ROLE OF NANOPARTICLES AS ROS -SCAVENGER

 

ABSTRACT

Reactive oxygen species generation is much critical in nanomaterial toxicity mechanisms. It is also involved in pathophysiology in reperfusion injury. Citric acid protected, two different sized platinum nanoparticles were produced to protect hepatocyte injury of mice. These particles exhibit ROS scavenging. Small size as well as larger size nanoparticles are formed with size 30 and 106 nm respectively. Superoxide anion, hydroxyl radicals and hydrogen peroxide, that are present in a glucose solution are reduced or scavenged by these particles. These Pt-NPs after injection into mice were accumulated in hepatic nonparenchymal cells of liver. More activity is showed by small Pt nanoparticles. Hepatic injury was induced in the mice. Pt-NPs were injected in bolus intravenously which inhibits the activities of aspartate and alanine aminotransferase i.e. AST and ALT. 6 hour reperfusion was followed before inhibition. As a result these platinum nanoparticles prevent hepatic  reperfusion/ischemic injury. This result shows the pharmacokinetic properties of nanoparticles.

INTRODUCTION

ROS are the unstable molecules that react with other molecules present in different cells. As the name indicates oxygen is the most important part of them. It was discovered in 1954 in a biological environment.  They can also generate radicals which are called as the oxygen radicals. Mostly they have damaging function in a cell. They cause damage to nucleic acids and proteins. At the same time they are playing the role of  messenger in cellular functioning and processes. All of its role is depending upon the equilibrium i.e. the production and scavenging of ROS. Their high level is extremely dangerous for organisms.  They contain unpaired of oxygen, for example hydroxyl and superoxide.

Cells have ability to maintain a intracellular ROS level by many regulatory strategies and other antioxidant defense system. For example superoxide dismutase is mostly used to degrade O₂^-. Many other methods have also been adopted to maintain ROS levels. By using nanotechnology we are able to deal with these by using certain in vivo techniques because these particles can control and influence many antioxidants. There are many types of nanoparticles, they could be inorganic and may posses catalytic properties. Such particles are influenced positively by large surface areas. Nanoparticles also include platinum, carbon, manganese and ceria. These are not limited to this but are also extended further i.e. they are associated with diseases such as cancer, diabetes and many inflammatory and neurogenerative diseases.

Here a short experiment is discussed to explain ROS scavenging by nanoparticles with reference to a disease. Hepatic reperfusion is such an example which occur in many clinical practices. This  practice is used in transplantation, shock and liver surgery. It may results in injuries and sometimes can cause death in such cases ( Thurman et al. 1988)( McCord et al. 1985). In hepatic reperfusion injury, oxidative stress is involved in pathogenesis. ROS is generated and accumulated by the activation of NADPH oxidase that is present in the Kupffer cells. The function of this NADPH is to activate the transcription of many different genes. This activation is achieved by nuclear factor-_KB mediated pathway (Granger et al. 1995) (Zhou et al. 2012).

Hepatocytes are extremely damaged with sudden increase in inducible nitric-oxide synthase, tumor necrosis factor i.e. IL-8 and interleukin, IL-6. After many studies and research it is declared that in order to inhibit hepatic reperfusion injury, we must eliminate ROS , for that reason its scavenging is a better therapeutic agent for this injury. Many scavengers have been used for this such as tocopherol, N-acetylcysteine and super oxide dismutase. In most recent studies ROS scavenging properties have be shown in platinum nanoparticles (Yoshihisa et al. 2011).  There are certain characteristics associated with these nanoparticles which help them to be more effective in ROS-mediated diseases and injuries. First is, they have high ratio of electron catalyze. Secondly there work as to quench hydrogen peroxides in a technique known as in vitro, they also quench superoxide anions. Due to presence of superoxide they interact with superoxide dismutase as  well (Honda et al. 2010).

Most of the studies have shown the beneficial effects of  platinum nanoparticles but  in recent times they are found to have  their function to act as pharmacokinetic and is much  more effective to treat these  kinds of reperfusions. In liver and spleen many nanoparticles and microparticles have been cleared by applying and using different techniques  of scavenging systems (Takino et al. 1994). For Pt-NPs to have more and more therapeutic effect they must be distributed evenly and correctly on tissue distribution. This distribution is helpful to know about many aspects of their pharmacokinetic properties as well as physiochemical properties. Due to that reason the aim is to study therapeutic potential and tissue distribution of Pt-NPs in these kids ofinjuries.

In order to perform more effective scavenging Pt-NPs are produced which are contain citric acids. These protected nanoparticles can be produced by two ways one method is to react platinumhydrochlride ions to citric acid and other way is by reacting it to ascorbic acid. Many aspects of Pt-NPs have been analyzed that determine the tissue distribution i.e. ROS-scavenging. In this regard we are going to study this in mice.

EXPERIMENTAL EVALUATION

The animal that was used for the experiment was a mice, a male ddY mice. It was weighing about 25-27 g. Certain chemicals were also required to perform this experiment. Few of them are discussed here. 2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one a typical chemical with most importance was used. More over, _L-ascorbic acid trisodium citrate anhydrous, potassium tetrachloroplatinate, collagenase type II, perchloric acid 60%, hydroxyphenyl fluorescein and hydrogen peroxides 30% were overall used in that experiment.

PLATINUM NANOPARTICLES PREPARATION

Many methods of Zhang et al. had been adopted to perform that experiment with little or more modifications. For that purpose these particles are cut in to many sizes. All this happened by reacting with K₂PtCl₄ by the  main chemical ingredients such as citric acid and ascorbic acid (Chang et al. 2006). We wee supposed to get 025mM concentration of K₂PtCl₄ for that reason we mixed it with ultrapure water and it was allowed to be stirred at room temperature. 30 nm  size of Pt particles was obtained when different concentration of ascorbic and citric acid were inserted. It was kept stirred for 5 minutes and left unless it becomes dark. It was analyzed by using microparticle analyzer. The result that we obtained showed that their size could be 106 nm or it could be 30 nm small. Pt particle diameter ranges between 20-200 nm and this is also proved by using Transmission electron microscope.

SUPEROIDE ANIONS SCAVENGING

Its scavenging was also addressed in previous modifications of Zhang (Hakozaki et al. 2008). To achieve scavenging 1000 micro-M hydrogen peroxide is required in which Pt-NPs were added to a glucose solution with 5% concentration. During this mix up platinum concentration was varying. This mixture was stirred at 37 degrees and then it was allowed to react with BES-H2O2 and the final concentration was 4.4 µM. After that measure the fluorescence intensity at emission wavelength of 535 nm and 485 nm as well by using spectrofluorometer.

HYDROXYL RADICALS SCAVENGING

The scavenging methodology of these are quite different from previous as it uses the methods of Setsukinai et al in which certain modifications were also present (Setsukinai et al. 2003).  A probe of hydroxyl radicals HPF was dissolved in a glucose solution ( 5%) where hydrogen peroxide was also present and its concentration was 890µM and the overall final conc. Becomes 3,0 µM. Like previously described Pt conc. Varies and this mixture was stirred at room temperature for 10 minutes in UV radiations and fluorescence intensity was measured.

TISSUE DISTRIBUTION IN MICE

50µg platinum was injected per kg in the tail of the mice. After blood sample was collected from vena cava the mice was killed and the blood was preserved using heparin sulfate and by using centrifugation plasma was collected. Many important body parts were removed and was rinsed out using saline and by the use of lyophilization plasma was dried. Dried tissues and plasma under the presence of HNO₃, H₂O₂ and HClO₄ was heated at 200 degrees in an 50mL beaker. The residue was then heated with aqua regia at 120 degrees, which further dissolved in nitric acid (7%) in 10 ml. Inductively coupled-mass spectrometer was used to measure the concentration of platinum (Yasuno et al. 2011).

HEPATIC CELLULAR LOCALIZATION

Collagenase perfusion methods are used to separate parenchymal and non-parenchymal cells of liver of mice (Managit et al. 2003). Mice was injected with Pt-NPs in the tail by using pentobarbital sodium and 37 degrees temperature was maintained by burning a lamp. After that Mg²⁺ and Ca²⁺-free perfusion buffer was added to perfuse the liver. After perfusion was started it is mandatory to maintain perfusion rate at 2 ml per min by cutting vena cava when perfusion is completed capsular membranes are removed then it was stirred with Hank’s HEPES buffer that contains 0.1% BSA. Centrifugation at 50 rpm took place then and parenchyma cells are washed twice with meshed cotton.  In supernatant non-parenchyma cells were present and they were also washed twice by Hank’s HEPES buffer. Cell numbers was also determined and platinum concentration was calculated by using ICP-MS.

HEPATIC ISCHEMIA

In order to induce hepatic ischemia, hepatic artery and portal vein were occluded for 15 minutes in vascular clamp by the incision in abdomen (Katsumi et al. 2008 and 2009) (Singal et al 2011). Then reperfusion was allowed i.e. blood reflow for this to happen 50µg per kg platinum dose was given through tail vein. After reperfusion again blood was collected  and its plasma was driven off through vena cava. Then liver was rinsed with saline. By using thiobarbituric acid method lipid peroxide level was determined in liver (Yagi et al. 1976).

RESULTS OF EXPERIMENT

ROS SCAVENGING BY PLATINUM NANOPARTICLES

In the following diagram effects of Pt-NPs are being shown on the concentrations of hydrogen peroxides, superoxide anions and hydroxyl radicals that are dissolved/ present in glucose solution (5%).  Their level is continuously reduced when small or large Pt-NPs are added to them. This reduction took place in concentration-dependent manner. An interesting fact that revealed was that, with an addition of small amount of Pt-NPs a clear reduction in ROS level was shown. Similarly, its opposite happened when large amount or concentration was added to these ROS. But hydrogen peroxide was not much affected with large concentration of these nanoparticles and it maintained its level.

DISTRIBUTION OF Pt-NPs

The distribution of these nanoparticles is shown in a time course it also explains whether its distribution is in large or small concentration. At this course of time their location is in plasma or in major organs of the mice after we had injected it with them. Shockingly we observe that these nanoparticles will disappear either they are small or large, from the plasma and platinum will accumulate in the liver in just 10 minutes and its concentration will be 80-100%.  Accumulation is not the same in each of the vital body part of mice i.e. large Pt particles are present in spleen whereas no such particles were found in heart or kidney of the mice.

 

 In the next figure we would be able to see hepatic cellular localization that suddenly taken place after the mice was being injected intravenously. This could take up to 30 minutes. Non parenchyma cells can accumulate these nanoparticles with a ratio of 14.0 and 21.4 for the PC/NPC respectively.

 

USE OF Pt-NPs TO PREVENT HEPATIC REPERFUSION INJURY

ALT and AST, that are present in mice , activity has been shown in the following diagram that was observed in injected mice. These were taken under study for 6 hour and an increase in the reperfusion activity of AST and ALT  was observed in injected mice from 100 to 650 IU ml^-1. But when we studied the normal mice i.e. untreated mice increase was many times less as it ranges from 15 to 290 IU ml^-1. These nanoparticles played a vital role to prevent elevation in activities. Small Pt-NPs were more effective in reducing ALT and AST activity levels. Large Pt-NPs had a bit less effect on AST activity. It was of no wonder that both of these small and large particles did not have any statistical effect on their activities.

 

 

EFFECT ON LIPID PEROXIDE AFTER REPERFUSION

Lipid content of treated and untreated mice with hepatic reperfusion is shown in the following diagram. It was seen that after 6 hour of reperfusion, there was a raise in lipid content present in the liver by 1.8 times. These nanoparticles was proved effective to reduce lipid content. By using small Pt-NPs we can reach at statistical significance of this reduction (Owen et al. 2010).

DISCUSSION

Nanoparticles are now being used in drug and medicine delivery (Akiyama et al. 2012). In this field gold nanoparticles are most recent in use for drug delivery (Jung et al. 2013).  ROS scavenging is also involved in suppression of cellular toxicity by using yttrium and cerium nanoparticles (Schubert et al. 2006). These particles are supposed to be the basic ingredient in food after being approved from food ministry of Japan (Onizawa et al. 2009). ROS-mediated diseases are also suppressed by these nanoparticles and these Pt-NPs have very high rate of ROS-scavenging activities (Yoshihisa et al. 2010). A lot of work has to be done to improve tissue distribution more effectively. Here we have studied its pharmacokinetics approaches and how they can treat reperfusion injury (Bowie et al. 2000).

We have seen the scavenging activity of ROS using these nanoparticles, small Pt-NPs show more activity. This scavenging is due to presence of electron on the surface of particles and are readily available for transformation (Kajita et al. 2007). Small nanoparticles have more catalytic activity due to more surface area and are more involved in ROS scavenging (Park et al. 2001) (Sun et al. 2000). Kupffer cells of ROS was generated and accumulated by the activation of NADPH oxidase activation in case of hepatic reperfusion that causes the activation of neutrophils. Tis activated neutrophil cab bind to endothelial cell and can generate ROS such as collagenase (Jaeschke et al 1991).

In that situation ROS scavenger should be deliver to the non-parenchyma cells of the liver to avoid  hepatic injury, such as Kupffer cells (Yabe et sl. 2001). Reticuloendothelial system can easily trap these micro or nanoparticles (Takino et al. 1994).  Particles size is supposed to be increased when it is taken by macrophages (Chono et al. 2007). We know intravenous injection accumulate these particles in liver. Larger particles are accumulated in greater number as compared to smaller one. Membrane lipid peroxidation is induced by ROS in Hepatic reperfusion (Nguyen et al. 1999). Detoxification mechanism by use cascade way is used to GSH to GSSH which is neutralized by ROS (Owen et al. 2010). ROS generation is maintained by the ratio of GSSH, that has been reduced from GSH and lipid peroxide content.  Many transcription factors have been used in this technology to treat this hepatic ischemia.. These particles are used to prevent inflammatory-related cellular events and oxidative stress. All this happens by the prevention of transcription factor NF-_KB in liver. Gene expression play a regulatory role, such as  cell adhesion molecules in neutrophils and endothelial cells (Li et al. 19990 (Sun et al. 2012).

 

CITATION TO ARTICLE

“Pharmacokinetics and preventive effects of platinum

nanoparticles as reactive oxygen species scavengers

on hepatic ischemia/reperfusion injury in mice”

(Katsumi, 2014)