 3, without magnetic field application but incubated with SPNPs (500 μg/mL). (4) NP(+)/MF(+) group, RGCs incubated with SPNPs (500 μg/mL) and with the application of AC magnetic field (f
appl
=
140 kHz and H
appl
=
140 Oe).
(a)
(b)As can be seen in Figure6, we confirmed that HSP72 was successfully induced in RGCs incubated with Mn0.5Zn0.5Fe2O4 nanoparticles when an appropriate magnetic field was applied. The results of Western blot analyses also showed a strong increase in the immunoreactivity of similarly treated RGCs. However, induction of HSP72 was not observed in the control group 3, indicating that mechanical stress due to incubation with Mn0.5Zn0.5Fe2O4 nanoparticles does not contribute to the induction of HSP72. In addition, the application of only the external magnetic field without SPNP agents (control group 2) does not induce HSP72 in cultured RGCs. Furthermore, it was found that the induction efficiency of HSP72 and the cell death rate are highly dependent on the temperature ramp rate. We speculate that a too rapid increase in temperature by using SPNP agents may inhibit RGCs from keeping enough thermal energy to induce stress proteins such as HSP72. Moreover, the more rapid temperature rise might lead to higher RGC cell death during HSP induction.Based on these results, the application of SPNP agents could be a promising biotechnical method for inducing localized HSP72. Because of specific limitations to intravenous injection of nanoparticles into the choroid, the SPNP agents can be injected into the intravitreal space of the eye. In a study performed by our group, the injected nanoparticles successfully diffused into the retina, with most found in the inner plexiform layer [12]. This is the layer most adjacent to the RGCs; therefore, highly effective heat transfer to the RGCs can be expected. These findings suggest that SPNP agents could potentially be used as neuroprotective mediators in glaucoma treatment.


## 4. Conclusions

We successfully induced HSP72 in cultured RGCs with hyperthermia via SPNPs, PEG 500-coated Mn0.5Zn0.5Fe2O4. Considering that glaucoma ultimately causes RGC death, an endogenous neuroprotection strategy through induction of an HSP response can widen the field of glaucoma treatment. Furthermore, our results suggest that the Mn0.5Zn0.5Fe2O4 nanoparticles could be used as a neuroprotective treatment method for cerebral ischemia and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Further studies with animal models and functional evaluations will be necessary to confirm Mn0.5Zn0.5Fe2O4 nanoparticles as potential novel neuroprotective therapeutic agents.



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*Source: 142387-2015-04-08.xml*