extfieldsofstudy: Chemistry Medicine

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Hypoxia Stress Modifies Na+/K+-ATPase, H+/K+-ATPase, Na+/NH4+-ATPase, and nkaα1 Isoform Expression in the Brain of Immune-Challenged Air-Breathing Fish

Fishes are equipped to sense stressful stimuli and are able to respond to environmental stressor such as hypoxia with varying pattern of stress response. The functional attributes of brain to hypoxia stress in relation to ion transport and its interaction during immune challenge have not yet delineated in fish. We, therefore, explored the pattern of ion transporter functions and messenger RNA (mRNA) expression of α1-subunit isoforms of Na+/K+-ATPase (NKA) in the brain segments, namely, prosencephalon (PC), mesencephalon (MC), and metencephalon (MeC) in an obligate air-breathing fish exposed either to hypoxia stress (30 minutes forced immersion in water) or challenged with zymosan treatment (25-200 ng g−1 for 24 hours) or both. Zymosan that produced nonspecific immune responses evoked differential regulation of NKA, H+/K+-ATPase (HKA), and Na+/NH4+-ATPase (NNA) in the varied brain segments. On the contrary, hypoxia stress that demanded activation of NKA in PC and MeC showed a reversed NKA activity pattern in MeC of immune-challenged fish. A compromised HKA and NNA regulation during hypoxia stress was found in immune-challenged fish, indicating the role of these brain ion transporters to hypoxia stress and immune challenges. The differential mRNA expression of α1-subunit isoforms of NKA, nkaα1a, nkaα1b, and nkaα1c, in hypoxia-stressed brain showed a shift in its expression pattern during hypoxia stress-immune interaction in PC and MC. Evidence is thus presented for the first time that ion transporters such as HKA and NNA along with NKA act as functional brain markers which respond differentially to both hypoxia stress and immune challenges. Taken together, the data further provide evidence for a differential Na+, K+, H+, and NH4+ ion signaling that exists in brain neuronal clusters during hypoxia stress-immune interaction as a result of modified regulations of NKA, HKA, and NNA transporter functions and nkaα1 isoform regulation.

Introduction
Fishes possess both innate and adaptive immune responses and are able to respond to environmental perturbations. 1 The nonspecific immune response in fish acts as a powerful attribute with versatility due to their opposing and slower specific immune response compared with mammals. 2,3 Furthermore, nonspecific immunity that produces inflammatory mediators and acute phase proteins shows upregulation during an active response. 4 For example, zymosan treatment that evokes nonspecific immune response can activate phagocytic and lysozomal functions in air-breathing fish, providing evidence for a direct association of ion transporter functions with phagocytic and lysozomal activities in this fish. 5 As a consequence of physiological reaction to stressor, stress response alters the capacity of fish to maintain their homeostasis. 6,7 They respond to stressors with complex and adaptive mechanisms that constitute a network of neuroendocrine and physiological interactions. [8][9][10] Activation of neuroendocrine axis that releases catecholamines and corticosteroids from the brain-sympathetic nerves-chromaffin cell and hypothalamo-pituitary-interrenal (HPI) axes occurs during stress response. [6][7][8] For example, a rise in cortisol after exposure to nontoxic stressor such as net confinement has been observed in a number of fishes, 6,11 including the airbreathing fish Anabas testudineus. 9 In contrast, a lowered plasma cortisol level after zymosan treatment has been found in this fish and that indicates suppression of a cortisol-driven stress response during immune challenge. 5 A bidirectional communication that operates between the brain and immune components has been established in fish. 12 Here, a network of ligands and their receptors together with cytokines, chemokines, and neural pathways interact at all levels of neuroendocrine and immune components. 12 As a key event during this bidirectional communication, sharing of receptors, production of hormonal ligands, and cytokines occur in these cellular systems. 13,14 Traditionally, as a sensor, immune system component informs the central nervous system (CNS) to trigger a neuroendocrine response. 15 On the contrary, stress that demands neuroendocrine interference communicates with central and autonomic nervous system and modifies HPI axis 6,9 and the components of immune system. 12,16 Consequently, a change in the activity of discrete populations of brain neurons may occur during immune system activation. 17 It is likely that the sensitivity of fish brain to both immune and neuroendocrine 2 Journal of Experimental Neuroscience challenges may thus demand integrative functions of ion transporters that coordinate neuronal functions.
Teleost fish brain comprises 3 major segments, namely, prosencephalon (PC) (forebrain), mesencephalon (MC) (midbrain), and metencephalon (MeC) (hindbrain). Prosencephalon has telencephalon that constitutes olfactory lobes located rostroventrally to pair cerebral hemispheres 18,19 and diencephalon that constitutes thalamus, epithalamus, and hypothalamus with the preoptic, postoptic, and the tuberal areas. 19 Mesencephalon that constitutes the optic tectum, torus longitudinalis, and tegmentum continues caudally into the MeC. 20 The MeC represents the most caudal part of the brain with the cerebellum and the brain stem. 19,21 The 3 major serotonergic neural groups that exist in the teleost brain as pretectal population, posterior tuberculum/hypothalamic populations, and raphe populations make a network of neuronal circuitries that are sensitive to physiological challenges. 22,23 Serotonergic neurons are also present in the pineal gland, area postrema, medulla oblongata, and spinal cord in the brain of teleosts. 23 In teleosts, serotonergic fibers from the brain directly project to the pituitary, 22 whereas in some teleost species, serotonin-immunoreactive cells are also present in the pituitary indicating its local production. 22,24 Serotonergic neurons that are distributed in the superior raphe project to the forebrain and midbrain, extend to inferior raphe, and project to hindbrain-spinal cord region in the brain of the teleosts. 23 Dopaminergic neurons are typically located in defined groups of forebrain and midbrain 25 and possess catecholaminecontaining neurons in the diencephalon. 26 Dopaminergic and noradrenergic neurons that constitute the major hindbrain of the teleost fish have been shown activated during stressful challenges. [27][28][29] The glutamatergic neurons while distributing in the granular layers of the cerebellum, and several other diencephalic, mesencephalic, and rhombencephalic regions, dominate also in the nuclei of the dorsal telencephalon. 30 γ-aminobutyric acid (GABA)-mediated (GABAergic) neurons, however, dominate the nuclei of the ventral telencephalon. 18 Na + /K + -ATPase (NKA) that establishes electrochemical gradient of Na + and K + across the plasma membrane maintains resting membrane potential and electrical activity of neurons for neurotransmitter uptake and its osmotic balance. 31 This transporter that regulates Na + and K + transport acts as a measurable indicator of neuronal activity and excitability 32,33 and its high concentrations in brain cellular membranes demands 40% to 50% of the adenosine triphosphate (ATP). 34 As a ubiquitous protein, NKA is abundant in brain regions and plays a fundamental role in maintaining cellular ionic gradients that are required for neural activity, transport of amino acids and glucose, and regulation of cell pH and volume. [34][35][36] In addition, NKA acts as a key contributor to management of extracellular [K + ] following activity-induced K + release. [37][38][39] Given its important role in regulating neural excitability, impairment of NKA function might lead to neural dysfunction. The activity of neuronal NKA that concentrates in the surrounding nerve ending membranes interacts with the CNS through specific receptors for classical neurotransmitter and neuropeptides. 40 For example, many neuromodulators including dopamine have been shown to regulate NKA activity, 41 probably by regulating the levels of cyclic adenosine monophosphate 42 and free radicals. 36,43 Na + /K + -ATPase contains 2 major subunits, α and β, which function as a αβ heterodimer. The α-subunit is a large (110)(111)(112)(113)(114)(115)(116)(117)(118)(119)(120) protein that contains all the functional sites and is responsible for the catalytic functioning of the enzyme. The different subunit composition and cellular/subcellular distribution of NKA may confer cell type-specific properties. 44 Four isoforms of the NKA α-subunit, nkaα1, nkaα2, nkaα3, and nkaα4, have been identified in mammals, 45 and 3 isoforms, nkaα1, nkaα2, and nkaα3, have been identified in mammalian brain. 45,46 The presence of distinct cellular NKA isoforms (α1, α3, β1 in neurons and α1, α2, β1, β2 in astrocytes) provides clues on distinct physiological roles of these isoforms in the CNS, 37 which could be attributed to the distinct apparent ion affinities (Km) of the different α and β isoform combinations and their subsequent neurophysical functions. 47 Na + /K + -ATPase that expresses in all cell types 45 can substitute NH 4 + for K + . 48 In fish brain, NKA is known to effectively differentiate K + from NH 4 + for maintaining intracellular K + homeostasis and stabilizing resting membrane potential. 49,50 Furthermore, in mammals, ammonia intoxication has been shown to increase NKA activity resulting in ATP depletion. 51 Modification of messenger RNA (mRNA) and/or protein expression of NKA α-subunit isoforms in brain ammonia transport have been proposed in swamp eel, 50 although its mode of action has not yet identified. Recently, we have found evidence for the presence of a Na /NH -ATPase + 4 + (NNA) transporter system in the osmoregulatory epithelia of air-breathing fish. 5 Ammonia rapidly compromises astrocyte K + buffering, increasing its extracellular concentration and over activating the Na + -K + -2Cl − cotransporter isoform 1 (NKCC1) in neurons. 52 NH 4 + increases Na + uptake into all areas of the brain which indicates that NH 4 + could stimulate NKA function and increases extracellular Na + concentrations and possibly intracellular concentrations with a concomitant decrease in K + concentrations. These observations have been shown to be linked with NH 3 + toxicity leading to hepatic encephalopathy and liver failure-induced cerebral edema. 53 H + /K + -ATPase (HKA), a member of P-type transport adenosine triphosphatase (ATPase), is responsible for ATP-dependent exchange of H + for K + across plasma membranes. 54 H + /K + -ATPase is expressed at a very high level in mammalian gastric parietal cells, where it functions primarily as a proton pump transporting H + in exchange for K + into secretory canaliculi and, from there, into the gastric lumen. 55,56 In addition to parietal cells, HKA has been shown to present in a variety of other cells including colonic enterocytes and renal tubular cells. 54 Presence of nongastric HKA has been found in elasmobranch gills of Atlantic stingray 57 and in the osmoregulatory epithelia of air-breathing fish A testudineus. 5 However, its role in the brain ion transport has not been looked into except a study that reported the presence of 3 HKA in microglia of brain. 58 A critical role for HKA in the regulation of K + and H + gradients in the brain especially in the regulation of K + homeostasis in microglia shows its higher magnitude for K + transport compared with epithelial HKA. 58 It is thus plausible that the dynamics of HKA in brain would reflect the functionality of glia cells as it can contribute to glial cell volume and pH regulation which would modulate the neuronal functions in general. 58 In this context, it is reasonable to presume that HKA may act as glial marker that appears to be sensitive to both hypoxia and immune challenges at least in fish brain.
It is not certain that how these ion transporter functions operate during hypoxia stress-immune interaction in the varied neuronal clusters in fish brain. We, therefore, examined the response of ion transporters in the brain segments of an obligate air-breathing fish A testudineus Bloch to either zymosan-induced immune challenge or water immersion-induced hypoxia stress or both. The response pattern of Na + /K + , H + /K + , and Na /NH specific ATPase transport systems and the transcriptomic analysis of nkaα1-subunit isoforms were subsequently done in PC, MC, and MeC segments of fish brain. The pattern of ion transporter regulation was analyzed, and the functional regulation of neuronal clusters in the brain segments during hypoxia stress-immune interaction was identified.

Fish holding conditions
The climbing perch (A testudineus Bloch), a tropical freshwater air-breathing fish, was used as the test species. This native teleost fish, belonging to order Perciformes and family Anabantidae, inhabits the backwaters of Kerala in Southern India. They are equipped to live in demanding environmental conditions with their well-defined physiological and biochemical mechanisms. 59,60 Due to its sensitivity to various biotic and abiotic stressors, this obligate air-breathing fish serves as an excellent model to study the advanced physiological mechanisms including gas transport, acid/base, ionic, and metabolic regulations. 7,60-62 These fish, approximately 40 ± 5 g, were collected from the wild conditions in their postspawning phase and held in the laboratory conditions for 3 weeks under natural photoperiod (12 hours L:12 hours D) at water temperature ranging from 28°C to 29°C with a mean water pH of 6.2. Fish were fed with dry commercial fish feed at 1.5% of body mass and were transferred to 50-L glass tanks for 2 independent static experimentations. Fish ate their normal food and there was no mortality during experimentation. The regulations of Animal Ethical Committee of the University were followed.

Experimental design
Dose-responsive effects of zymosan on brain segments. The doseresponsive in vivo effect of zymosan-induced experimental peritonitis (25-200 ng g −1 ) within 24 hours 63 was tested for the activity of Na + /K + -ATPase (NKA), HKA, NNA, and nkaα1-subunit isoform transcripts in PC, MC, and MeC of the fish. In total, 32 laboratory-acclimated fish kept in cement tanks were transferred into glass tanks (30 cm × 30 cm × 60 cm) and kept as groups with 8 fish in each group for another 2 weeks. The first fish group was received intraperitoneal injection of 0.65% NaCl as vehicle, which served as sham control. The remaining 3 groups of fish were given varied doses of zymosan (25,50, and 200 ng g −1 ) for 24 hours.
Effect of hypoxia stress in immune-challenged fish. A selected zymosan dose was employed in another set of experiment that tested the impact of hypoxia stress on brain ion transport functions in immune-challenged fish. For that purpose, 2 subsets of fish groups, namely, nonstressed fish (I) and hypoxia-stressed fish (II), were held with 8 fish in each group. The first fish group in nonstressed subset I that served as sham controls received saline (0.65% NaCl) as vehicle. The second group fish were given intraperitoneal injection of a selected dose of zymosan (200 ng g −1 ) that showed substantial acid/base responses after 24 hours (unpublished data, 2017). Each fish group in the subset II that comprised 2 groups was given saline injection (control) and zymosan, respectively, for 24 hours. Before sampling, these fish groups were given hypoxia stress for 30 minutes.
Water immersion was practiced to induce hypoxia stress in these fish by keeping them under water for 30 minutes without allowing gulping or breathing air. All fish groups were sampled together after the stipulated time interval.

Sampling and analysis
Feeding was discontinued for 24 hours prior to experiment to ensure standardized experimental conditions. While handling for injection, care was taken to ensure minimum disturbance to fish. After the experimental treatment, the fish were quickly netted and dipped in 0.2% 2-phenoxyethanol (SRL, Mumbai, India) solution for 2 minutes. These anesthetized fish were held on a wet towel and blood was immediately drawn from the caudal artery. The fish were killed immediately by spinal trans-section, and the whole brain was then segmented into 3 portions based on the morphological and functional properties. The first segment, the PC, including the telencephalon and diencephalon; the second segment, the MC, including optic tectum, torus longitudinalis, and tegmentum; and the third segment, the MeC, comprising cerebellum, medulla, and a part of the spinal cord were separated. These 3 brain segments were blotted, weighed, and kept in icecold brain mitochondrial extraction (BME) buffer (pH 7.4) containing 0.25 M sucrose, 10 mM HEPES, 0.5 mM EDTA, and 0.5 mM EGTA and stored at −80°C for further analysis. Samples were extracted for mRNA expression analysis and were aseptically isolated and preserved in RNAlater solution (Ambion, Invitrogen, USA.) at −80°C for further analysis.

Quantification of ion-specific ATPases
NKA, HKA, and NNA-specific activities. Frozen PC, MC, and MeC were thawed and a homogenate (50 mg/500 μL) was prepared in BME buffer (pH 7.4) using glass homogenizer fitted with Teflon pestle giving 3 strokes as described previously (Samuel et al., 2014). 64 Homogenates were centrifuged at 2000g for 3 minutes at 4°C (Eppendorf 5430R, Germany.), and the membrane fraction prepared (Samuel et al., 2014) was used for quantifying the activity of ATPases, namely, NKA, HKA, and NNA. The protein concentrations in the tissues were determined using modified biuret assay 65 with bovine serum albumin as standard.
NKA-specific activity. The ouabain-sensitive NKA-specific activity in the PC, MC, and MeC was quantified adopting the method of Peter et al. (2000) modified for microplate assay. 60 Saponin (0.2 mg protein −1 ) was routinely added to optimize substrate accessibility. Samples in duplicates containing 1.0 μg protein were added to a 96-well microplate containing 100 mM NaCl, 30 mM imidazole (pH 7.4), 0.1 mM EDTA, and 5 mM MgCl 2 . About 0.13 mM KCl was used as the promoter and 0.14 mM ouabain was used as the inhibitor. After vortexing, the assay mixture was incubated at 37°C for 15 minutes. The reaction was initiated by the addition of 0.3 mM ATP and was terminated with addition of 8.6% trichloroacetic acid (TCA). The liberated inorganic phosphate was measured against phosphate standard at 700 nm in Synergy HT Biotek Microplate Reader, USA. The change in absorbance between promoter and inhibitor assays was calculated and regression analysis was employed to derive the rate of activity of NKA and expressed in micromoles of Pi liberated per hour per milligram of protein.
HKA-specific activity. SCH28080-sensitive HKA assay for brain homogenates was developed after extensive standardization including kinetics (data not shown) based on NKA microplate assay, 60 but SCH28080 was used as inhibitor instead of ouabain. As inhibitor, 0.1 mM SCH28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile) was used, whereas 0.13 mM KCl was used as promoter to measure HKA activity. Saponin-treated samples in duplicates (1.0 μg protein) were added to a 96-well microplate containing above reaction mixture, and the reaction was initiated by the addition of 0.3 mM ATP and incubated at 37°C for 15 minutes. The reaction was terminated by adding 8.6% TCA, and the inorganic phosphate content was measured in Synergy HT Biotek Microplate Reader. The change in absorbance at 700 nm between promoter and inhibitor assays was calculated using regression analysis, and the rate of transport activity of H + /K + -ATPase was expressed in micromoles of Pi liberated per hour per milligram of protein.
NNA-specific activity. An ouabain-sensitive NNA assay system has been developed for brain based on the previously described NKA microplate assay 60 but used 0.02 mM NH 4 Cl instead of 0.13 mM KCl. Extensive standardization of this assay system including kinetics was done (data not shown). Samples were treated with saponin (0.2 mg protein −1 ) to optimize substrate accessibility. Samples in duplicates containing 1.0 μg protein were added to a 96-well microplate containing 100 mM NaCl, 30 mM imidazole, 0.1 mM EDTA, and 5 mM MgCl 2 . As promoter, 0.02 mM NH 4 Cl and 0.14 mM ouabain were used in the assay as the inhibitor. After vortexing, the assay mixture was incubated at 37°C for 15 minutes. The reaction was initiated by the addition of 0.3 mM ATP and was terminated with addition of 8.6% TCA. The liberated inorganic phosphate was measured against phosphate standard at 700 nm in Synergy HT Biotek Microplate Reader. The change in absorbance between promoter and inhibitor assays was calculated, and regression analysis was employed to derive the rate of activity of NNA and expressed in micromoles of Pi liberated per hour per milligram of protein. Quantitative reverse transcription-polymerase chain reaction was performed in duplicates using the StepOne Real-Time qPCR system (Applied Biosystems) using TaqMan assays (Applied Biosystems, Life Technologies). The reactions contained 5 μL of 2× TaqMan Universal Master Mix, 0.5 μL 20× custom-made primers (Table 1), and cDNA (1 ng) or standard (1 μL) in a total volume of 10 μL. Cycling conditions were kept as 50°C for 2 minutes and 95°C for 10 minutes for 1 cycle followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. 18S ribosomal RNA was used as the endogenous control (Applied Biosystems). Data (threshold cycle [Ct] values) were collected at real time. The Ct, slope, PCR efficiency, y-intercept, and correlation coefficient (R 2 ) were calculated using the default setting of StepOne Software (Applied Biosystems). The amplification efficiency for nkaα isoforms was 95% to 100%, and the mRNA expression was calculated by adopting 2 −ΔΔCt comparative Ct method (where ΔCt = ΔCt sample − ΔCt control. 66 The results were statistically analyzed using Relative Expression Software Tool 2009 (REST), and the values were then expressed relative to the control sample.

Statistics
Data were collected from 8 animals in each group. Before statistical analyses, data were checked for normal distribution and variance homogeneity. Data were subjected to 1-way analysis of variance followed by Student-Newman-Keuls test (GraphPad InStat-3; GraphPad Software, Inc., San Diego, CA, USA). Stastistical changes between the means were accepted as significant if P < .05. In dose-responsive zymosan treatments, the significance levels were represented as "a" (P < .05), "b" (P < .01), and "c" (P < .001) compared with sham control (0 dose). Likewise, in hypoxia stress experiment, the differences between the means were accepted as statistically significant if P < .05.
The relative mRNA expression of NKA α1-subunit isoforms nkaα1a, nkaα1b, and nkaα1c in brain regions after varied doses of zymosan treatment was statistically analyzed using REST, and the significant upregulation (x) and downregulation (y) between fish groups were recorded after depicting significance at P < .05.

Dose-responsive effect of zymosan in intact fish brain
A dose-dependent rise (P < .001) in the NKA activity was found after zymosan challenge in PC, where serotonergic neurons were abundant ( Figure 1A). The NKA activity that showed a rise (P < .05) in MC after a medium dose of zymosan treatment elevated (P < .001) its activity in MeC after a low dose of zymosan treatment ( Figure 1A). Dose-dependent sensitivity of serotonergic neurons in the MC and dopaminergic neurons in the MeC to zymosan was observed in these fish brain segments. Likewise, a dose-dependent rise (P < .001) in HKA activity after zymosan treatment was observed in PC ( Figure 2A). A low dose of zymosan treatment was necessary to produce a rise (P < .001) in HKA activity in MeC, where it showed a dosedependent decline (P < .001) after medium and high dose of zymosan treatment (Figure 2A). On the contrary, the HKA activity of the MC remained unaffected after zymosan treatment ( Figure 2A). A high dose of zymosan treatment was necessary to produce a rise (P < .01) in NNA activity in PC, where it declined (P < .01, P < .001) after low and medium doses of zymosan treatment ( Figure 3A). A dose-dependent rise (P < .01, P < .001) in NNA activity after zymosan challenge was found in MC ( Figure 3A). Likewise, NNA activity increased (P < .01, P < .001) after low and high doses of zymosan treatment in MeC ( Figure 3A). Spatial and differential participation of both serotonergic and dopaminergic neurons could be found in these segments that transport NH 4 + ions in fish brain during zymosan challenge. The analysis of the activity pattern of NKA, HKA, and NNA after zymosan treatment revealed dosedependent spatial and functional differentiation of these serotonergic and dopaminergic neurons in their tested brain segments ( Table 2). The analysis of the activity pattern of NKA during hypoxia-immune challenges showed differential functional and spatial responses in the varied neuronal clusters (Table 3). This further provides evidence for the activation of serotonergic, dopaminergic, glutamergic, and GABAergic neurons after hypoxia stress in PC and its inhibition in immunechallenged fish brain (Table 3). However, absence of these neuronal responses was evident in PC of fish brain during hypoxia stress-immune interaction (Table 3). It is also evident in the analysis pattern that dopaminergic neurons in the MeC segment showed a reversal during hypoxia stress-immune interaction, thus pointing to a role for this neuronal cluster during hypoxia stress-immune interaction ( Table 3).

Effect of hypoxia in immune-challenged fish brain
A rise (P < .05) in NKA activity after hypoxia stress was observed in the PC and MeC of intact brain, whereas its activity decreased (P < .05) after hypoxia stress in MeC of immune-challenged brain ( Figure 1B and D). A compensatory role of dopaminergic neurons in these brain segments could maintain Na + and K + homeostasis after hypoxia stress and immune challenge. On the contrary, the NKA activity in the MC remained unaffected after hypoxia stress in the intact and immune-challenged brain ( Figure  1C). The analysis of activity pattern of NKA revealed a shift in its activity pattern after hypoxia stress in the PC and MeC of the immune-challenged fish (Table 4).

Journal of Experimental Neuroscience
A rise (P < .05) in HKA activity occurred after hypoxia stress in the MeC of intact fish brain ( Figure 2D). An increased sensitivity of the dopaminergic neurons to hypoxia stress could be found in these neuronal cells. On the contrary, the HKA activity in PC and MC remained unaffected after hypoxia stress in intact and immune-challenged fish brain ( Figure 2B and C). The analysis of activity pattern of HKA revealed that alterations in the MeC of intact fish brain showed a reversed pattern after hypoxia stress in immunechallenged fish, although its activity remained unaffected in the PC and MC (Table 4).
A rise in NNA activity occurred after hypoxia stress in PC and MC of the intact fish brain ( Figure 3B and C). Activated serotonergic neurons that showed a demand for transporting NH 4 + ions in exchange of Na + ions could be attributed to these brain segments. However, hypoxia stress declined (P < .05) the NNA activity in the MeC of immune-challenged fish brain ( Figure 3D), which may indicate a compensatory role for dopaminergic neuron in excreting NH 4 + ions from the neuronal membranes. The analysis of the activity pattern of NNA showed a disturbance after hypoxia stress in the MeC of immune-challenged fish brain. However, these alterations in the PC and MC were modified by hypoxia stress in immunechallenged fish brain (Table 4).

Dose-responsive effect of zymosan challenge on nkaα1 mRNA expression in intact fish brain
The mRNA expression of nkaα1a showed an upregulation after low dose of zymosan treatment in PC, whereas medium and high doses of zymosan downregulated its expression ( Figure 4A). On the contrary, low and high doses of zymosan treatment upregulated the mRNA expression of nkaα1a in MC, whereas a medium dose of zymosan produced a downregulation ( Figure 4A). An upregulation in the mRNA expression of nkaα1a after medium and high doses of zymosan treatment was found in the MeC ( Figure 4A). Likewise, an upregulation of nkaα1b mRNA expression in PC was observed only at low dose of zymosan treatment, whereas medium and high doses of zymosan downregulated its expression ( Figure 5A). On the contrary, low and high doses of zymosan treatment upregulated the mRNA expression of nkaα1b in MC, whereas medium dose produced a downregulation ( Figure 5A). A downregulation of nkaα1b  Figure 1A, the significance levels are represented as "a" (P < .05) and "c" (P < .001) when compared with control fish (0 ng g −1 ). The significance levels of Figure 1B to D are represented as "x" (P < .05) when compared between control and immersed fish (IMR). "y" (P < .05) represents significance between zymosan-treated (ZYM) fish and immune-challenged hypoxia-stressed (ZYM + IMR) fish. 7 mRNA expression was found in MeC after zymosan challenge ( Figure 5A). Likewise, a downregulation in the mRNA expression of nkaα1c after medium and high doses of zymosan treatment was found in PC ( Figure 6A). Low and high doses of zymosan treatment upregulated the mRNA expression of nkaα1c in MC, whereas medium dose of zymosan downregulated its expression ( Figure 6A). An upregulation in the mRNA expression of nkaα1c after low dose of zymosan treatment was observed in MeC, whereas its medium and high doses downregulated its expression ( Figure 6A). The analysis of the expression pattern of nkaα1 subunit isoforms after zymosan treatment showed a doseresponsive pattern in the varied brain segments. A substantial sensitivity of MC that possesses more dopaminergic neurons to immune challenge was also observed ( Table 5).

mRNA expression of nkaα1 subunit isoforms in fish brain after hypoxia in zymosan-challenged fish
An upregulated nkaα1a mRNA expression after hypoxia stress was found in PC of immune-challenged fish ( Figure 4B). But, hypoxia stress downregulated its mRNA expression in MC of intact fish brain, whereas upregulating its expression after hypoxia stress in immune-challenged fish brain ( Figure 4C). On the contrary, hypoxia stress produced an upregulation in nkaα1a expression in the MeC of both intact and immunechallenged fish brain ( Figure 4D). A downregulation of nkaα1b mRNA expression after hypoxia stress was found in PC and MC of intact fish brain, whereas an upregulation of nkaα1b after hypoxia stress was found in immune-challenged fish ( Figure 5B and C). On the contrary, an upregulation in the mRNA expression of nkaα1b was found after hypoxia stress in the MeC in both intact and immune-challenged fish brain ( Figure 5D). Likewise, a downregulation of nkaα1c mRNA expression was observed after hypoxia stress in PC of intact fish brain, whereas it upregulated after hypoxia stress in immune-challenged fish brain ( Figure 6B). On the contrary, hypoxia stress in intact and immune-challenged fish produced a downregulation of nkaα1c mRNA expression in MC and MeC ( Figure 6C and D).
The mRNA expression analysis of nkaα1 isoforms showed a shift in the pattern of nkaα1a and nkaα1b after hypoxia stress in the PC and MC of immune-challenged fish (Tables  6 and 7). Likewise, the analysis of nkaα1c pattern showed a shift in its expression after hypoxia stress in PC of immunechallenged fish brain (Tables 6 and 7). The mRNA expression  Figure 2A, the significance levels are represented as "c" (P < .001) compared with control fish (0 ng g −1 ). The significance level of Figure 2D is represented as "x" (P < .05) when compared between control and immersed fish (IMR).  Figure 3A, the significance levels are represented as "b" (P < .01) and "c" (P < .001) compared with control fish (0 ng g −1 ). The significance levels of Figure 3B to D are represented as "x" (P < .05) when compared between control and immersed fish (IMR). "y" (P < .05) represents significance between zymosan-treated (ZYM) fish and immune-challenged hypoxia-stressed (ZYM + IMR) fish. Table 2. Glowchart showing the differential activity pattern of Na + /K + -ATPase (NKA), H + /K + -ATPase (HKA), and Na /NH -ATPase

Journal of Experimental Neuroscience
 indicates activation,  indicates inactivation, andindicates unaffected.
Peter and Simi 9 analysis of nkaα1 isoforms showed differential pattern during hypoxia-immune challenges in the varied neuronal clusters, indicating a spatio-temporal regulation of NKA function in the neuronal clusters.

Discussion
Similar to vertebrate brain, fish brain also coordinates the spatial and temporal physiological activities through the activation/inactivation of their specific neuronal groups. The membrane-bound NKA that maintains the asymmetric distribution of Na + and K + in neuronal cells shows synaptic and neuronal activity and is connected to neuronal functions. 67 In our study, we found a differential pattern of NKA regulation in both immune-challenged and hypoxia-stressed fish brain which suggests a differential sensitivity of brain NKA to these challenges. This further points to the differential regulation of neuronal groups including serotonergic and dopaminergic neurons to immune challenge as these neuronal clusters are more abundant in the PC and MeC of fish brain. 22,23 The increased NKA activity after hypoxia stress in the PC and MeC of the intact fish brain showed a decrease in MeC of immune-challenged brain, indicating a spatial sensitivity of NKA to hypoxia stress and immune challenge. A reversal of this activation was found in the MeC during hypoxia stress-immune interaction. This deactivation of dopaminergic neurons during hypoxia stress-immune interaction probably points to a compromised dopamine release, which appears to be a protective action against hypoxia-stress and immune challenges. Induction of hypoxia stress in immune-challenged fish brain, on the contrary, shows a shift in the pattern of NKA activity, and this further points that hypoxia stress has a direct control on brain Na + /K + -pump function. This would probably be due to a differential activation of functional neuronal clusters in the brain segments to the challenges in oxygen delivery and Na + availability. This view is consistent with the fact that NKA shows more sensitivity to intracellular Na + concentration besides its K + -dependent activation. 68 The dose-responsive inhibition of NKA activity to zymosan challenge in the PC indicates inactivation of neuronal clusters such as serotonergic, dopaminergic, glutamergic, and GABAergic neurons. On the contrary, hypoxia stress demands an activated NKA pattern in the PC that reveals activation of these neuronal clusters. These differential sensitivities of neuronal clusters further support the view that immune challenge could disturb the neuronal activity of neuronal clusters in the brain that shows spatial and differential responses. This further provides evidence for the critical role of NKA in the regulation of Na + and K + transport as a major target Na + transporter. Interestingly, the pattern of NKA activity that showed little response to hypoxia stress in immune-challenged fish brain further points that hypoxia stress demands a modified NKA function during hypoxia stress-immune interaction. This classic NKA response pattern in fish brain presents clues on the specific target response of NKA to both hypoxia stress and immune-induced disturbance in ion transport. Consistent observation has also been found in the osmoregulatory epithelia of air-breathing fish where NKA responded differentially to both immune challenge and hypoxia stress. 5 Neuronal Na + accumulation occurs gradually via ligandgated and voltage-gated Na + channels and in parallel to the release of K + to the extracellular space. 67 It is evident in our study that both immune and hypoxia stress target intracellular Na + signals that prompt an enhanced activation of neuronal NKA as reported earlier. [69][70][71][72] It is likely that the immune challenge or hypoxia stress can extrude Na + and uptake of K + and that permits the neurons to reestablish the concentration gradients of these ions. This activation of the neuronal clusters in the tested segments of brain further indicates its differential sensitivity of varied types of neuronal clusters including serotonergic and dopaminergic neurons to hypoxia stress or immune challenges. Moreover, it is likely that the differential and spatial responses of these neurons appear to be due to the altered neuronal membrane potential. It is known that the release of dopamine from the dopaminergic neuronal synaptosomes is sensitive to pH changes. 73,74 Similar sensitivity of neuronal clusters to acid/base variables after zymosan challenge has been found in our test species (Peter et al, unpublished data, 2017).

11
The abundant dopaminergic neurons in the MeC that respond to immune challenge as well as to hypoxic stress further provide clues that NKA in this brain segment could function as a neuronal marker that could fine-tune its neuronal activity during hypoxia stress-immune interaction. It has been further shown that the failure of NKA that leads to dissipated transmembrane H + and Na + gradients may contribute to unregulated dopamine efflux. 75,76 It is further plausible to assume that the differential sensitivity of nkaα1 isoform expression may also be related to the varied demands for intracellular Na + concentration in brain segments. Similar changes in Na + signaling and its sensitivity have been attributed to a modified NKA function as it has been linked with neuronal activity. 77-79 A lead role for NKA in the regulation of Na + and K + gradients has been well established in neurons where any alterations of this pump could lead to its altered physiological functions. Likewise, changes in the neuronal crosstalk with astrocytes could also lead to changes in brain function as neuronal activity requires association with astrocytes for the clearance of K + from the extracellular space. 80 H + /K + -ATPase possesses a catalytic α-subunit and a regulatory β-subunit that use energy from ATP hydrolysis to transport H + in exchange for K + . 81 The large, ~100-kDa, α-subunit with 10 transmembrane helices contains the catalytic site and translocates H + and K + . A second, smaller glycosylated β-subunit, ~30 kDa, is required for proper trafficking and processing of the enzyme. 81 The HKα 1 H + -K + -ATPase, also known as the gastric HKA, is sensitive to micromolar concentrations of imidazopyridine, 2-methyl-8-(phenylmethoxy)imidazole[1,2a]-pyridine-3-acetonitrile (SCH-28080), 81 whereas the HKα 2 H + -K + -ATPase is sensitive to relatively high concentrations of ouabain. 82 Studies have shown that respiratory/metabolic acidosis [82][83][84] and ammonia toxicity could stimulate SCH-28080-sensitive HKA activity in mice. 85 Recently, we found that SCH-28080-sensitive HKA transport system in the osmoregulatory epithelia of immune-challenged air-breathing fish responds to hypoxia stress. 5 The presence of this ion transporter in the varied brain segments that possess serotonergic and dopaminergic neurons provides evidence that the membrane potential of these neurons sensitizes both H + and K + ions. It appears that immune challenge that modifies HKA activity activates the serotonergic neurons in the PC while inactivating the dopaminergic neurons in the MeC. This spatial and differential regulation thus for the first

12
Journal of Experimental Neuroscience time provides evidence for a role of H + and K + gradients in integrating ion signals in the brain neuronal clusters. There are reports showing that dopamine release could modify the neuronal transmembrane H + and Na + gradients due to its  indicates activation,  indicates inactivation, andindicates unaffected.
Peter and Simi 13 dependence on energy metabolism and Na +/H + Exchanger (NHE) activity. 74 HKA activity pattern in the MeC during hypoxia stress and immune challenge in our test species also supports this view. The rise in HKA activity in the MeC of hypoxia-stressed fish showed a reversal after hypoxia stress in immune-challenged fish, indicating that hypoxia stress may activate the dopamine release from the MeC during these challenges. The unaffected HKA activity in the PC and MC also support this view. Overall, our HKA data indicate that this transporter is sensitive to both hypoxia stress and immune challenge as neurons are shown to be more permeable to K + than to any other cation, and the reduction in its ion permeability ("channel arrest") can be considered as a major strategy for saving energy during anoxia as reported earlier. [86][87][88] Studies have found that anoxia can induce reduction in K + permeability in the brain of the freshwater turtle Trachemys scripta. Likewise, in crucian carp, hypoxia can reduce the neural membrane "leakiness" for ions such as Na + and Ca 2+ , a strategy that should lead to reduce neuronal excitability. 89 Fish can tolerate high internal ammonia compared with humans, and at high blood ammonia concentrations, NH 3 probably enters the brain via diffusion. 90 Diffusion of ammonia into the brain is promoted by the lower intracellular pH of brain tissue, in which NH 3 is trapped as ionized NH 4 + and unable to move back across the blood-brain barrier due to its positive charge. 91,92 We have seen that both zymosan and hypoxia stress alter the acid/base indices of our test species (Peter et al., unpublished). This in turn may promote the diffusion of NH 3 into the neuronal tissues during nontoxic stressor exposure such as hypoxia and immune challenge and trap it as NH 4 + ion. An ouabain-sensitive ATPase system that is activated by NH 4 + , could direct the transport of NH 4 + against Na + because hydrated NH 4 + and K + ions can compete each other because of the same ionic radius. 93 Our recent study provided evidence that a NNA system exists in the osmoregulatory tissues of our air-breathing fish, 5 and it is likely that it may have a functional role in the fish brain segments that transport NH 4 + in exchange of Na + . However, traditionally, in brain, excess NH 4 + has been converted to glutamine by glutamine synthetase via an ATP-dependent reaction of NH 4 + and glutamate. 92,[94][95][96][97][98] This protection works against ammonia toxicity by causing osmotic shifts of water into the intracellular space of astrocytes by increasing the osmolarity of the cell cytosol. 99 Krogh proposed that Na + absorption by fish is linked with NH 4 + excretion. [100][101] As NH 4 + is the major end product of nitrogen metabolism in teleosts that accumulates in the bloodstream, it is likely that NH 4 + efflux gradient exists as a driving force to absorb Na + . 102 Moreover, as ammonotelic animals, teleosts use internal NH 4 + as an ideal driving force for Na /NH + 4 + exchange especially during stressed conditions. 103 In our study, evidence is presented that serotonergic and dopaminergic neuronal clusters in the varied brain segments are involved in zymosan-induced immune challenge. We found that Na /NH + 4 + -dependent ATPase transport, which is more active in PC and MC of intact fish brain, showed a reversed switching pattern in MeC on hypoxia stress. This further indicates an increased sensitivity of the serotonergic neurons to NH 4 + transport in the PC and MC during hypoxia stress. The decreased NNA activity in the MeC after hypoxia stress in immune-challenged fish suggests a compromised activity of the dopaminergic neurons to NH 4 + ion transport in this brain segment. This further points to a major role of Na /NH + 4 + transport system in the different brain segments and its sensitivity to immune challenge and stress response. This would imply that when confronted with high brain ammonia concentration, neuronal cells could maintain intracellular K + homeostasis and low intracellular ammonia concentration through the differential regulation of NNA with only a low level of K + being substituted by NH 4 + . Furthermore, a downregulation of the mRNA and protein expression of NKA α-subunit isoforms would be essential to ameliorate the deleterious effects of ammonia under hyperammonia conditions. 49,50 Alternatively, it seems that the disturbances to intracellular ion balance arising from changes in the kinetics of different ion transporters could also contribute to the varied patterns of ion transport functions during hypoxia stress in immune-challenged fish. However, an increase in brain ammonia concentration that induces higher NKA gene expression and NKA activity 51 increases in the production of ouabain-like substances and NKA activity in cultured mouse astrocytes. 104 Ion-dependent ATPase plays an important role in intracellular functions and is considered as a sensitive indicator. There are 3 subunits of NKA, α, β, and γ, and each subunit has its isoforms. The minimum functional unit is a heterodimer of the α-subunit and β-subunit, which may also be co-expressed with the γ-subunit. 105 The NKA α-subunit (110-120 kDa) contains the catalytic unit responsible for binding and transporting Na + out and K + into the cell and the binding site for ATP. 45 Four NKA α-subunit isoforms (α1, α2, α3, and α4) have been identified in mammals 45 and nkaα1, nkaα2, and nkaα3 are expressed in the brain of all mammalian species examined. 45,46,106,107 Here, we present evidence for the first time that 3 α1 isoforms such as nkaα1a, nkaα1b, and nkaα1c are expressed in brain segments of air-breathing fish as reported for the osmoregulatory tissues of this fish. 5 We also found that hypoxia stress and immune challenge can modify the mRNA expression of nkaα1 isoforms, indicating the major functional role of these isoforms in brain NKA regulation during immune and hypoxia stress challenges. The shift in the mRNA expression of nkaα1a and nkaα1b in the PC and MC to hypoxia stress indicates a differential regulation of these nka1 isoforms to both hypoxia stress and immune challenge, which in turn is associated with the differential response of serotonergic neurons that are abundant in the PC and MC of fish brain. These transcriptomic data further confirm the physiological role of brain NKA as a major Na + transporter of brain that coordinates many physiological activities and also account for its observed spatial and differential regulation during hypoxia stress-immune interaction. In brain, NKA α1-subunit isoforms are found both in neuronal cells and astrocytes contributing to the control of membrane potential and K + re-uptake after depolarization. 44,108,109 In this sense, NKA is a crucial transporter that controls neuronal excitability and its dysregulation may lead to several dysfunctions including neurological and physiological and behavioral disturbances. The activity patterns of the NKA ion transporter seem to be opposite to the expression pattern of the nkaα1 isoforms in the brain segments after challenges. However, a general shift in the pattern of the mRNA expression was noticed during hypoxia stress-immune interaction, suggesting the transcriptomic regulation of NKA during hypoxia stress-immune interaction. This further provides evidence for the differential sensitivity of neuronal clusters that are distributed abundantly in the PC and MC, whereas dopaminergic neurons in the MeC may play a compensatory role. While analyzing the differential expression pattern of the nkaα1 subunit isoforms, it is evident that MeC is less sensitive to the hypoxia stress in immune-challenged fish. Furthermore, the switching of nkaα1a and nkaα1b isoforms in the PC and MC clearly indicates a differential modification of sodium pump in these brain segments during hypoxia stress in immune-challenged fish. This spatial and differential genomic regulations of nkaα1 isoforms in these brain segments further demand transcriptomic regulations, which would alter the NKA activity during hypoxia stressimmune interaction. The differential activity of nkaα1 isoforms to bind with K + or NH 4 + makes us to believe that immune challenge and hypoxia stress demand differential regulation of Na + , K + , and NH 4 + ions in the different neuronal clusters of brain. The brain serotonergic system is crucial for the development of divergent stress coping styles and plays a central role in the processes of neuronal plasticity. 110 Activation of brain serotonergic system (serotonin, 5-hydroxytryptamine, 5-HT) along with catecholamines (norepinephrine and dopamine) has been found associated with stressful conditions in fish. 111,112 We have found the involvement of many neuronal clusters mainly serotonergic and dopaminergic neuronal clusters during hypoxia stress-immune interaction in the fish brain. Similarly, stressful conditions that induce hyperactivity in the metabolism of brain serotonin both in mammals [113][114][115] and in fish 116,117 have been implicated to hormonal control. 117 Likewise, dopamine, primarily synthesized in the brain and kidney, has direct action on brain functions, where it is important in the control of locomotion, cognition, and affect neuroendocrine secretion. [119][120][121] The involvement of dopaminergic neuronal clusters in fish brain could point to a role for dopamine in Na + transport as reported earlier. 122 Hypothalamic proopiomelanocortin neurons that release GABA as excitatory transmitter and glutamate as inhibitory transmitter 123 are involved in neuronal control of physiological mechanisms. 124,125 Furthermore, in fish, the transport of Na + and K + has been associated with either 5-HT 3 receptor channel function 126 or stress-induced immunosuppressive effects in fish via glucocorticoids or serotonin. 127 It is thus obvious that crosstalks between neural, immune, and endocrine systems occur during immune-hypoxia stress interaction in fish.
In summary, this study explored the mechanism of hypoxia stress-immune interactions during ion transporter function in the brain segments of an air-breathing fish. We found for the first time that immune challenge evoked a varied response pattern of ion transporters that include NKA, HKA, and NNA in the different regions of fish brain that showed differential regulation to hypoxia stress. The differential and spatial modifications of these ion transporters along with transcriptomic analysis of nkaα1 isoforms in fish brain provide evidence for the regulatory functions of the serotonergic and dopaminergic neuronal clusters in fish brain to hypoxia stress and immune challenge. Our data provide evidence for the first time that highlighted the critical role of functional neuronal clusters in the integrative physiology of brain activities, where signaling of Na + , K + , H + , and NH 4 + ions occurs through NKA, HKA, and NNA transporters to maintain the neuronal membrane potential. Furthermore, the tested ion transporters that show sensitivity to both immune challenge and hypoxia stress can act as functional brain markers, thus providing validity for assessing the magnitude of stress-immune interaction in our airbreathing fish.


New Developments in Long-Acting Injectable Nanoformulations

Nanomedicine is an emerging field that combines nanotechnology with pharmaceutical and biomedical sciences, with the goal of developing drugs and imaging agents with higher efficacy and improved safety and toxicological profiles. According to BCC Research (www.bccresearch.com), the global nanotechnology market should reach $90.5 billion by 2021, from 39.2 billion in 2016 at a compound annual growth rate (CAGR) of 18.2%. The nanomaterials market (which includes nanomedicines) should reach $77.3 billion by 2021 from $32.5 billion in 2016 at a CAGR of 18.9% (Figure 1).

their therapeutic categories. The principal areas in which nanomedical products have made an impact are cancer, CNS disease, cardiovascular disease and infection control. Figure 2 below presents the trends in the development of nanomedicines in terms of drug products approved by FDA or currently under clinical investigation. Different types of long acting injectable nanoparticles are included, although liposomal and polymeric nanoparticles are the most commonly used. Nanomedicines under clinical trial investigation stratified by category overall [1].

Liposomes
Liposomes as nanopharmaceuticals are formed from phospholipids and cholesterol in aqueous medium. Liposomes have a spherical phospholipid liquid crystalline phase, and are simply produced by dispersion of phospholipid in water by shaking. This results in the formation of multilayer structures consisting of several bilayers of lipids. After sonication, these multilayer structures produce unilamellar structures that are referred to as vesicles. Liposomes can entrap both hydrophilic and hydrophobic drugs and drug release can be targeted to specific sites. Biocompatibility, biodegradability and low toxicity are the main advantages of liposomal delivery systems. Since injected liposomes can avoid uptake by the reticuloendothelial system (RES), the particles remain in circulation for a prolonged period of time. Typically, the particle sizes of liposomes range from 50 to 200nm. To make liposomes suitable for therapeutic applications, their size distribution has to be controlled, which can be realized by passing them repeatedly, under elevated pressure, through membranes with defined pore size.
The concept of liposomal drug delivery system has had a revolutionary effect on the pharmaceutical field. Since Alec Bangham first described liposomes in 1961, a massive amount of research has been carried out, and their applications are now well established in various areas, such as drug, biomolecules and gene delivery. Due to extensive developments in liposome technology, a number of long-acting liposome-based drug formulations are available for human use and many products are under clinical trials. Most commercial available liposomal drug formulations are listed in Table 1 [2,3]. Liposome technologies for long acting drug delivery: Several widely used liposome technologies include Stealth Liposome Technology (SLT), Non-PEGylated Technology (NPL), DepoFoamTM Technology and Lysolipid Thermally Sensitive Technology (LTST) [4]. In SLT, strands of the polymer(s) are attached to drug molecules, or to a system that can improve the safety and efficacy of the therapeutic agents.In general, PEGylation is attained by the incubation of a reactive derivative of PEG with the target moiety. Covalent linkage of the liposome to a PEG protects the active moiety from the recipient's immune system, which results in reduced immunogenicity and antigenicity [3]. It also produces alterations in the physiochemical properties of the active moiety, including changes in the hydrodynamic size, which further reduce its renal clearance and thereby prolongs its circulatory time. Also, it provides hydrophilicity to hydrophobic drugs and reduces dosage frequency.
NPL is a unique drug-delivery system that came as a breakthrough in cancer therapy by offering the benefits of PEGylated-liposome while eliminating the side effects associated with PEG such as hand-foot syndrome [2]. NPL Doxorubicin (NPLD) injection provides a better safety profile over conventional DOX and Doxil®. NPLD not only reduces the cardiac toxicity associated with DOX, but also the doselimiting toxicity linked with the use of Doxil®. DepoFoamTM technology encapsulates drugs in its multivesicular liposomal platform without modification of their molecular structure. The multivesicular liposomes releases drug(s) over a required period of time ranging from 1 to 30 days. Upon administration, DepoFoam™ particles release the drug over a period of hours to weeks following erosion and/or reorganization of the lipid membranes [4].
DepoFoam™ technology improved the properties of both small and large molecules. This technology considerably improved patient care by providing a remarkable solution for medications that require frequent multiple injections and have a short period of action or side effects. Thermosensitive liposomes have been studied for drug release at sites of elevated temperature. These novel liposomes are being developed to exhibit temperature-dependent release of encapsulated drug(s). Local tissue temperature is generally elevated to 42 0 C by radiofrequency ablation, a technique based on the application of radiofrequency [4]. Lipid components present in the liposome undergo a gel to liquid transition at elevated temperatures, making it more permeable, and thus releasing the drug.

Polymeric nanoparticles
Polymeric nanoparticles possess the advantage of stabilityboth in storage and in vivo application. For polymeric particles, the drug is entrapped within the polymer matrix, usually a biodegradable polymeric matrix. Polymer nanomedines typically fall into one of two categories:

I.
Polymer-drug conjugates for increased drug half-life and bioavailability, and II. Degradable polymer architectures for controlled release applications [5].
However, it should be noted that aspects of polymer chemistry are emerging in nearly all of the categories, because many of the components required (e.g. amphiphilic block copolymers) can be designed and controlled through organic synthesis methods.
Polymeric nanoparticles are recognized as foreign bodies and they can be removed from the blood circulation by the phagocyte cells of the RES. A majority of the nanoparticles can be phagocytosed by the macrophages of the liver and spleen shortly after intravenous (i.v.) injection. The nanoparticle clearance is mediated by adsorption of blood components to the surface of the particles, namely opsonization.
The polymers themselves include those that are synthetic, pseudo-synthetic or those that arise from natural sources. Their application has spanned the full nanomaterial size-scale, from single polymer chains up to large aggregates, depending on the required therapeutic outcome. To realize long-acting attributes, the polymeric nanoparticles can protect the drug from degradation, thus achieving prolonged drug delivery (and a long shelf life). For therapeutic purposes, the most commonly used polymers include polyethylene glycol (PEG), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), alginate, chitosan and gelatin base. Polymeric nanoparticles comprise a very heterogeneous group of nanosized therapeutics (Table 2) [2,6]. No mechanism attributable to nanosize. Based on its resemblance to myelin basic protein, glatiramer is thought to divert as a "decoy" an autoimmune response against myelin. The most basic class of polymeric nanomedicines utilize single polymer chains, either directly as the therapeutic itself, or as a modifying agent for a drug or diagnostic agent. More frequently in terms of polymeric nanomedicines, drugs are attached to a hydrophilic polymer to increase circulation or to improve biocompatibility/solubility. The most well-established polymer is poly (ethylene glycol) (PEG), and PEGylation results in a significant increase in biological half-life in plasma. In addition to PEGylation, other hydrophiles can be utilized to increase circulation half-life. A polymer-drug conjugate of paclitaxel and polyglutamic acide (poliglumex (PPX)) has entered phase III trials and is showing significantly improved standard of living for patients who undergo paclitaxel therapy for non-small cell lung cancer.
Beyond just extending the circulation time of established drugs, polymeric nanoparticles can be developed based on hydrophobic materials that facilitate controlled release of the therapeutic. This is achieved by using slowly-degradable functionality that subsequently leads to kinetically-driven release of the drug. A long established polymer nanoparticle that has had significant success is based upon incorporation of leuprolide (a testosterone inhibiting drug) into a polylactide-coglycolic acid (PLGA) nanoparticle.

Nanocrystals
Nanocrystals are composed of mainly hydrophobic drug with a small amount of excipient or surfactant, and different kinds of hydrophobic drugs can be nanoformulated into nanocrystals with high loading and encapsulation efficacy. Micronization is widely used as a common formulation method for sparingly soluble compounds. The saturation solubility of the nanocrystals is highly related to the particle size, and solubility increases with particle size decrease due to the increased surface area, especially when the nanocrystals are below 300nm. Consequently, the concentration gradient between gut lumen and blood is increased, which usually results in improved absorption by passive diffusion. In these suspension formulations, the ratelimiting step for drug absorption is the speed for drug particle dissolution in the formulation or in the in vivo fluid surrounding the drug formulation. In many formulations, a fatty acid ester of a drug is used to prepare an oil-based parenteral solution, and the drug-release rate from the solution is determined by the drug partitioning between the oil vehicle and the tissue fluid, also it can be influenced by the drug bioconversion rate from the prodrugs to the parent drugs.
To increase the circulation half-life, it is a widely accepted way in long-acting formulations for the parent drug to be synthesized into a prodrug through use of long-chain fatty acids (esterification). Due to their extremely low water solubility, the fatty acid ester of a drug dissolves slowly at the injection site after intramuscular (IM) injection. With the help of in vivo hydrolase, the prodrug is hydrolyzed into the parent drug and becomes available in the systemic circulation. Several other factors such as injection site, injection volume, the extent of spreading of the drug depot at the injection site, and the absorption and distribution of the oil vehicle can also affect the overall pharmacokinetic profile of the drug.
Most of the nanocrystals approved by FDA are used as oral formulations, and there are also some approved as a bone substitute. For long acting injectables, FDA has approved Invega® Sustenna® for schizophrenia and schizoaffective disorder treatment in 2009, and Ryanodex® for malignant hypothermia treatment in 2014. Invega® Sustenna® is a good example of long acting injectable prodrug formulation (paliperidone palmitate is the prodrug of paliperidone palmitoyl ester). This long acting injectable formulation is indicatedas a once-every 28 days injection after an initial titration period.
The production of nanocrystals has been applied to both organic drugs as well as inorganic materials. Synthesis methods include either "top-down"diminution approaches which are often employed for the organic compounds, and "bottom-up"precipitation methods that are more commonly applied to inorganic materials. Wet milling and high pressure homogenization technologies are widely used for nanocrystal preparation.
Wet milling technology: Wet milling (a top-down process) is a relatively effective milling technique for nanocrystal preparation. The process is done in a media milling, and it treats a dispersion of concentrated drug in an aqueous or nonaqueous liquid medium with milling balls. Wet milling has several advantages in its economic value and ease of scaling up. With the right media milling equipment, manufacturers can cost-effectively create uniformly fine particles with limited or no contamination. However, due to the intensive mixing forces in the vessel, erosion of the milling balls is a common occurrence, and must be monitored appropriately.

High Pressure homogenization technology:
High pressure homogenization (also a top-down process) can also achieve suspensions with narrow particle size distribution. It is a purely mechanical process, which is evoked by forcing a fluidic product through a narrow gap (the homogenizing nozzle) at high pressure (150-200MPa, or 350-400MPa for ultra high pressure homogenization, UHPH) [6]. The liquid product is subjected to very high shear stress causing the formation of very fine particles. High pressure homogenization can effectively process large volumes of liquid suspension sample thoroughly and reproducibly. Since it doesn't use milling balls, contamination of the final product is much less; however, the high pressures applied cause a temperature increase (due to the heat of compression), and this needs to be controlled, especially for athermally labile drug substance. Alternatively, a combination of bottom-up and top-down processes can be employed, e.g. solvent dissolution of API, crystallization via a non-solvent, and then homogenization of freshly formed particles

Antibody-drug conjugates
Antibody-drug conjugates (ADCs), a form of immunoconjugate or bio-conjugate, are an emerging class of medicines designed for high-specificity targeting and destruction of cancer cells. The mechanism of action is targeted delivery of a cytotoxic agent to the cancer cell via monoclonal antibody targeting of a specific cell surface marker. Upon binding, a biochemical reaction activates internalization of the ADC into the cell cytoplasm, where the drug becomes active, killing the cancer cell [7]. The ultimate advance with ADC therapeutics is that targeting and release of the drug specifically within the cancer cell means that healthy cells are not adversely affected and cancer cells can be more effectively destroyed.
Success in ADC therapeutics stems from a deep understanding around each of the trilogy 'Antibody-Linker-Payload (drug)'technologies, with having a complementary optimization of all three to generate an effective and potent ADC. With non-cleavable ADCs, the linker unit remains attached to the drug, which mitigates externalization and the resulting side effect of the drug entering healthy neighboring cells [8,9]. With cleavable ADCs, the drug is completely cleaved from the linker unit upon internalization, the antibody is degraded to its amino acid form, and the entire complex becomes active drug. Innovation in linking technology aspires to improve the coupling of payloads as well as to improve cleavage reactions, allowing improvements in payload delivery. The majority of ADC payloads are small molecules, which act via disruption of microtubules or inducing DNA damage.
To date, there are only four ADCs that have received market approval. However, after a request from the U.S. Food and Drug Administration (FDA), Pfizer/Wyeth, the developer and marketer of the first ADC to receive marketing approval (in 2001 for the treatment of patients with acute myelogenous leukemia (Gemtuzumabozogamicin, trade name: Mylotarg®)), withdrew the drug from the market in June 2010 (although it is still marketed in Japan). It was re-introduced into the US market in 2017. The second and third marketed ADCs are Brentuximab vedotin (trade name: Adcetris®, marketed by Seattle Genetics and Millennium/Takeda) and Trastuzumab emtansine (trade name: Kadcyla, marketed by Genentech and Roche). They were approved by U.S. FDA in 2011 and 2013 respectively. The newest ADC, Inotuzumabozogamicin, was approved by the European Commission as monotherapy for the treatment of adults with relapsed or refractory CD22-positive-B-cell precursor acute lymphoblastic leukemia on June 30, 2017 under the trade name Besponse® (Pfize/Wyeth) and approved for the treatment of adults with relapsed or refractory B-cell precursor acute lymphoblastic leukemia by the U.S. FDA on August 17, 2017.
Technical and manufacturing challenges: The biggest technical challenge associated with any drug manufacturing process is to provide consistently efficacious drug product, which is of the required purity and is safe from environmental and process related contamination. This challenge must be accomplished in a manner that protects employees, operators and the general environment from the harmful substances inherent in the process, and at a cost that makes the final drug marketable. The starting point for ADC manufacturing is the parent monoclonal antibody (mAb). Often the supply of mAbs of suitable quality for therapeutic purposes is taken for granted due to the successful developments in purification templates and platform processes in previous years.

Conclusion
The revolutionary development of nanoscience and nanotechnology in pharmaceutical research and development opens new horizons for the creation of novel drug products, which will utilize the unique characteristics of nanosized long acting injectable formulations. The application of engineered nanomaterials to medicine will produce nanomedicines with unprecedented benefits for the clinical outcome in many therapeutic indications.