Antimicrobial activity of the PEGylated antibiotic enrofloxacin and its functional and structural effect on the liver in rats

strain from microbial culture collection: Escherichia coli ATCC 11105 and Staphylococcus aureus ATCC 6538P. ItwasfoundthattheMICofenrofloxacinagainst


INTRODUCTION
Fluoroquinolones are considered to be the most promising group of antibacterial drugs due to a wide range of activity against a number of Gram-negative and Gram-positive bacteria (Wright et al., 2000). Enrofloxacin is an antibiotic agent in the fluoroquinoloneclassusedtotreatinfectiousdiseasesoftheurinary tract, pyelonephritis, sexually transmitted diseases, prostatitis, and skin and tissue infections (Tarushi et al., 2010). However, many experts point to increased development of microbial resistance to enrofloxacin (Cohn et al., 2003;Cummings et al., 2014 ;Pereira et al., 2014).Inaddition,theantibioticenrofloxacinissparingly soluble in water, is hygroscopic, and has a bitter taste which limits its use (Hewitt et al., 2009). Therefore, it is important to search for newenrofloxacincompoundswithimprovedtherapeuticefficacy andminimalsideeffects (Dron et al., 2018). Polyethylene glycol (PEG) as a carrier of the active substance is promising for the development of new antibiotic compounds (Вruсе,2001). PEG is water-soluble, biodegradable, and biocompatible because it does not form toxic metabolites and is commercially available (Mozar and Chowdhury, 2018;Wanget al., 2018). The process of chemical attachment of PEG to a native drug molecule is called PEGylation. PEGylation is one of the most successful ways to improve drug delivery to the cell (Barry, 2007;Nikitin et al., 2005). In addition, PEG is a hydrophilic polymer that promotes resistance to plasma protein binding and inhibits aggregation caused by salts and serum *Corresponding Author Mariya Kozak, Institute of Animal Biology of National Academy of Agrarian Sciences of Ukraine, Lviv, Ukraine. E-mail: mariyarkozak @ gmail.com proteins. Due to this, PEGylated peptides are more protected from opsonization and active phago-and endocytosis of cellular structures of the macroorganism (Avgoustakis, 2004;Otsuka et al., 2003). An important step in preclinical studies is toxicity testing (Chekh et al., 2017;Kozak et al., 2020). Among therapeutic agents, antibacterialdrugshavethemostpronouncedsideeffects,which limit their use (Kovtun et al., 2011). One of the manifestations of the side effects of antibiotics is their hepatotoxicity (Polson, 2007).
The aim of the study was to PEGylate the antibiotic enrofloxacin,investigateitsantimicrobialactivityin vitro, evaluate the effect of intramuscular administration to laboratory rats of PEGylated enrofloxacin on the functional state and structure of the liver, and to assess hepatotoxicity.

PEGylation
PEGylation was conducted in two steps. In the first step, thionyl chloride in dimethylformamide (DMFA) was used toobtainenrofloxacinchloridebyinfluenceonthecarboxylgroup in the composition. The reaction was carried out for 12 hours at 45℃-50°C. In the second step, the solution of enrofloxacin chloride was added dropwise to a water-dried solution of PEG-400for3hoursat20℃-25°C.Thereactionschemeisshownin Figure 1.
In the first step, a 1.3-1.35 molar excess of thionyl chloride was used. For use in the second step, the acid chloride was not isolated from the solution; only partially (not more than half) was the solvent evaporated in vacuo to evacuate hydrochloric acid and sulfur dioxide from the solution. The second step was carriedoutina2.3-2.5molarexcessofacidchloride(basedon loaded enrofloxacin). To isolate the product from the reaction mass in a vacuum, DMFA was removed, and the resulting viscous mixture was dispersed in a 5% soda solution. The dispersion product was extracted with methyl ethyl ketone. The methyl ethyl ketone was evaporated from the extract; the product was redispersed in water and reextracted with dimethyl ketone. The last procedure was repeated twice. After the last extraction and evaporation of methyl ethyl ketone, a yellowish paste-like mass oftheproductwasobtained.Thecontentandbindingefficiencyof PEG-400toenrofloxacinweremeasuredusinghigh-performance liquid chromatography (HPLC).

Chromatography
Chromatography of the test products was carried out on aWatersliquidchromatographwithanAlliance2690separation moduleandaWaters996diodearraydetector.LunaC18,250× 4.6mm,5μmwasusedasachromatographiccolumn.Themobile phase was a mixture of acetonitrile and 0.2% phosphoric acid in a ratio of 1:1. Enrofloxacin was detected (Fig. 2) at 280 nm (Anacleto, 2018). The flow rate of the mobile phase was 1 ml/ minute; the injection volume was 10 μl. Standard solutions of enrofloxacin (concentration100 µg/ml) were prepared using the mobilephaseasasolvent.Samplesofpolymer+enrofloxacinwere diluted with the same solvent to the same concentration.

Hepatotoxicity
Three-month-old healthy male Wistar rats weighing 180-200gwererandomlydividedinto4groups,1controland3 experimental, 12 animals in each group. All rats were given free access to a normal diet and water.
The rats of the control group were injected intramuscularly with saline in a volume of 0.03 ml. Rats of the first experimental group were injected intramuscularly with enrofloxacininavolumeof0.03ml,thesecondgroupreceived 0.03 ml of PEG-400, and the third received 0.03 ml of PEGylated enrofloxacin. The dose of the antibiotic enrofloxacin (EMEA, 1995)inthefirstandthirdexperimentalgroupswas2.7mg/kgof body weight of rat per day. The dose of PEG-400 in the second and third experimental groups was 1.5 mg/kg of body weight of rat per day. The drugs were administered daily for a period of 4 days. Animals were removed from the experiment on days 7, 14, and 21 after drug administration by an overdose of thiopental anesthesia (25 mg/kg). Blood was collected from rats during anesthesia, and the liver tissue was collected immediately after death.

Histology
The liver specimens were placed in a formaldehyde solution(liquidfixingagent).Subsequently,atypicaldehydration sequence in ethanol and clearing in xylene was carried out (Vlizlo, 2012).Tissuesweretheninfiltratedwithhistologicalwax andformedintoblocks.Thinsections(6µm)oftheliverswere mounted on glass slides and stained by Romanowsky-Giemsa's method to investigate normal and abnormal structures.

Ethical Conduct in the Care and Use of Animals
Three-month-old healthy male Wistar rats weighing 180-200gwerekindlyprovidedbytheStateScientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives.All animals were maintained in a specific pathogenfree animal facility with water and commercial food provided ad libitum. The protocol for animal experiments was approved by the Ethical Committee of the Institute of Animal Biology of NAAS of Ukraine, and the experiments were carried out in accordance with the European Convention for the Protection of Vertebrate Animals (Strasbourg, 1986), the principles of humanity set out in the European Union Directive (Directive 2010/63/EU), and the "General Ethical Principles of Animal Experiments," adopted by the First National Congress on Bioethics (Kyiv, 2001).

Statistical Analysis
Statisticalcalculationsofresults(М±m)werecarried out using Microsoft Excel 2007. The probability of differences was determined by Student's t-test with p < 0.05 accepted as statisticallysignificant.
Inthiscase,abifil-typemacromoleculewasmodeled, which was able to form a self-stabilized dispersion with particles of the nanometric size of the dispersed phase in aqueous solutions. Stabilization of the particles in the aqueous medium is due to the formation of a structural-mechanical barrier of hydrated polyoxyethylene chains around the nucleus, which contains the antibiotic. HPLC showed that the purity of the PEGylated enrofloxacinwas98%-99%.
PEGylated antibiotic enrofloxacin has good solubility in water and is stable. Other scientists have also pointed out the positiveeffectofPEGylationonstability,solubilityinbodyfluids, and increasing of the half-life (Chen еt al., 2008). PEGylation  facilitatestheeffectivenessofdrugdeliverytotheaffectedorgans, as well as minimizing the toxic effects on the body (Rafiei and Haddadi, 2017). Covalently linked enrofloxacin to PEG has the highest antibacterial activity. PEG has a positive impact on the cell membrane permeability, thereby increasing the uptake of the antibacterial drug by cells (Chakrabarty et al., 2008;Dron et al., 2018).

Study of Antimicrobial Action of "Pure" Substance and PEGylated Enrofloxacin
It was found that the MIC of enrofloxacin for E. coli ATCC 11105 was 0.31 μg/ml (0.86 μM/l) and the MIC of the PEGylated enrofloxacin 0.16 μg/ml (0.44 μM/l) (

Analysis of the Hepatotoxicity of Drugs
Animal studies indicated the absence of visible physiological changes in both the control and experimental groups. All rats had an appetite, and the dynamics of weight gain did not differ between the groups. However, morphological and functional changes in the organs caused by the drug are usually asymptomatic or subclinical (Babak, 2008). Hepatotoxicity is one of the most common side effects associated with the use of drugs (Chernushkin еt al., 2020;Polson, 2007). Liver function bloodtestsarerecommendedtomonitorthesideeffectsofcertain medications known to affect the liver (Ramadori and Cameron, 2010). One of the markers of hepatic cytolysis is increased activity of the liver enzyme (Simonov and Vlizlo, 2015). ALT activity in the serum of rats gradually decreased during the experiment in all the groups (Fig. 4).Whencomparingthelevelsoftheenzymeinthe serumextractedfromtheratsofthedifferentgroups,itwasfound thatALTactivitywassignificantlylowerinthegroupinjectedwith PEGylatedenrofloxacinthaninthecontrolgroupandintheother experimental groups (p <0.01-0.001).ALTactivityintheblood oftheratsinjectedwiththeantibioticenrofloxacinremainedhigh over three weeks, which may indicate liver cell damage.
AST activity in the serum taken from the rats during the experiment was higher in the experimental groups (p < 0.001) compared to that of the control group (Fig. 5). High levels of AST in the blood of the rats injected with the test substances may indicate their active penetration into cells and mitochondria, where this enzyme has high activity. Additionally, due to its branched molecular structure, PEG may slow down the active metabolism ofthedrug,increasingthecirculationtimeofenrofloxacininthe blood (Вruсе, 2001;Kozlowski and Harris, 2001). This may be the reason for elevated AST levels in the blood of the rats injected withPEGylatedenrofloxacinandPEG-400.Ondays14and21, AST activity in the blood of the rats injected with PEGylated enrofloxacindecreasedascomparedwiththoseinjectedwiththe traditionalantibioticenrofloxacin(p < 0.001; 0.05).   According to the dynamics of ALT and AST activity in the blood of animals, it can be concluded that the structure restoration of the liver cells appeared to be faster in the rats injected withPEGylatedenrofloxacinascomparedtotheratsinjectedwith traditionalenrofloxacin.
Seven days after the last administration of drugs, the GGTP activity in the serum of the rats from all the experimental groups was higher (p < 0.05; 0.001) than in the control group (Fig.  6). High GGTP activity in the blood may indicate intrahepatic cholestasis and damage to the liver cells that form intrahepatic bile ducts (Simonov and Vlizlo, 2015). Fourteen days after the end of the administration of the test substances, GGTP activities in the serum collected from the rats injected with the traditional antibiotic enrofloxacin and PEGylated enrofloxacin were higher than in the control group (p < 0.01 and <0.001, respectively). Three weeks after the last administration, blood GGTP activity in theratsinjectedwiththePEGylatedenrofloxacinwaslowerthan in both the control group (p < 0.001) and the first experimental group,whichwasinjectedwithtraditionalenrofloxacin(p < 0.05).
On day 7 after the end of the administration of the test drugs, alkaline phosphatase activity was higher in the blood of the control group (p < 0.001) than in all three experimental groups (Fig. 7). In the next experimental periods (days 14 and 21), blood alkaline phosphatase activity in the rats treated with the traditional antibiotic enrofloxacin and PEG-400 was higher than in the rats from these two groups: control (p < 0.001) and the third experimental group, which was treated with PEGylated enrofloxacin.
Cholesterolcontentinthebloodoftheratsdifferedlittle between all groups (Fig. 8). Cholesterol levels were elevated in the ratsinjectedintramuscularlywithPEGylatedenrofloxacin,which can be considered as the stability of its synthesis by liver cells (Janičkoet al., 2013).
Three weeks after treatment, albumin content in the bloodoftheratswas41.5−54.5g/linthecontrolandexperimental groupsanddidnotdifferbetweeneither (Fig. 9). The high albumin concentration in the blood indicated the stability of the proteinsynthesizing function of the liver (Carvalho and Machado, 2018).   The total protein content in the serum collected from the rats differedlittlebetweenallthegroups (Fig. 10).
On the seventh day after the end of treatment, microscopic examination was conducted on the liver tissue from the rats injected with the traditional antibiotic enrofloxacin. The examination showed signs of granular dystrophy, lysis, and nuclear pyknosis. There were foci characteristic of paranecrosis and necrosis of liver parenchyma (Fig. 11). The livers of the rats treated with PEG-400 did not display any histological changes, but the cytoplasm of some cells was foamy and granular on days 7, 14, and 21. On the seventh day of the experiment, in the livers of rats treated with PEGylated enrofloxacin,itwasfoundthatindividuallivercellswereshowing signs of atrophy, granular dystrophy, paranecrosis, and necrosis. On days 14 and 21 of the experiment, the liver morphology was unchanged and identical to the control group.

CONFLICT OF INTERESTS
The authors declared that they have no conflicts of interest.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

FUNDING
There is no funding to report.

DATA AVAILABILITY
All data generated and analyzed are included within this research article.

PUBLISHER'S NOTE
This journal remains neutral with regard to jurisdictional claimsinpublishedinstitutionalaffiliation.
The protocol for animal experiments was approved by the Ethical Committee of Institute of Animal Biology of NAAS of Ukraine.