Magnetically softened iron oxide (MSIO) nanofluid and its application to thermally-induced heat shock proteins for ocular neuroprotection

Abstract

Magnetically softened iron oxide (MSIO) nanofluid, PEGylated (Mn_0.5Zn_0.5)Fe₂O₄, was successfully developed for local induction of heat shock proteins (HSPs) 72 in retinal ganglion cells (RGCs) for ocular neuroprotection. The MSIO nanofluid showed significantly enhanced alternating current (AC) magnetic heat induction characteristics including exceptionally high SLP (Specific Loss Power, > 2000 W/g). This phenomenon was resulted from the dramatically improved AC magnetic softness of MSIO caused by the magnetically tailored Mn²⁺ and Zn²⁺ distributions in Fe₃O₄. In addition, the MSIO nanofluid with ultra-thin surface coating layer thickness and high monodispersity allowed for a higher cellular uptake up to a 52.5% with RGCs and enhancing “relaxation power” for higher AC heating capability. The RGCs cultured with MSIO nanofluid successfully induced HSPs 72 by magnetic nanofluid hyperthermia (MNFH). Moreover, it was interestingly observed that systematic control of “AC magnetically-induced heating up rate” reaching to a constant heating temperature of HSPs 72 induction allowed to achieve maximized induction efficiency at the slowest AC heating up rate during MNFH. In addition to in-vitro experimental verification, the studies of MSIO infusion behavior using animal models and a newly designed magnetic coil system demonstrated that the MSIO has promising biotechnical feasibility for thermally-induced HSPs agents in future glaucoma clinics.

Introduction

Neurodegeneration has been considered as a main cause for different types of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, dementia, and stroke. According to the previous reports, the progressive loss of function or programmed death (apoptosis) of neurons in the central nervous system (CNS) was revealed to primarily result in the neurodegeneration [1], [2]. Therefore, the interests on the protection of neurons against programmed death or continuous damage, so called “neuroprotection”, performed by different biotechnical approaches, such as direct introduction of genes, induction of heat shock proteins (HSPs), and infusion of stem cells or drugs, have been paid considerable attentions to achieve an effective treatment modality for the neurodegenerative diseases [2], [3], [4], [5], [6].

Among the various eye diseases, glaucoma is considered as a notoriously well-known neurodegenerative disease, in which the progressive loss or death of retinal ganglion cells (RGCs) due to the increased intraocular pressure (IOP), is generally recognized as the main physiological reason for the ocular neurodegeneration [7], [8], [9]. Up to now, dropping IOP by taking a medicine or doing surgery has been typically carried out as a primary clinical treatment to cure glaucoma [10], [11]. However, since these treatments have been revealed not to be effective for ocular neuroprotection, the interests in utilizing previously developed “neuroprotection” modalities to prevent the RGCs from glaucoma-induced progressive loss (death) have been rapidly increased in glaucoma clinics [12], [13], [14]. Correspondingly, various neuroprotective drug based modalities have been developed to prevent the damaged RGCs from the progressive loss or death; however, although they were proven to reduce the death rate of damaged RGCs [12], the high toxicity and the unclear mechanism of some of the drugs for protecting RGCs were revealed to be critical limitations for clinical use [1], [14], [15]. Thus, alternatively, the induction of HSPs, particularly HSPs 72 family, has been recently considered as a new powerful clinical modality for ocular neuroprotection to treat glaucoma, since its physiological effectiveness has been empirically demonstrated from the damaged rat eye [16], [17]. Several experimental approaches such as zinc injection, whole body hyperthermia, and thermotherapy using laser etc. have been introduced and executed to induce HSPs 72 for ocular neuroprotection [18], [19], [20]. However, although the research efforts made so far had demonstrated to successfully identify the induction of HSPs 72 in RGCs, these biotechnical approaches were found to cause systemic or chemical side effects such as over-expression of HSPs in the entire body, and death or deformation of RGCs (or optic nerve). Moreover, some of the critical issues relevant to real clinical applications – 1) how the HSPs 72 can be locally induced at targeted sites, 2) how the induction efficiency of localized HSPs 72 can be accurately controlled and significantly enhanced, and 3) how the death rate of healthy cells, which is caused by applied AC thermal stress, can be minimized during the induction process of HSPs 72 in RGCs – have been urgently raised to be solved in order to achieve much physiologically and biologically safer and more effective local induction of HSPs. Therefore, to settle down these current biotechnical challenges, the development of new biotechnical, or biomedical engineering approaches enabling to provide highly efficient local induction of HSPs 72 in RGCs is inevitably required for the effective and safe ocular neuroprotection in modern glaucoma clinics.

In this study, we employed magnetic nanofluid hyperthermia (MNFH) using magnetically softened iron oxide (MSIO) nanofluid, which is PEGylated (Mn_0.5Zn_0.5)Fe₂O₄ nanoparticles with magnetic core and polyethylene glycol (PEG) surface coating dispersed in water, as a new nano-biotechnical approach to effectively induce and control the localized HSPs 72 as well as to minimize the death rate of healthy cells during the induction process in RGCs. The main physical reason to employ MNFH for local induction of HSPs is that it can allow to locally generate the thermal stress (“AC magnetically-induced heating stress”) in RGCs during the induction process of HSPs. Particularly, another crucial reason is that the “AC magnetically-induced heating stress”, which is directly relevant to the biochemical behavior of HSPs as well as the HSPs induction characteristics, can be controlled by tuning the AC magnetically-induced heating-up rate of MNFH agents by changing the externally applied AC magnetic field. The systematically controllable “AC magnetically-induced heating stress” during HSPs induction process is expected to enhance the efficiency of HSPs induction, i.e. high induction rate of HSPs and minimal death rate of healthy cells, because the change of thermal stress in cells including RGCs directly influences on their cytological behaviors such as the induction of HSPs and apoptosis [20], [21], [22]. Accordingly, to explore the effects of electromagnetically controlled strength of thermal stress (“AC magnetically-induced heating stress”) on the efficiency of local induction of HSPs 72 in RGCs, the AC heating-up rate, which is defined as increasing rate of the temperature reaching to a typical HSPs induction temperature, was systematically controlled by changing the applied AC magnetic field, H_appl, at the fixed applied frequency, f_appl, in the biologically tolerable and physiologically safe rage (H_appl·f_appl < 3 × 10⁹ A m⁻¹ s⁻¹) [23]. Particularly, for this experimental work, in order to prevent the undesirable induction of HSPs generated by the inaccurately controlled AC heating temperature and time, the saturated AC magnetically-induced heating temperature (T_AC,mag) was constantly kept at a typical HSPs induction temperature of 40.5 ± 0.3 °C with a fixed heating time of 900 s [24].

However, for the successful demonstration of highly efficient local induction of HSPs 72 in RGCs by “AC magnetically-induced heating stress” controllable MNFH, the most crucial issue to be satisfied is to develop a MNFH agent with high performance that can exhibit sufficiently high enough T_AC,mag and specific loss power (SLP) along with a higher biocompatibility (higher cellular uptake and lower cytotoxicity). Therefore, in order to address such an issue, we designed and developed MSIO nanofluid. The main physical and physiological reason to develop MSIO nanofluid as a MNFH agent for the local induction of HSPs is that both Mn and Zn are biocompatible elements (essential trace nutrients). Furthermore, another is that the magnetization (M_s = χ_mH) including magnetic susceptibility (χ_m = χ´_m (in-phase) + χ″_m (out-of-phase)) and correspondingly the AC hysteresis loss power or AC hysteresis loss area (A), which is directly proportional to the total power of AC magnetically-induced heat generation [25], can be readily modified and significantly improved by thermally tailoring the concentration or the distribution of Mn²⁺ and Zn²⁺ in A (tetrahedral) and B (octahedral) sites of Fe₃O₄ [26], [27].