Nanoparticle-Mediated Therapeutic Application for Modulation of Lysosomal Ion Channels and Functions

Applications of nanoparticles in various fields have been addressed. Nanomaterials serve as carriers for transporting conventional drugs or proteins through lysosomes to various cellular targets. The basic function of lysosomes is to trigger degradation of proteins and lipids. Understanding of lysosomal functions is essential for enhancing the efficacy of nanoparticles-mediated therapy and reducing the malfunctions of cellular metabolism. The lysosomal function is modulated by the movement of ions through various ion channels. Thus, in this review, we have focused on the recruited ion channels for lysosomal function, to understand the lysosomal modulation through the nanoparticles and its applications. In the future, lysosomal channels-based targets will expand the therapeutic application of nanoparticles-associated drugs.

further by movement of other ions [5]. Thus, for effective and continuous movement of H + into the lysosome, an accompanying counter-ion movement is necessary [5].
The lysosomal V-ATPases consists of two domains: V 1 domain, which hydrolyses ATP, and the V 0 domain, which translocates H + ions across the lysosomal membrane [60]. The catalytic domain V 1, drives a rotary H + transport motor by hydrolyzing ATP with translocation of H + [61,62]. In this case, the V-ATPase rotor is operated in only one direction with an irreversible ATP hydrolysis due to the movement of H + from cytosol to the lysosomal lumen [5]. The continuous V-ATPase-mediated H + pumping generates a positive charge in the lysosomal lumen, which inhibits any further movement of H + [63]. To dissipate this membrane potential, other ions have to be transferred in the opposite direction, and this process is referred to as the counterion flux [5,63]. Counter ion movement is suggested as both entering anions and exiting cations through the lysosomal lumen [5]. One important counter ionic candidate is chloride, transferred by CLC-7, as attenuation of CLC-7 leads to lysosomal dysfunction such as LSD and osteopetrosis [25,64]. Another candidate counter ion is K + , transferred by TMEM175. Its mutation induces neuronal degeneration and LSD [65]. The R740S mutant osteoclasts, mutated in the V-ATPase α3 subunit, possess a higher lysosomal pH, and shows altered mTORC expression (increase in basal protein level and decrease of gene expression) and activity, which, in turn, plays a key role in cell proliferation [57,66]. Additionally, acidification of lysosomes can induce macrophages to secrete N-acetyl-β-D-glucosaminidase through lysosomal exocytosis [67,68], which includes absorption of cytochrome c in rat kidney during renal metabolism [69], and transport of cystine, the product of protein degradation by cathepsin, from lysosomes to cytosol [70]. Thus, alteration of lysosomal pH can be like a commander's order to modulate the cellular life cycle.

Lysosome-Associated Ion Channels for Lysosomal Function
The lysosomal function is modulated by the ion movement and subsequent pH regulation. This movement is accomplished through various ion channels ( Figure 1). We have previously reported application of NPs on various channels [71]. In this section, we summarize the recruited channels for lysosomal function to understand the lysosomal modulation through the NPs (Table 1).

CLC
CLC channels are the chloride channels that play a critical role in lysosomal function. CLC channels consist of two major isotypes: plasma membrane-associated (CLC-1, -2, and -Ka/-Kb) and intracellular organelle-associated (CLC-3 to CLC-7) [114,115]. Among the intracellular organelle- Figure 1. The channels localized in lysosomal membrane to transport ions. These channels and transporters can regulate lysosomal and cellular functions through transporting and maintaining hydrogen, chloride, Ca 2+ , and potassium which indicated in Table 1. Table 1. The relationship between lysosomal ion channels and cellular functions.

Channels
Mechanisms and Related Diseases Ref.

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A prevalent cause of CF results from a deletion of the 508th positioned phenylalanine (∆F508) even though several other mutations have been identified in CF [120,121,125]. CFTR has been reported to support lysosomal acidification and is localized in intra-organellar components, including ER, Golgi, and endo/lysosomes [126,127]. In CF cells, which have a ∆F508 mutation in CFTR, lysosomal pH is higher than in normal cells [85]. CFTR-null macrophages showed a defective killing function of internalized bacteria by inhibiting phago-lysosomal fusion [86]. Typically, these macrophages kill bacteria by phago-lysosomal ingestion, which is followed by lysosomal acidification [86,127]. This suggests that CFTR-mediated lysosomal acidification can regulate bacteria-killing activity of macrophages. Additionally, activation of CFTR leads to re-acidification of alkalinized lysosomes in retinal pigmented epithelial cells, which suggests it is a useful target for lysosomal clearance [128].

TMEM175
Intra-organelle K + channel TMEM175 was recently identified in endosomes and lysosomes and is involved in the modulation of luminal pH stability and autophagosomes [165]. Deficiency of TMEM175 results in dysregulated lysosomal pH, impaired autophagosome clearance, and mitochondrial dysfunction in the neuronal system [166]. In addition to TMEM175, Ca 2+ -activated large conductance K + channel also localizes to the lysosome and is involved in lysosomal Ca 2+ signaling and lipid accumulation [104,105], which suggests lysosomal K + channels can be considered the new target of neurodegenerative diseases such as LSD. TPCs are the key components of Ca 2+ signaling in the endo-lysosomal system including TRPML and TRP channels and have been extensively reviewed in various studies [167][168][169]. The TPC1-3 are identified in the endo-lysosome [170][171][172][173][174] and stimulated by nicotinic acid adenine dinucleotide phosphate and phosphatidylinositol 3,5-bisphosphate [171,[175][176][177][178]. The roles and pathways of TPCs have been addressed in various organs and biological systems. The inhibition of the TPC channel abolishes the migration of metastatic cancer cells by disrupting the trafficking mechanism of β1integrin and the formation of leading edges [179]. The TPCs are involved in the autophagic flux of

TMEM175
Intra-organelle K + channel TMEM175 was recently identified in endosomes and lysosomes and is involved in the modulation of luminal pH stability and autophagosomes [165]. Deficiency of TMEM175 results in dysregulated lysosomal pH, impaired autophagosome clearance, and mitochondrial dysfunction in the neuronal system [166]. In addition to TMEM175, Ca 2+ -activated large conductance K + channel also localizes to the lysosome and is involved in lysosomal Ca 2+ signaling and lipid accumulation [104,105], which suggests lysosomal K + channels can be considered the new target of neurodegenerative diseases such as LSD.

Two Pore Channels (TPCs)
TPCs are the key components of Ca 2+ signaling in the endo-lysosomal system including TRPML and TRP channels and have been extensively reviewed in various studies [167][168][169]. The TPC1-3 are identified in the endo-lysosome [170][171][172][173][174] and stimulated by nicotinic acid adenine dinucleotide phosphate and phosphatidylinositol 3,5-bisphosphate [171,[175][176][177][178]. The roles and pathways of TPCs have been addressed in various organs and biological systems. The inhibition of the TPC channel abolishes the migration of metastatic cancer cells by disrupting the trafficking mechanism of β1-integrin and the formation of leading edges [179]. The TPCs are involved in the autophagic flux of mouse cardiomyocytes [180]. It has been discussed that TPC2 is involved in autophagy progression, cancer cell migration, and cellular pigmentation [106][107][108]. Additionally, signaling events of Parkinson's disease involve the regulation of TPCs in trafficking [109,110].

P2X4
The P2X4 receptor is expressed ubiquitously in cells from immune, nervous, muscle, and vascular systems [181][182][183]. The P2X4 is stable within the acidic environment of the lysosome and also traffics to the plasma membrane to enhance the phagocytic function [181,184]. P2X4 is activated by ATP and inhibited by the luminal acidic pH in the lysosome [185]. P2X4 consist of an ATP-activated Ca 2+ channel and is involved in calmodulin activation to promote endo-lysosomal fusion of intracellular organelles [111,112]. P2X4 is also involved in liver fibrogenesis [113] and alcohol-induced microglial damage [186]. Although P2X4 has been associated with ATP-dependent signaling in the endo-lysosome, further studies are still needed in the future.

NP-Induced Proton Sponge Effect through Ion Channels in the Tumor System
Swelling of lysosomes has the potential to increase cellular toxicity by releasing lysosomal compartments and nanoparticles [187,188]. The lysosomal 'proton sponge effect' is triggered by the influx of cationic nanoparticles with hydrogen and chloride ions to lysosomes [188]. Accumulated ions in the lysosome may trigger water intake to equilibrate the physiological osmolarity and, subsequently, induce lysosomal rupture [188]. It has been addressed that conceptual use of the lysosomal pH-dependent system and lysosomal rupture develops the self-assembled luminescent AuNPs by the swelling property [189]. In a previous study, we reported that the cationic nanorod conjugated with doxorubicin (DOX) (AuNR-DOX) induced lysosomal swelling and rupture with increased apoptosis (Figure 3) [190]. Lee et al. reported that encapsulated AuNR-DOX in lysosomes is dissociated with DOX by lysosomal hydrolases. A charged linker of AuNR is opened and then recruited negative charged ions such as chloride into the lysosome. The ionic accumulation is developed, and lysosomal rupture occurred. Released chloride from the lysosome through lysosomal rupture activates Ca 2+ influx channel TRPM2 in the plasma membrane and, lastly, overload of Ca 2+ triggers the enhanced apoptotic effect including the effect of DOX in cancer cells [190]. The intracellular mechanism of nanomaterials and its related channels is now started. However, the effect of nanoparticles on lysosomal ion channels and transporters has still been poorly studied. To use nanomaterials for medicines, understanding the relationship between nanoparticles and lysosomal ion channels has to be expanded.  Nanomaterials can act as the carrier for conventional drugs by transporting drugs or proteins through lysosomes such as AuNRs conjugated with Naja kaouthia protein toxin 1 (NKCT1) (one of the snake toxin protein) [191], silk NPs conjugated with doxorubicin (anti-cancer drugs) [192], and AgNPs conjugated with salinomycin (killing agent for cancer stem cells) [193]. These nanomaterials can maximize drug delivery to reach the lysosome easily, and, subsequently, kill the cancer cells from leukemia [191], breast cancer [192], and ovarian cancer [193]. The "small size" of NPs, which is one of the typical characteristics, can be used to penetrate obstacles that conventional drugs cannot cross, especially the blood brain barrier (BBB) [194]. One of the LSD, Gaucher's type 3 disease, which occurs by accumulation of glucocerebroside in the brain can be cured by transporting enzymes into the brain

Clinical Application and Limitation of Nanomaterials
As mentioned earlier, NPs have a bio-toxic effect on lysosomes by triggering pH alteration, malfunctions of protein activity, accumulation in lysosomes, and subsequent cell death. We summarized the effect of NPs on cellular functions in Table 2. Accordingly, application of NPs has limitations for nanodrugs and nano-therapies. Thus, recent efforts have challenged to overcome these limitations by maximizing transport ability or reducing cytotoxicity.
Nanomaterials can act as the carrier for conventional drugs by transporting drugs or proteins through lysosomes such as AuNRs conjugated with Naja kaouthia protein toxin 1 (NKCT1) (one of the snake toxin protein) [191], silk NPs conjugated with doxorubicin (anti-cancer drugs) [192], and AgNPs conjugated with salinomycin (killing agent for cancer stem cells) [193]. These nanomaterials can maximize drug delivery to reach the lysosome easily, and, subsequently, kill the cancer cells from leukemia [191], breast cancer [192], and ovarian cancer [193]. The "small size" of NPs, which is one of the typical characteristics, can be used to penetrate obstacles that conventional drugs cannot cross, especially the blood brain barrier (BBB) [194]. One of the LSD, Gaucher's type 3 disease, which occurs by accumulation of glucocerebroside in the brain can be cured by transporting enzymes into the brain [194]. A recent study demonstrated the potential for transporting enzymes across the BBB by using a recombinant arylsulfatase enzyme with polysorbate 80 coated poly-butyl cyanoacrylates NPs [195].
Although biocompatible nanodrugs have been developed, which are made of albumin-based [196][197][198] and lipid-based [199,200] nanoparticles, various studies have attempted to eliminate the toxicity of NPs via conjugation with other materials. For example, iron oxide NPs that induce autophagosome accumulation and impair lysosomes can be rendered bio-safe by coating with poly(lactic-co-glycolic acid) (PLGA) [193]. ZnO NPs and Quantum Dots that induce lysosomal damage with the generation of ROS can be stabilized by coating with α-linolenic acid [201] and 3-mercaptopropionic acid [202]. There are non-toxic nanomaterials that can be degraded into lysosomes, such as nano-diamonds, which are delivered to lysosomes by coating with ubiquitin, to associate with autophagy receptors: sequestosome 1 [203], Ca 2+ binding and coiled-coil domain 2 [204,205], and optineurin [206]. Additionally, PLGA NPs are degraded easily in the autophagy pathway [207]. Adjustment of the NPs size can avoid lysosomal accumulation: 60 nm-sized TiO 2 NPs Pharmaceutics 2020, 12, 217 9 of 19 are more aggregated and more destabilized in the lysosomal membrane than 180 nm-sized TiO 2 in the lysosomes and endosomes [208].

Future Perspectives
The primary lysosomal function is to maintain cellular homeostasis. Various attempts of drug delivery systems including nanomaterials and other new paradigms against diseases were engaged ( Figure 4). However, a plethora of questions should be answered about nano therapy against lysosomal targets or lysosomal pathways. Although our limited knowledge about the effect of nanomaterials on lysosomal function has been posted, its therapeutic potential cannot be neglected. Nanomaterials are attractive machinery, as carriers for conventional drugs for therapeutic purposes. In addition to the role of the attractive carrier, other unfavorable characteristics of nanomaterials including toxicity should be considered while developing the therapeutic strategies. Understanding the functional support of ion channels or transporters on the lysosome will be expanded further in the coming years and, subsequently, favorable potential of nanomaterial-based therapy will also improve.