research article superoxide dismutase 1 nanozyme for...

Research ArticleSuperoxide Dismutase 1 Nanozyme for Treatment ofEye Inflammation

Olga A Kost1 Olga V Beznos2 Nina G Davydova2 Devika S Manickam3

Irina I Nikolskaya1 Anna E Guller45 Petr V Binevski1 Natalia B Chesnokova2

Anatoly B Shekhter5 Natalia L Klyachko136 and Alexander V Kabanov13

1Chemistry Faculty MV Lomonosov Moscow State University Moscow 119991 Russia2Helmholtz Institute for Eye Disease Moscow 105062 Russia3UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill NC 27599-7362 USA4Macquarie University Sydney NSW 2109 Australia5Research Institute for Molecular Medicine IM Sechenov First Moscow State Medical University Moscow 119992 Russia6Pirogov Russian National Research Medical University Moscow 117997 Russia

Correspondence should be addressed to Olga A Kost olgaakostgmailcom and Alexander V Kabanov skabanovmecom

Received 9 April 2015 Revised 6 August 2015 Accepted 12 August 2015

Academic Editor Felipe Simon

Copyright copy 2016 Olga A Kost et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential This workexamines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme superoxide dismutase 1 (SOD1) termedldquonanozymerdquo The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer poly(L-lysine)-poly(ethyleneglycol) followed by covalent cross-linking of the complexes with 331015840-dithiobis(sulfosuccinimidylpropionate) sodiumsalt The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined invivo in rabbits with immunogenic uveitis Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity however the nanozyme was much more effective compared to the free enzyme in decreasing uveitismanifestations In particular we noted statistically significant differences in such inflammatory signs in the eye as the intensities ofcorneal and iris edema hyperemia of conjunctiva lens opacity fibrin clots and the protein content in aqueous humor Clinicalfindings were confirmed by histological data Thus SOD1-containing nanozyme is potentially useful therapeutic agent for thetreatment of ocular inflammatory disorders

1 Introduction

Uveitis is an inflammatory disease of the uvea a section ofthe eye which consists of the middle pigmented vascularstructures of the eye and includes the iris ciliary body andchoroid Common causes of uveitis include infections mul-tisystem disorders such as sarcoidosis and Behcetrsquos diseaseand autoimmune disorders such as rheumatoid arthritis orankylosing spondylitis [1ndash4] Uveitis is a severe sight threat-ening disease frequently leading to vision loss and blindnesswith retinal vasculitis retinal detachment and glaucomaUveitis accounts for 5ndash20 of legal blindness in United Statesand in Europe and perhaps as much as 25 of blindness

in the developing world [1] Severe cases of uveitis need tobe treated aggressively to prevent damage caused by chronicinflammation Corticosteroids constitute the first line oftherapy for patients with noninfectious ocular inflammatorydisease However as the use of corticosteroids became moreprevalent in treating ocular inflammation the side effects ofthis treatment became more prevalent as well Another classof compounds known as ldquoimmunosuppressive drugsrdquo suchas cyclosporine A was found to be successful in treatinguveitis However such treatment is also complicated by sideeffects associated with immunosuppression [5ndash7] Patientswho cannot take medications because of the side effects orpatients who are not responsive to the existing medications

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5194239 13 pageshttpdxdoiorg10115520165194239

2 Oxidative Medicine and Cellular Longevity

experience unavoidable impaired visual function Thus itis important to investigate alternative approaches for thetreatment of uveitis

Inflammatory diseases including ocular ones are accom-panied by excessive production of reactive oxygen species(ROS) and by depletion of endogenous antioxidants Antiox-idant enzymes superoxide dismutase 1 (SOD1 also knownas CuZn SOD) catalase and glutathione peroxidase areknown to be very effective scavengers of ROSThese enzymeswere shown to be effective in the treatment of various eyediseases associated with oxidative stress Thus SOD1 wasused for the treatment of lens-induced and bovine albumin-induced uveitis in rabbits [8 9] as well as for the treatmentof acute corneal inflammation in animals induced by sodiumhydroxide [10 11] Both SOD1 and glutathione peroxidasewere employed for the treatment of severe experimentalallergic uveitis induced by retinal S antigen in rats [12] whilepoly(ethylene glycol)- (PEG-) modified catalase and PEG-SOD were employed for the treatment of the same type ofuveitis in guinea pigs [13] We have shown recently [14] thatSOD1 instillations may help to reduce clinical presentationsof immunogenic uveitis in rabbits

Eye diseases are most commonly and preferably treatedby topical instillations of eye drops These formulationsface technical and clinical problems such as solubility ofthe components and instability of drug solutions limitedefficacy and limited cornealsclera permeability and localand systemic toxicity Moreover 2min after instillation themajor part of the topical drug solution is eliminated via thenasolacrimal drainage system limiting ocular penetration ofthe drug to less than 5 of the administered dose [15]

Nanoparticles are colloidal drug carrier systems that canimprove the efficacy of drug delivery into the eye by overcom-ing cornealsclera diffusion barrier Drug loaded polymericnanoparticles offer several favorable biological propertiessuch as biocompatibility and mucoadhesiveness enhancingbioavailability without blurring the vision The use of drug-containing nanoparticles can decrease the dose of the drugand diminish side effects So nanoparticles are a promisingdrug delivery system which fulfills the requirements forophthalmic application (for reviews see [16 17])

Recently new formulations of antioxidant enzymesSOD1 and catalase were prepared by electrostatic couplingof these negatively charged enzymes (pI values are 495 and58 for SOD1 and catalase resp) with cationic block copoly-mers such as methoxy-PEG-block-poly(L-lysine hydrochlo-ride) block copolymer (PEG-pLL

50) followed by covalent

cross-linking to stabilize nanoparticles Catalytic nanopar-ticles based on polyion complexes of enzymes with blockcopolymers of opposite charge were termed ldquonanozymesrdquo[18ndash22] Spontaneous self-assembly of oppositely chargedproteins and polymers results in stoichiometric complexeswith 100 loading efficiency These nanozymes were shownto be prospective agents for the treatment of various diseasesof the central nervous system due to prolonged ability toscavenge experimentally induced ROS in cultured brainmicrovessel endothelial cells and central neurons increasedstability in both blood and brain enhanced penetrationthrough the blood-brain barrier and therefore increased

accumulation in brain tissues in comparisonwith non-cross-linked complexes and native enzyme [18 21 22]

In the current study we demonstrate the advantages oftopical instillations of superoxide dismutase 1 in the formof ldquonanozymerdquo in the treatment of ocular inflammation in arabbit model of immunogenic uveitis

2 Methods21 Preparation of and Characteristics of Nanozyme SOD1nanozyme was synthesized by self-assembly of recombinantSOD1 (ldquoEnzyme Technologiesrdquo St Petersburg Russia) withcationic block copolymer PEG-pLL

50(MW 13 kDa poly-

dispersity index 109 Alamanda Polymers Huntsville AL)in aqueous solution followed by cross-linking with 331015840-dithiobis(sulfosuccinimidylpropionate) (DTSSP) as in [21]Unreacted cross-linker was desalted using NAP-25 columnand cross-linked nanozymes were purified using a 100 kDaMWCO filter Purified particles were then lyophilized from005M Hepes-buffer pH 75 containing 015M NaCl andstored at minus20∘C For further experiments precalculatedquantity of lyophilized nanozyme was dissolved in deion-ized water and gently vortexed for 2min until sample dis-solved completely Intensity-mean z-averaged particle diam-eter (effective diameter) polydispersity index (PDI) and 120577-potential were measured after filtration via a 02 120583m filterusing a Zetasizer Nano ZS (Malvern Instruments Ltd MA)Aliquots of nanozyme solution required for daily experi-ments were then frozen and kept at minus20∘C

22 Enzyme Activity SOD1 activity was determined usingSOD1 ability to inhibit autooxidation of quercetin as in [23]with detection kit (Belarusian State University Belarus) Theexperimental sample in phosphate buffer pH 78 contain-ing 008M EDTA 0125 (vv) TEMED was mixed withquercetin solution in DMSO The absorbance was measuredat 406 nm immediately after addition of quercetin (119863

0) and

after 20min (11986320) In control phosphate buffer was used

instead of the sample and the absorbances 119888119863

0and 119888119863

20

were measured correspondinglyThe percent of inhibition ofquercetin autoxidation by SOD1 in experimental samples wascalculated by the formula [(119888

119863

0minus

119888

119863

20) minus (119863

0minus119863

20)](

119888119863

0minus

119888

119863

20) times 100 One unit of SOD1 activity was defined as the

amount of SOD1 which inhibits the quercetin autoxidationby 50 The protein content was determined using MicroBCA Protein Assay Kit (Pierce Rockford IL)

23 In Vitro Drug Release Study Onemg of SOD1 nanozymewas dispersed in 05mL of PBS transferred to 100 kDaMWCO membrane and centrifuged for 5min at 1200timesgSupernatant (about 005mL) was diluted by PBS to theinitial volume and centrifuged againThe ldquofiltration-dilutionrdquoprocedure was repeated 5 times SOD1 activity and proteinconcentration were measured in the initial nanozyme solu-tion in each filtrate and in the final supernatant In anotherseries of experiments equal amounts of freshly dissolvednanozyme in PBS were incubated at room temperature fordifferent time periods Then the solutions were filtered

Oxidative Medicine and Cellular Longevity 3

through 100 kDamembrane and SOD1 activity in the filtrateswas determined

24 Animals A randomized and double-blinded study wasconducted using adult Chinchilla rabbits weighing 20ndash25 kg All experiments with live rabbits were carried outin strict accordance with the Association for Research inVision and Ophthalmology (ARVO) statement for the Use ofAnimals in Ophthalmic and Vision Research The protocolwas approved by the Committee on the Ethics of AnimalExperiments of the Helmholtz Institute for Eye Disease(Permit number 222) All efforts were made to minimizerabbits suffering After the end of experiments the rabbitswere sacrificed by lethal pentobarbital injection

25 In Vivo Studies Immunogenic uveitis was induced asdescribed in [24] Briefly rabbits were initially injectedsubcutaneously with 5mL of normal horse serum for sensiti-zation Ten days later 5 anesthetic Alcain (Alcon Belgium)was instilled into each eye before the intravitreal injections of70 120583L of the same serum in the eyes to induce acute uveitis

Rabbits received 30 120583L of the drug solutions as eye dropstopically in each eye three times a day for 14 days Threeindependent series of experiments for clinical estimationof uveitis were performed In each series animals wererandomly divided into 4 groups (119899 = 5 per each group ie10 eyes) and treated as follows (1) control (healthy) groupwithout uveitis and (2) placebo group with uveitis received10mM Hepes buffer pH 74 containing 015M NaCl (3)SOD1 group with uveitis received 1mgmL SOD1 solutionin the same buffer (4) treatment (nanozyme) group withuveitis received 8ndash10mgmL SOD1 nanozyme solution in thesame buffer Hepes buffer did not cause any irritation of theeye SOD1 dose was chosen in accordance with what wasrecommended in [25] The dose of nanozyme solution wascalculated based on enzymatic activity of nanozyme (unitspermg) so that the activities of SOD1 andnanozyme solutionswere equal that is 8ndash10mg nanozyme corresponded to 1mgSOD1 by specific activity Eyes were examined in a double-blinded trial by indirect ophthalmoscopy using a slit lamp(Zeiss slit lamp 30SL USA) Clinical symptoms of uveitisincluding eyelid and conjunctival edema and hyperemiacorneal edema and neovascularization iris edema and hyper-emia fibrin clots and precipitates on the iris and on thelens lens opacity presence of synechiae (cohesions betweenthe pupillary margin of iris and anterior part of the lens)which lead to immobilization of the pupil and presence ofpurulent exudate (hypopyon) and blood (hyphema) in theanterior chamber of the eye were estimated Evaluation ofinflammation scores was performed using a conventionalscale (0) no symptom (1) low degree of manifestation (2)medium (3) strong

26 Analyses of Aqueous Humor Aqueous humor (intraocu-lar fluid) from anterior chamber of the eye was collected byparacentesis in limb area under topical anesthesia on day 8of uveitis (that is 16 h after last instillations of the drugs) andon day 4 in the separate experiment on two rabbits (4 eyes)in each group The samples were centrifuged at 21000timesg

for 10min and the supernatant was stored at minus20∘C Theamount of leukocytes was determined microscopically The120572

2-macroglobulin content (in arbitrary units) was estimated

indirectly as previously described [26 27] based on theability of the complex of 120572

2-macroglobulin with trypsin to

react with benzoyl-L-arginine-p-nitroanilide as a substrateAntioxidant activity was determined by chemiluminescencekinetics in hemoglobin-H

2O2-luminol system as described in

[28] with Trolox a water-soluble analog of vitamin E as astandard antioxidant Antioxidant activity of the sample wasexpressed as trolox-equivalents calculated on the basis of atrolox standard curve

27 Histopathology Histopathological analysis was per-formed in a double-blinded fashion For this experimentwe used 10 rabbits (20 eyes) 9 rabbits with uveitis and 1healthy rabbit For the topical treatment rabbits with uveitiswere randomly divided into three groups The 1st group of 3rabbits received placebo as described above the 2nd group of3 rabbits received native SOD1 and the 3rd group of 3 rabbitsreceived SOD1 nanozyme with the same SOD1 activityOn day 4 rabbits were sacrificed by sodium pentobarbitalinjection (100mgkg) and the eyes were enucleated Sampleswere fixed in 10 neutral buffered formalin dehydrated ina graded series of alcohol embedded in paraffin and cutinto 4-5 120583m serial sections The sections were stained withhaematoxylin and eosin (HampE) and examined in uprightlight microscope (Olympus BX51) using dry-air (4NA01010NA025 20NA040) and oil-immersion (100NA125oil) objectives (Olympus Optical Tokyo Japan) Histologyimages were recorded in a single-frame mode using a digitalvideo camera SDU-252 (2048 times 1536 ldquoSpetsteletechnikardquoRussia) integrated into the microscope optical path

28 Statistical Analysis All data are means plusmn SEM Signif-icance was analyzed using the Mann-Whitney U test withSTATISTICA 6 (StatSoft Inc OK)

3 Results

31 Synthesis and Characterization of Nanozyme SOD1nanozymewas synthesized as described earlier [21] bymixingof aqueous solutions of SOD1 and block copolymer PEG-pLL50

at pH 74 followed by cross-linking and purificationSOD1 retained 100 its catalytic activity in polyion complexbefore cross-linking consistent with previous report [21 22]but partly lost activity as a result of cross-linking with DTSSPand filtering through 100 kDa membrane Altogether thelyophilized dry nanozyme samples displayed the specificactivity about 30 kUmg while the activity of the unmodifiedpure recombinant SOD1 was ca 250 kUmg The observeddecrease in the specific activity was mainly due to thepresence of the bulk of polymer in nanozyme as well asto the presence of buffer substance and salt in the finallyophilized preparation The DLS analysis revealed that theparticles of SOD1 nanozyme had an effective diameter of35 nm (compared to about 5 nm for native SOD1 as reportedin [21]) narrow particle size distribution (PDI ca 01) andnearly neutral (zero) 120577-potential


0

5

10

15

20

25

Initi

al ac

tivity

()

2 3 4 51Number of filtrations

(a)

0

5

10

15

20

25

Initi

al p

rote

in co

ncen

trat

ion

()


(b)

Figure 1 The release of SOD1 activity and protein upon subsequent dilutions and filtrations of nanozyme Nanozyme was dissolved indeionized water and immediately filtered through 100 kDaMWCO filter supernatant was diluted using PBS to the initial volume and filteredagain ldquoDilution-filtrationrdquo steps were repeated and SOD activity (a) and protein content (b) were determined in each filtrateThe experimentwas performed in duplicate

To examine whether SOD1 can be released from thenanozyme we determined the activity of SOD1 in the filtratesand supernatant (1) after repeated centrifugal filtration ofthe nanozyme solution using 100 kDa MWCO filters and(2) after centrifugal filtration of nanozyme aqueous solutionincubated for different periods of time In the first experi-ment the lyophilized nanozyme was dissolved in deionizedwater so that the final concentration of NaCl was 015M andimmediately filtered through 100 kDa membrane for 5minAfter this first filtration step about 24 of the protein andabout 27 of SOD1 activity (Figure 1) were found in thefiltrate Further dilutions of supernatant to the initial volumeand subsequent filtration resulted in additional release ofSOD1 activity and protein from the nanozyme albeit toa lesser extent than the initial filtration step from 2 to7 of the initial amount After 5 subsequent dilutions andfiltrations the nanozyme retained about 55ndash65 of bothSOD1 activity and protein It is interesting that the release ofSOD1 from the nanozyme occurs not only after the ldquodilution-filtration-dilutionrdquo procedures but also upon incubation of itsaqueous solution for various time periods after preparationSpecifically freshly prepared solution of 2mg nanozyme in05mL 015M NaCl contained about 25 to 30 free SOD1while after 2 hr incubation the same solution contained 40of the free SOD1 and 4 hr 40 to 50 of the free SOD1 Furtherincubation of the nanozyme solution did not result in thefurther release of the free SOD1 These data suggest that thenanozyme synthesis process and specific chemistries usedin this work produce nanozymes encapsulating significantportion of SOD1 that is not chemically coupled to the blockcopolymer and can be released in the surrounding media asit was observed previously [22]

32 Effects of Topical Instillations of SOD1 and SOD1Nanozyme on Clinical Manifestations of Immunogenic Uveitisin Rabbits We induced immunogenic uveitis in rabbits inthree independent series of experiments In one series theuveitis appeared to show severe manifestations of inflam-mation in the outer part of the eye (eyelid cornea andconjunctiva) while in the other two inflammation of thesetissues was rather moderate Manifestations of inflammationin the inner part of the anterior segment of the eye howeverwere significant in all three series of experiments It isnoteworthy that in all series uveitis developed similarly withthe most acute phase on days 3ndash5 and fading till the end ofsecond week

321 Rabbits without Treatment (Placebo) Three days pastintravitreal injection of horse serum the eyes of animalsshowed classical clinical symptoms of anterior uveitis whichintensified on day 4 Edema of the eyelid cornea andconjunctiva were observed Hyperemia of conjunctiva wassignificant Iris had both edema and hyperemia its structurewas changedTherewas a lot of fibrin clots in the anterior partof the eye which in several cases formed massive clouds Inmost eyes there were multiple synechiae which resulted inpupil immobilization improper pupil form and the lack ofreaction of pupil to light Fibrin clots were also found on thesurface of lens of all eyes lenses itself were characterized bysignificant opacity which thwartedmicroscopic investigationof the vitreous body Many animals (about half) had mas-sive purulent exudates (so-called hypopyon) in the anteriorcamera of the eye which was formed by leukocytes anddetritus Neovascularization of the cornea which is knownto be a result of oxidative stress [29] was observed in half


Day 5

PlaceboSODNanozyme

NS NS

NSNS

0

1

2

3

Scor

e

Day 8Day 3 Day 4

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 2The effect of instillations of nanozyme and SOD solutionsin the rabbit eye on conjunctival hyperemia at uveitis The datafrom three series of experiments were analyzed each experimentincluding 5 animals (10 eyes) in each group control placebo SOD1-treated and SOD1 nanozyme-treated group Thus 119899 = 30 foreach group The scores were estimated as a degree of manifestationof hyperemia of conjunctiva 0 no symptom 1 low degree ofmanifestation 2 medium degree 3 strong degree Symbols lowast thelevel of significance of differences by the Mann-Whitney U test119901 lt 005 lowastlowast the level of significance of differences by the Mann-Whitney U test 119901 lt 001 NS not significant differences

of the eyes Some animals exhibited symptoms of elevatedintraocular blood pressure (from 8th day) which indicatedthe development of common uveitis complication secondaryglaucoma

322 SOD1-Treated Rabbits The development of uveitis inthis group remarkably differed from that in placebo groupEyelid edema was much less pronounced and hypopyonwas absent at all times during the disease Corneal andiris edema were only local and diminished in time Con-junctival edema and hyperemia were less pronounced aswell Neovascularization of the cornea in the acute phase ofuveitis was observed in 20ndash30 of eyes During treatmentwe observed regress of synechiae formation (from day 4 today 8) and partial restoration of the reaction of the pupilto light At the end of the treatment however lens opacitydecreased insignificantly Many eyes retained precipitates onthe lens Figures 2 and 3 are representative examples of thecomparative effects of SOD1 and placebo instillations in therabbit eye on conjunctival hyperemia and formation of fibrinclots at different times during uveitis It was seen that whilethere was no statistical difference between the extents ofhyperemia of conjunctiva in the eyes of SOD-treated and

Day 2 Day 4 Day 7 Day 9

NSNSNS

NS

NS

NS

PlaceboSODNanozyme

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

0

1

2

3

Scor

eFigure 3The effect of instillations of nanozyme and SOD solutionsin the rabbit eye on the formation of fibrin clots at uveitis The datafrom three series of experiments were analyzed each experimentincluding 5 animals (10 eyes) in each group control placebo SOD1-treated and SOD1 nanozyme-treated group Thus 119899 = 30 for eachgroupThe scores were estimated as degree of fibrin clots formation0 no clots 1 low degree of clot formation 2 medium degree 3strong degree Symbols lowast the level of significance of differences bythe Mann-Whitney U test 119901 lt 005 lowastlowast the level of significanceof differences by the Mann-Whitney U test 119901 lt 001 NS notsignificant differences

placebo-treated eyes on day 3 later (on days 4 and 5) thisdifference became statistically significant and on day 8 thisdifference is remarkable (Figure 2) On the contrary therewas no difference between the amount of fibrin clots observedin SOD-treated and placebo-treated rabbits (Figure 3)

323 SOD1 Nanozyme-Treated Rabbits Clinical manifesta-tions of uveitis in this group were less pronounced andappeared later than that in placebo- and SOD1-treatedgroups Most importantly hyperemia of conjunctiva cornealedema iris edema and lens opacity were significantly lesspronounced than in the two other groups There were noeyes with neovascularization of the cornea in this groupSynechiae were lower by 20ndash25 which improved the pupilreaction to light Fibrin clots were less intense aswell Figure 2demonstrates the effect of SOD1 nanozyme instillations onthe conjunctival hyperemia in comparison with the effects ofinstillations of placebo and native SOD1 There was a clearstatistical difference (119901 lt 001) between SOD1 nanozymeand placebo groups at all times of the disease Moreovernanozyme was statistically more effective than native SOD1on day 3 (in acute phase of uveitis) The formation of fibrinclots in the case of nanozyme-treated groups was statistically


Table 1 Biochemical parameters of aqueous humor on day 8 of uveitis in rabbits

Biochemical parameter TreatmentControl Placebo SOD1 Nanozyme

Total protein concentration mgmL 22 plusmn 03 193 plusmn 32 156 plusmn 23 105 plusmn 11lowastlowast

120572

2-Macroglobulin arb UmL 06 plusmn 01 108 plusmn 10 87 plusmn 15 72 plusmn 16lowast

Antioxidant activity UmL 144 plusmn 10 23 plusmn 09 33 plusmn 06 37 plusmn 02SOD1 activity trolox-equivalentsmL 280 plusmn 40 460 plusmn 100 370 plusmn 80 330 plusmn 50lowastlowastSignificant difference between nanozyme and placebo by the Mann-Whitney U test 119901 lt 005 lowastlowastSignificant difference between nanozyme and placebo andnanozyme and native SOD1 by the Mann-Whitney U test 119901 lt 001

less pronounced (Figure 3) than in the placebo- and SOD1-treated groups at every time point of uveitis starting fromday4 Thus we demonstrated that nanozyme treatment resultedin the considerable improvement of uveitis condition inrabbits compared not only with the untreated animals butwith the native SOD1-treated group as well

33 Effects of Topical Instillations of SOD1 and SOD1Nanozyme on Clinical Symptoms of Uveitis in the Acute Phaseof the Disease Most clearly the differences in the effects oftopical instillations of SOD1 nanozyme SOD1 and placeboare seen in the acute phase of the disease that is on days3-4 We compared the efficacy of these treatments using thesums of the scores for the manifestations of inflammation inthe outer and inner parts of the anterior eye segment This isa common approach in ophthalmology to test drug efficacy[30] The manifestations of inflammation were assessed by(1) the eyelid edema corneal edema and hyperemia ofconjunctiva in the outer part and (2) the iris edema lensopacity and fibrin clots in the inner part The results areshown in Figures 4 and 5 While native SOD1 seems to havea healing effect on inflammation in the outer part of theeye this effect was not statistically significant In contrastSOD1 nanozyme showed statistically significant healing effect(Figure 4) in comparison with both placebo (119901 lt 001)and native SOD1 (119901 lt 005) The difference between SOD1formats was even more pronounced when we comparedtheir effect on the inflammation in the inner part of theanterior segment of the eye (Figure 5) While the healingeffect of native SOD1 was not statistically different from thatof placebo the SOD1 nanozyme showed remarkable healingeffect which was significantly distinct from the effects ofnative SOD1 and placebo (119901 lt 001 in both cases)

34 Effect of Treatments on the Leukocyte Counts and Bio-chemical Parameter of the Aqueous Humor of the Eye ROSmetabolites are predominantly produced by polymorphonu-clear leukocytes which migrate to inflamed tissues and canserve as an indication of the inflammation The aqueoushumors from the eyes of all rabbits with uveitis containedconsiderable amount of leukocytes both in the acute (day 4)and later (day 8 eg after 16 h after the last instillations ofthe drugs) phases of the disease On day 4 the SOD1-treatedgroup displayed approximately the same leukocyte countsas placebo group while SOD1 nanozyme-treated groupexhibited decrease in leukocyte counts although statistically

Median

Range

Placebo SOD Nanozyme

NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75

Figure 4 Comparison of the clinical symptoms of uveitis in externalrabbit eye structures as a sum of the scores for eyelid edemaconjunctival hyperemia and corneal edema in the acute phaseof the uveitis The data from three series of experiments wereanalyzed each experiment including 5 animals (10 eyes) in eachgroup control placebo SOD1-treated and SOD1 nanozyme-treatedgroup Thus 119899 = 30 for each group The scores were estimatedas degrees of manifestations of clinical symptoms of the disease 0no symptom 1 low degree of manifestation 2 medium degree 3strong degree Symbols lowast the level of significance of differences bythe Mann-Whitney U test 119901 lt 005 lowastlowast the level of significanceof differences by the Mann-Whitney U test 119901 lt 001 NS notsignificant differences

insignificant On day 8 both SOD-treated groups showedthe decrease in leukocyte counts SOD1 nanozyme-treatedgroup exhibiting statistically significant effect compared withplacebo (Figure 6)

Tissue inflammation is characterized by elevated totalprotein concentration in biological fluids along with theincrease in proteinase inhibitor 120572

2-macroglobulin and

decrease of overall antioxidant activity as well as increasein endogenous SOD1 [31ndash34] Therefore we determinedthese biochemical parameters in aqueous humor on day 8after the disease onset (Table 1) The protein concentration



NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75

Figure 5 Comparison of the clinical symptoms of uveitis in internalrabbit eye structures as a sumof the scores for iris edema fibrin clotsand lens opacity in the acute phase of the uveitisThe data from threeseries of experiments were analyzed each experiment including 5animals (10 eyes) in each group control placebo SOD1-treated andSOD1 nanozyme-treated group Thus 119899 = 30 for each group Thescores were estimated as in the legend of Figure 3 Symbols lowastlowast thelevel of significance of differences by the Mann-Whitney U test 119901 lt001 NS not significant differences

Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast

Figure 6 Leukocyte numbers in aqueous humor of the eye of uveitisrabbits as percentage from the value for control (healthy) rabbits ondifferent times of the disease 119899 = 4 in each group on day 8 119899 = 10in each group on day 8

in nontreated group increased 8-fold compared to healthyanimals and it was only slightly affected after the nativeSOD1 treatment In contrast SOD1 nanozyme treatmentresulted in nearly 2-fold decrease in this parameter Theeffect of nanozyme was significant compared to bothplacebo and native SOD1 (119901 lt 001) The development ofuveitis also resulted in drastic increase of 120572

2-macroglobulin

activity in aqueous humor however it was mitigated afterboth the native SOD1 and especially SOD1 nanozymetreatments (Table 1) Antioxidant activity increase in bothSOD1 and nanozyme-treated groups was rather small andnot significant (Table 1) The endogeneous SOD1 activityin aqueous humor of placebo-treated eyes with uveitis wasincreased by more than in 15 times compared to controlgroup suggesting a compensatory reaction of the eye to theuveitis-induced oxidative stress The native SOD1 treatmentsdecreased the enzyme activity in aqueous humor albeitnonsignificant while SOD1 nanozyme treatment decreasedthis parameter significantly (Table 1) Notably in the healthyand placebo-treated rabbits SOD1 activity in aqueous humorrepresents only the endogenous enzyme while in SOD1- andnanozyme-treated rabbits it may contain contributions ofexogenous SOD1 as well Still the decrease in the measuredSOD1 activity in treated eyes clearly shows that SOD1 andespecially nanozyme treatments decrease inflammationduring experimental uveitis

35 Histology Examination of the Disease Manifestation Theeyes of the control healthy rabbits were unchanged Thecornea displayed its common structure with thin multilayerepithelium on the outer side (cells form 2 or 3 layers) andsingle-layer endothelium on the inner side (Figure 7(a))The epithelium and endothelium cells had normal structurethe main part of the stroma could be clearly seen Theconjunctival tissue was loose moderately full-blooded Thevessels in the region of conjunction of cornea conjunctivaand sclera were wide and full-blooded as well The sclera andciliary body (Figure 8(a)) possessed their normal structureas well The retina in the eyes of normal rabbits was alsounchanged

351 Rabbits without Treatment (Placebo) In the placebogroup having uveitis one eye contained white thick non-transparent expandingmassThismass is known to representa purulent exudate consisting of leukocytes (some of themin a stage of disintegration) and small amount of fibrin Thevitreous body in this eye was in the state of destruction andcell infiltration Another eye of the same animal as wellas the eyes of other rabbits in this group maintained thevitreous body but the inner part of choroid contained whiteprecipitates Choroid in these eyes was thickened knownto be due to infiltration of neutrophils macrophages andlymphocytes Some destruction of the pigment cells layerwas also observed The vessels within choroid were full-blooded but with some extent of erythrocyte aggregationThe cornea in this group exhibited edema swelling andloosening of collagen fibers aswell as partial desquamation ofendothelium and partial destruction of Descemet membrane


(a) (b)

(c) (d)

Figure 7 Histology picture of the cornea stained with hematoxylin and eosin magnification times200 on day 4 of uveitis (a) Control (119899 = 2)cornea is lined with epithelium and endothelium collagen fibers and keratocytes (specialized corneal fibroblasts) are visible (b) placebo(119899 = 6) swelling and loosening of corneal collagen fibers endothelial desquamation partial destruction of Descemetrsquos membrane epitheliumis not changed (c) SOD1 (119899 = 6)moderate loosening of the corneal collagen fibers partial endothelial desquamation cell infiltration is absent(d) SOD1 nanozyme (119899 = 6) normal structure of the cornea

(Figure 7(b)) Epithelium of the cornea was not changedThesclera in almost all eyes exhibited pronounced edema andcontained sporadic neutrophil-macrophage infiltrates Thebundles of collagen fibers within sclera were loosened Therewere also significant edema and inflammatory infiltration inciliary body (Figure 8(b)) while loosened stroma of ciliarybody exhibited signs of cellular dystrophy characterized bythe formation of cytoplasm vacuoles Partial destruction anddesquamation of epithelium of ciliary body and its processeswith deposits of purulent exudates was also observed Retinain the eyes of rabbits from this group had regions ofdestruction and exhibited signs of dystrophy of cells elements

352 SOD1-Treated Rabbits The eyes of rabbits in the SOD1treatment group had only moderate inflammation manifes-tation in uveal tract The cornea was lined by unchangedepithelium The Descemet membrane did not have defectshowever some regions of the cornea contained loosened col-lagen fibrils and the endothelium was partially desquamated(Figure 7(c)) The sclera exhibited moderate edema andlooseness with some inflammatory infiltration The choroidwas relatively thin without purulent exudates but with slightinfiltration by neutrophils lymphocytes and macrophagesSome eyes however contained thickened regions of choroidwith more pronounced infiltrates especially in the posteriorsegment of the eye The ciliary body retained its ordinary

structure but exhibited some regions of edema and inflam-matory cell infiltration (Figure 8(c))

353 SOD1 Nanozyme-Treated Rabbits In the SOD1 nanoz-yme-treated group 2 eyes (from 6 eyes examined) hardlyshowed any inflammatory symptomsThese eyes appeared tobe unaffected by the disease and were indistinguishable of theeyes of the healthy rabbits In the remaining eyes the corneawas laid by epithelium and endothelium without desquama-tion the collagen fibers and keratocytes of stroma showedno changes as well (Figure 7(d)) The sclera in the eyes ofthis group was of common thickness without inflammationand looseningThe choroid was thin without destruction andcell infiltration The retina was also unchanged The ciliarybody also displayed its natural structure without signs ofinfiltration (Figure 8(d))

Thus while native SOD1 showed a pronounced thera-peutic effect in the treatment of experimental immunogenicuveitis in rabbits the nanoformulated form SOD1 nanozymeprovided much more remarkable effect as revealed by thehistopathology analysis

4 Discussion

ROS are excessively produced in many disease statesand contribute to tissue degeneration and pathogenesis of


(a) (b)

(c) (d)

Figure 8 Histological picture of the ciliary body stained with hematoxylin and eosin magnification times200 on day 4 of uveitis (a) Control(119899 = 2) ciliary body with processes moderate plethora (b) placebo (119899 = 6) edema and infiltration of the ciliary body deposition of purulentexudates (c) SOD1 (119899 = 6) edema and inflammatory infiltration of the ciliary body (d) SOD1 nanozyme (119899 = 6) ciliary body without cellularinfiltration

many clinical conditions including atherosclerosis strokeischemiareperfusion injury myocardial infarction centralnervous system disorders and wounds In particular ROSmetabolites may be important factors in the early tissue dam-age that develops from immunopathologic inflammations[35ndash37] Uncontrolled ROS production in acute inflamma-tion can lead to destruction of structural and functionalproteins as well as lipids in cell membranes Because of thenonspecific nature of ROS-induced tissue injury excessiverelease of these agents can cause substantial damage not onlyto the tissue in an inflamed state but also to the surroundingnormal tissue In particular this is very important for theeye as the transparency of the cornea and lens as well asthe functioning of photoreceptor apparatus relies on theirhighly ordered structures and excessive tissue damage willcompromise visual function

Antioxidants SOD1 in particular are known to be ben-eficial in the treatment of the various diseases connectedwith oxidative stress Thus SOD1 was reported to reduceinflammation [38] accelerate the healing of skin lesionscaused by burns systemic lupus erythematosus and herpes[39ndash41] protect cultured human neurons under oxidativestress [42] reduce ischemia-reperfusion injury [22 43 44]inhibit angiotensin II (AngII) intraneuronal signaling [19]prolong viability of 120573-cells [45] be effective in the treatmentof rat adjuvant arthritis [46] and so forth Most relevant

to this study antioxidants including SOD are also thoughtto be beneficial in the treatment of eye diseases The eyeis rather isolated organ and the pathological processeswithin it are preferably treated not via systemic but by localdrug intake SOD1 was found in the corneas of mammals[47] suggesting that the enzyme plays an important role inmaintaining homeostasis of the ocular surface The use oftopical subconjunctival parabulbar or intraocular injectionsof SOD1 could therefore provide a supplement for intrinsicantioxidants in eye tissues which may be depleted duringinflammation

According to official statistics inflammatory eye dis-eases are the most common eye pathologies which leadto partial disability and sometimes to the complete lossof vision Among these diseases the most severe one isuveitis inflammation of uveal tract involving both outerand inner structures of the eye Both noninfectious andinfectious uveitis are accompanied by the enhancement offree radicals formation and by the increase of the content ofthe products of lipid oxidation in eye tissues The major rolein ROS formation in the eye belongs to polymorphonuclearleukocytes which initiate as well as perpetuate themembraneoxidative processes at uveitis Thus uveitis is believed tobe strongly associated with overproduction of ROS in theeye tissues during inflammation and antioxidants can playbeneficial role in the treatment of this disease [48] It was


shown [9] that superoxide production by the leukocytes ofBehcet patients (with uveal inflammation) was significantlyhigher in the attack phase than in the remission phase Leuko-cyte superoxide generation was also enhanced in guinea pigswith S-antigen-induced experimental autoimmune uveitisThese observations indicate the perspectives of the use ofantioxidants SOD1 in particular as potential drugs in thetreatment of uveitis

Previous studies have shown beneficial effects of SOD1 inthe treatment of eye inflammation including uveitis and eyeburnsThus animals with phacoanaphylactic endophthalmi-tis (lens-induced uveitis) were treated with SOD1 [8] Thistreatment resulted in strong reduction of choroid inflamma-tion retinal edema and vasculitis In another study aqueoushumor cell quantity and infiltration of the inflammatory cellsin the anterior retina weremarkedly reduced in SOD1-treatedanimals with both S-antigen induced and bovine serumalbumin-induced passive Arthus type uveitis [9] Positiveeffect of SOD1 was also demonstrated on the rabbit modelof immunogenic uveitis [14] Moreover topical antioxidanttherapy by SOD1 in acute corneal inflammation (induced byalkali burn) was shown to be efficient in reduction of cornealulcers [11] while subconjunctival injections of SOD1 werereported to prevent tissue destruction after alkali burns of theeye and prevented corneal perforation [10]

However the methods of the treatment of the diseaseswhich include inner structures of the eye are relatively lesseffective due to the poor transport of proteins and otherdrugs into the eye In recent years there has been significantinterest in the developing nanosized drug delivery systemsto overcome the limitations of drug therapy Such nanosys-tems can improve the therapeutic efficacy of the drugs byovercoming diffusion barrier by increasing their stability inbiological tissues and fluids and enhancing cellulartissueuptake These nanosystems are attractive for the treatmentof various eye diseases including both acute and chronicconditions [17 49] So far SOD1 entrapped in liposomeswas previously shown to be effective in the treatment ofnoninfectious corneal ulcers [50]

Recently a cross-linked polyion complex of SOD1 witha cationic block copolymer PEG-pLL

50 termed ldquoSOD1

nanozymerdquo was developed [21 22]This SOD1 nanoformat ischaracterized by high dispersion stability small particle sizeparticle uniformity decreased cellular toxicity and efficienttransport into cells SOD1-nanozyme was shown to be able toeffectively scavenge ROS and decrease ischemiareperfusion-induced tissue injury and improve sensorimotor functionsin a rat middle cerebral artery occlusion model [22] Inthis study the therapeutic efficacy of SOD1 nanozyme forthe treatment of ophthalmic inflammatory diseases wasdemonstrated in a rabbit model of immunogenic uveitis

Immunogenic uveitis is an animal model of acute ocularinflammation induced by the intraocular injection of serumfrom a foreign animal after presensitization This type ofuveitis usually includes inflammation in the anterior inter-mediate and posterior segments of the eye thus representingpanuveitis We hypothesized that antioxidant agent SOD1in the form of nanozyme can attenuate oxidative stress andproduce a significant therapeutic effect For the treatment we

have chosen the most simple and convenient drug formula-tion aqueous solution of nanozyme as eye drops

In this study wemostly followed clinical and biochemicalparameters in the anterior segment of the eye in uveitiswhile further histological study allowed us to estimate uveitismanifestations in the posterior segment of the eye as wellClinical manifestations of uveitis in the anterior segment ofthe eye could be divided into two groups The first are man-ifestations in the outer part of the segment including eyelidedema eyelid hyperemia conjunctival edema conjunctivalhyperemia corneal edema and neovascularization of thecornea The second are manifestations in the inner part ofthe segment including iris edema iris hyperemia fibrin clotsand precipitates on the iris and on the lens lens opacity theexistence of synechiae and the existence of exudates in theanterior chamber of the eye

The major result of this study is a clear demonstrationthat topical instillations of SOD1 nanozyme solution into theeye exhibit remarkable effect on the clinical manifestationof the disease improve biochemical characteristics of theaqueous humor and help to maintain the cells of variouseye tissues in normal condition In the eyes of rabbits notreceiving any treatment but receiving placebo instead weobserved acute panuveitis Inflammation included entireuveal tract which in turn caused inflammation in other eyetissues Deformation of almost every eye tissue was observedretina (defects of photoreception apparatus) ciliary body(distortion in the aqueous humor formation and in accom-modation) iris (changes in the structure and immobilizationof the pupil) lens (opacity cataract) and cornea (decrease ofthe transparency) In the second group of rabbits receivingnative SOD1 solution the clinical manifestations of uveitiswere less severe However edema inflammatory infiltrationand endothelium desquamation were still observed In thethird group of rabbits receiving SOD1 nanozyme solutionthe therapeutic effect of antioxidant agent was much morepronounced We observed statistically significant differencesin those clinical manifestations of uveitis such as cornealand iris edema hyperemia of conjunctiva lens opacity andamount of fibrin clots between this group and native SOD1-treated groupThe biochemical characteristics of the aqueoushumor were also improved Moreover histological studydemonstrated almost normal structure of eye tissues fromthis group Remarkably the proposed therapy appears to bebeneficial for treatment of not only the surface but also innerareas of the eye

The current study did not allow precise delineating of themechanism by which the nanozyme formulation improvesthe therapeutic effect of SOD1 The effect of the topicalapplication of common drugs is greatly impeded by theprotective physiological barriers of the eye which effectivelydecrease the concentration of the drug in the site of theaction [15] Previous works have shown that incorporation ofSOD1 in nanozyme format increases the efficacy of nanozymedelivery in cells [19 51] Moreover the stability of the enzymetaken up into the cells within the nanozyme format is greatlyincreased presumably due to stabilization of the enzymemolecule against metabolic degradation andor lysosomalescape [22] Studies have also shown that nanozymes can


be taken up in macrophages when they can reside forconsiderable periods of time [18 20] Nanozymes can bealso transported with macrophages to distal disease siteswhere they are released into the extracellular media as wellas within other tissue cells and exerted the protective effectby scavenging the ROS [52 53] All these effects could inprinciple contribute to improved therapeutic effect of theSOD1 nanozyme during uveitis observed in this work

Altogether the results obtained demonstrate high poten-tial therapeutic efficacy of topical administration of SOD1nanozyme for the treatment of inflammatory eye diseasesMost current therapies of uveitis are predominantly based onsteroids and immunosuppressants [54 55] However steroidshave systemic side effects such as cataract glaucoma andsecondary ocular hypertension [56 57] while immunosup-pressive drugs are teratogenic and contraindicated duringpregnancy [55] Intraocular or periocular injections candeliver relatively high doses of drug to the eye with fewerside effects [54 55] however each such injection is in essencea minor surgical procedure that could be quite disruptiveand inconvenient to a patient Recently several sustained-release drug delivery implants have been developed to treatnoninfectious uveitis but such implantation requires surgicaloperation and the cost of this invasive treatment is high to thepatients and insurance companies [55]

Therefore a noninvasive topical SOD1 nanoformat thatcan be conveniently applied as eye drops by a patient ifshown successful as a therapeutic modality could be a majorbreakthrough in treatment of uveitis and possibly otherinflammatory conditions of the eye

5 Conclusions

In summary our work demonstrates that the nanozymeformed by self-assembly of the SOD1 with PEG-pLL

50block

copolymer and stabilized by cross-linking can be used as acarrier for sustained delivery of SOD1 into ocular tissues forthe treatment of inflammation processes in the eye Topi-cal instillations of SOD1-nanozyme significantly decreasedinflammation both in the outer and inner parts of the eyeas determined using scores of the clinical manifestations ofuveitis multiple biochemical parameters and histologicalanalysis These results may have broad clinical implicationsin the treatment of other disorders of the eye where oxidativestress contributes to pathology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding publication of this paper

Acknowledgments

This work was supported by the Government of Rus-sian Federation Grant no 11G34310004 Russian ScientificFoundation Grant 14-13-00731 an Institutional DevelopmentAward (IDeA) from theNational Institute of GeneralMedicalSciences of the National Institutes of Health under Grant

P20GM103480 and The Carolina Partnership a strategicpartnership between theUNCEshelman School of Pharmacyand The University Cancer Research Fund through theLineberger Comprehensive Cancer Center

References

[1] E Miserocchi G Fogliato G Modorati and F BandelloldquoReview on the worldwide epidemiology of uveitisrdquo EuropeanJournal of Ophthalmology vol 23 no 5 pp 705ndash717 2013

[2] R Bansal V Gupta and A Gupta ldquoCurrent approach in thediagnosis and management of panuveitisrdquo Indian Journal ofOphthalmology vol 58 no 1 pp 45ndash54 2010

[3] M Khairallah M Accorinti C Muccioli R Kahloun and J HKempen ldquoEpidemiology of Behcet diseaserdquoOcular Immunologyand Inflammation vol 20 no 5 pp 324ndash335 2012

[4] M Uyama ldquoUveitis in sarcoidosisrdquo Int Ophthalmol Clin vol 42no 1 pp 143ndash150 2002

[5] E T Cunningham Jr and J D Wender ldquoPractical approach tothe use of corticosteroids in patients with uveitisrdquo CanadianJournal of Ophthalmology vol 45 no 4 pp 352ndash358 2010

[6] J Kruh and C S Foster ldquoThe philosophy of treatment of uveitispast present and futurerdquo Developments in Ophthalmology vol51 pp 1ndash6 2012

[7] P Kulkarni ldquoReview uveitis and immunosuppressive drugsrdquoJournal of Ocular Pharmacology and Therapeutics vol 17 no 2pp 181ndash187 2001

[8] N A Rao A J Calandra A Sevanian B Bowe J M Delmageand G E Marak Jr ldquoModulation of lens-induced uveitis bysuperoxide dismutaserdquo Ophthalmic Research vol 18 no 1 pp41ndash46 1986

[9] M Yamada H Shichi T Yuasa Y Tanouchi and Y MimuraldquoSuperoxide in ocular inflammation human and experimentaluveitisrdquo Journal of Free Radicals in Biology and Medicine vol 2no 2 pp 111ndash117 1986

[10] V S Nirankari S D Varma V Lakhanpal and R D RichardsldquoSuperoxide radical scavenging agents in treatment of alkaliburns An experimental studyrdquo Archives of Ophthalmology vol99 no 5 pp 886ndash887 1981

[11] J L Alio M J Ayala M E Mulet A Artola J M Ruiz andJ Bellot ldquoAntioxidant therapy in the treatment of experimentalacute corneal inflammationrdquo Ophthalmic Research vol 27 no3 pp 136ndash143 1995

[12] Y de Kozak J P Nordman J-P Faure N A Rao and GE Marak Jr ldquoEffect of antioxidant enzymes on experimentaluveitis in ratsrdquoOphthalmic Research vol 21 no 3 pp 230ndash2341989

[13] N A Rao A Sevanian M A S Fernandez et al ldquoRole ofoxygen radicals in experimental allergic uveitisrdquo InvestigativeOphthalmology and Visual Science vol 28 no 5 pp 886ndash8921987

[14] N B Chesnokova B B Neroev O V Beznos et al ldquoOxidativestress in uveitis and its correction with superoxide dismutaseantioxidative enzyme (experimental study)rdquo Vestnik Oftal-mologii vol 130 pp 30ndash34 2014

[15] M R Prausnitz and J S Noonan ldquoPermeability of corneasclera and conjunctiva a literature analysis for drug delivery tothe eyerdquo Journal of Pharmaceutical Sciences vol 87 no 12 pp1479ndash1488 1998

[16] R C Nagarwal S Kant P N Singh P Maiti and J K PanditldquoPolymeric nanoparticulate system a potential approach for


ocular drug deliveryrdquo Journal of Controlled Release vol 136 no1 pp 2ndash13 2009

[17] M A Zarbin C Montemagno J F Leary and R RitchldquoNanotechnology in ophthalmologyrdquoCanadian Journal of Oph-thalmology vol 45 no 5 pp 457ndash476 2010

[18] E V Batrakova S Li A D Reynolds et al ldquoA macrophage-nanozyme delivery system for Parkinsonrsquos diseaserdquoBioconjugateChemistry vol 18 no 5 pp 1498ndash1506 2007

[19] E G Rosenbaugh J W Roat L Gao et al ldquoThe attenuationof central angiotensin II-dependent pressor response and intra-neuronal signaling by intracarotid injection of nanoformulatedcopperzinc superoxide dismutaserdquo Biomaterials vol 31 no 19pp 5218ndash5226 2010

[20] Y ZhaoM J Haney N L Klyachko et al ldquoPolyelectrolyte com-plex optimization for macrophage delivery of redox enzymenanoparticlesrdquo Nanomedicine vol 6 no 1 pp 25ndash42 2011

[21] N L Klyachko D S Manickam AM Brynskikh et al ldquoCross-linked antioxidant nanozymes for improved delivery to CNSrdquoNanomedicine Nanotechnology Biology and Medicine vol 8no 1 pp 119ndash129 2012

[22] D S Manickam A M Brynskikh J L Kopanic et al ldquoWell-defined cross-linked antioxidant nanozymes for treatment ofischemic brain injuryrdquo Journal of Controlled Release vol 162 no3 pp 636ndash645 2012

[23] V A Kostyuk and A I Potapovich ldquoSuperoxide-driven oxida-tion of quercetin and a simple sensitive assay for determinationof superoxide dismutaserdquoBiochemistry International vol 19 no5 pp 1117ndash1124 1989

[24] V V Neroev G A Davydova and T S Perova ldquoModelof immunogenic uveitis in rabbitsrdquo Bulletin of ExperimentalBiology and Medicine vol 142 no 5 pp 649ndash650 2006

[25] V N Alekseev E BMartynova and I V ChurilovaMethod fortreating primary open angle glaucoma RU2144343 (C1) 2000

[26] J Bieth P Metais and J Warter ldquoDetection and determinationof alpha 2-macroglobulin trypsin activity in pleural fluids andascitesrdquo Enzyme vol 12 no 1 pp 13ndash24 1971

[27] V F Nartikova and T S Paskhina ldquoA method for estimationof 1205721-antitrypsin and of 1205722-macroglobulin in human bloodserum (plasma) in normal state and under some pathologicalconditionsrdquoVoprosyMeditsinskoj Khimii vol 25 no 4 pp 494ndash499 1979

[28] O V Gulidova O B Lyubitskii G I Klebanov and N BChesnokova ldquoAntioxidant activity of tear fluid in experimentalalkali eye burnsrdquo Bulletin of Experimental Biology andMedicinevol 128 no 11 pp 1155ndash1158 1999

[29] A Dong B Xie J Shen et al ldquoOxidative stress promotes ocularneovascularizationrdquo Journal of Cellular Physiology vol 219 no3 pp 544ndash552 2009

[30] C-B Fang D-X Zhou S-X Zhan et al ldquoAmelioration ofexperimental autoimmune uveitis by leflunomide in Lewis ratsrdquoPLoS ONE vol 8 no 4 Article ID e62071 2013

[31] D E Panrucker and F L Lorscheider ldquoSynthesis of acute-phase120572

2-macroglobulin during inflammation and pregnancyrdquoAnnals

of the New York Academy of Sciences vol 417 pp 117ndash124 1983[32] T Kuribayashi M Tomizawa T Seita K Tagata and S

Yamamoto ldquoRelationship between production of acute-phaseproteins and strength of inflammatory stimulation in ratsrdquoLaboratory Animals vol 45 no 3 pp 215ndash218 2011

[33] S Saraswathy and N A Rao ldquoMitochondrial proteomics inexperimental autoimmune uveitis oxidative stressrdquo InvestigativeOphthalmology and Visual Science vol 50 no 12 pp 5559ndash5566 2009

[34] B K Singh A Kumar I Ahmad et al ldquoOxidative stress inzinc-induced dopaminergic neurodegeneration implicationsof superoxide dismutase and heme oxygenase-1rdquo Free RadicalResearch vol 45 no 10 pp 1207ndash1222 2011

[35] B Halliwell ldquoReactive oxygen species and the central nervoussystemrdquo Journal of Neurochemistry vol 59 no 5 pp 1609ndash16231992

[36] C A Papaharalambus and K K Griendling ldquoBasic mech-anisms of oxidative stress and reactive oxygen species incardiovascular injuryrdquo Trends in Cardiovascular Medicine vol17 no 2 pp 48ndash54 2007

[37] J Lugrin N Rosenblatt-Velin R Parapanov and L LiaudetldquoThe role of oxidative stress during inflammatory processesrdquoBiological Chemistry vol 395 no 2 pp 203ndash230 2014

[38] K Yasui and A Baba ldquoTherapeutic potential of superoxidedismutase (SOD) for resolution of inflammationrdquo InflammationResearch vol 55 no 9 pp 359ndash363 2006

[39] I V Churilova E V Zinovrsquoev B A Paramonov Y I DrozdovaV O Sidelrsquonikov and V Y Chebotarev ldquoEffect of erysod(erythrocyte superoxide dismutase) on blood concentration ofreactive oxygen species in patients with severe burns and burnshockrdquo Bulletin of Experimental Biology and Medicine vol 134no 5 pp 454ndash456 2002

[40] Y Mizushima K Hoshi A Yanagawa and K Takano ldquoTopicalapplication of superoxide dismutase creamrdquoDrugs under Exper-imental and Clinical Research vol 17 no 2 pp 127ndash131 1991

[41] K Vorauer-Uhl E Furnschlief A Wagner B Ferko and HKatinger ldquoTopically applied liposome encapsulated superoxidedismutase reduces postburn wound size and edema formationrdquoEuropean Journal of Pharmaceutical Sciences vol 14 no 1 pp63ndash67 2001

[42] M K Reddy L WuW Kou A Ghorpade and V LabhasetwarldquoSuperoxide dismutase-loadedPLGAnanoparticles protect cul-tured human neurons under oxidative stressrdquoApplied Biochem-istry and Biotechnology vol 151 no 2-3 pp 565ndash577 2008

[43] M K Reddy and V Labhasetwar ldquoNanoparticle-mediateddelivery of superoxide dismutase to the brain an effectivestrategy to reduce ischemia-reperfusion injuryrdquo The FASEBJournal vol 23 no 5 pp 1384ndash1395 2009

[44] M Hangaishi H Nakajima J-I Taguchi et al ldquoLecithinizedCu Zn-superoxide dismutase limits the infarct size followingischemia-reperfusion injury in rat hearts in vivordquo Biochemicaland Biophysical Research Communications vol 285 no 5 pp1220ndash1225 2001

[45] S Giovagnoli G Luca I Casaburi et al ldquoLong-term deliveryof superoxide dismutase and catalase entrapped in poly(lactide-co-glycolide) microspheres in vitro effects on isolated neonatalporcine pancreatic cell clustersrdquo Journal of Controlled Releasevol 107 no 1 pp 65ndash77 2005

[46] M L Corvo J C S Jorge R vanrsquot Hof M E M CruzD J A Crommelin and G Storm ldquoSuperoxide dismutaseentrapped in long-circulating liposomes formulation designand therapeutic activity in rat adjuvant arthritisrdquo Biochimica etBiophysica ActamdashBiomembranes vol 1564 no 1 pp 227ndash2362002

[47] T M Redmond E J Duke W H Coles J A V Simson andR K Crouch ldquoLocalization of corneal superoxide dismutase bybiochemical and histocytochemical techniquesrdquo ExperimentalEye Research vol 38 no 4 pp 369ndash378 1984

[48] U C S Yadav N M Kalariya and K V Ramana ldquoEmergingrole of antioxidants in the protection of uveitis complicationsrdquoCurrent Medicinal Chemistry vol 18 no 6 pp 931ndash942 2011


[49] Y Diebold and M Calonge ldquoApplications of nanoparticles inophthalmologyrdquo Progress in Retinal and Eye Research vol 29no 6 pp 596ndash609 2010

[50] S Shimmura R Igarashi H Yaguchi Y Ohashi J Shimazakiand K Tsubota ldquoLecithin-bound superoxide dismutase in thetreatment of noninfectious corneal ulcersrdquo American Journal ofOphthalmology vol 135 no 5 pp 613ndash619 2003

[51] X Yi M C Zimmerman R Yang J Tong S Vinogradovand A V Kabanov ldquoPluronic-modified superoxide dismutase1 attenuates angiotensin II-induced increase in intracellularsuperoxide in neuronsrdquo Free Radical Biology and Medicine vol49 no 4 pp 548ndash558 2010

[52] M JHaney P Suresh Y Zhao et al ldquoBlood-bornemacrophage-neural cell interactions hitchhike on endosome networks forcell-based nanozyme brain deliveryrdquo Nanomedicine vol 7 no6 pp 815ndash833 2012

[53] Y Zhao M J Haney V Mahajan et al ldquoActive tar-geted macrophage-mediated delivery of catalase to affectedbrain regions in models of Parkinsonrsquos diseaserdquo Journal ofNanomedicine amp Nanotechnology supplement 4 article 0032011

[54] YHe S-B JiaW Zhang and J-M Shi ldquoNewoptions for uveitistreatmentrdquo International Journal of Ophthalmology vol 6 no 5pp 702ndash707 2013

[55] N Haghjou M Soheilian and M J Abdekhodaie ldquoSustainedrelease intraocular drug delivery devices for treatment ofuveitisrdquo Journal of Ophthalmic and Vision Research vol 6 no4 pp 317ndash319 2011

[56] N V Saraiya and D A Goldstein ldquoDexamethasone for ocularinflammationrdquo Expert Opinion on Pharmacotherapy vol 12 no7 pp 1127ndash1131 2011

[57] D S Friedman J T Holbrook H Ansari et al ldquoRisk ofelevated intraocular pressure and glaucoma in patients withuveitis results of the multicenter uveitis steroid treatment trialrdquoOphthalmology vol 120 no 8 pp 1571ndash1579 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


experience unavoidable impaired visual function Thus itis important to investigate alternative approaches for thetreatment of uveitis

Inflammatory diseases including ocular ones are accom-panied by excessive production of reactive oxygen species(ROS) and by depletion of endogenous antioxidants Antiox-idant enzymes superoxide dismutase 1 (SOD1 also knownas CuZn SOD) catalase and glutathione peroxidase areknown to be very effective scavengers of ROSThese enzymeswere shown to be effective in the treatment of various eyediseases associated with oxidative stress Thus SOD1 wasused for the treatment of lens-induced and bovine albumin-induced uveitis in rabbits [8 9] as well as for the treatmentof acute corneal inflammation in animals induced by sodiumhydroxide [10 11] Both SOD1 and glutathione peroxidasewere employed for the treatment of severe experimentalallergic uveitis induced by retinal S antigen in rats [12] whilepoly(ethylene glycol)- (PEG-) modified catalase and PEG-SOD were employed for the treatment of the same type ofuveitis in guinea pigs [13] We have shown recently [14] thatSOD1 instillations may help to reduce clinical presentationsof immunogenic uveitis in rabbits

Eye diseases are most commonly and preferably treatedby topical instillations of eye drops These formulationsface technical and clinical problems such as solubility ofthe components and instability of drug solutions limitedefficacy and limited cornealsclera permeability and localand systemic toxicity Moreover 2min after instillation themajor part of the topical drug solution is eliminated via thenasolacrimal drainage system limiting ocular penetration ofthe drug to less than 5 of the administered dose [15]

Nanoparticles are colloidal drug carrier systems that canimprove the efficacy of drug delivery into the eye by overcom-ing cornealsclera diffusion barrier Drug loaded polymericnanoparticles offer several favorable biological propertiessuch as biocompatibility and mucoadhesiveness enhancingbioavailability without blurring the vision The use of drug-containing nanoparticles can decrease the dose of the drugand diminish side effects So nanoparticles are a promisingdrug delivery system which fulfills the requirements forophthalmic application (for reviews see [16 17])

Recently new formulations of antioxidant enzymesSOD1 and catalase were prepared by electrostatic couplingof these negatively charged enzymes (pI values are 495 and58 for SOD1 and catalase resp) with cationic block copoly-mers such as methoxy-PEG-block-poly(L-lysine hydrochlo-ride) block copolymer (PEG-pLL

50) followed by covalent

cross-linking to stabilize nanoparticles Catalytic nanopar-ticles based on polyion complexes of enzymes with blockcopolymers of opposite charge were termed ldquonanozymesrdquo[18ndash22] Spontaneous self-assembly of oppositely chargedproteins and polymers results in stoichiometric complexeswith 100 loading efficiency These nanozymes were shownto be prospective agents for the treatment of various diseasesof the central nervous system due to prolonged ability toscavenge experimentally induced ROS in cultured brainmicrovessel endothelial cells and central neurons increasedstability in both blood and brain enhanced penetrationthrough the blood-brain barrier and therefore increased

accumulation in brain tissues in comparisonwith non-cross-linked complexes and native enzyme [18 21 22]

In the current study we demonstrate the advantages oftopical instillations of superoxide dismutase 1 in the formof ldquonanozymerdquo in the treatment of ocular inflammation in arabbit model of immunogenic uveitis

2 Methods21 Preparation of and Characteristics of Nanozyme SOD1nanozyme was synthesized by self-assembly of recombinantSOD1 (ldquoEnzyme Technologiesrdquo St Petersburg Russia) withcationic block copolymer PEG-pLL

50(MW 13 kDa poly-

dispersity index 109 Alamanda Polymers Huntsville AL)in aqueous solution followed by cross-linking with 331015840-dithiobis(sulfosuccinimidylpropionate) (DTSSP) as in [21]Unreacted cross-linker was desalted using NAP-25 columnand cross-linked nanozymes were purified using a 100 kDaMWCO filter Purified particles were then lyophilized from005M Hepes-buffer pH 75 containing 015M NaCl andstored at minus20∘C For further experiments precalculatedquantity of lyophilized nanozyme was dissolved in deion-ized water and gently vortexed for 2min until sample dis-solved completely Intensity-mean z-averaged particle diam-eter (effective diameter) polydispersity index (PDI) and 120577-potential were measured after filtration via a 02 120583m filterusing a Zetasizer Nano ZS (Malvern Instruments Ltd MA)Aliquots of nanozyme solution required for daily experi-ments were then frozen and kept at minus20∘C

22 Enzyme Activity SOD1 activity was determined usingSOD1 ability to inhibit autooxidation of quercetin as in [23]with detection kit (Belarusian State University Belarus) Theexperimental sample in phosphate buffer pH 78 contain-ing 008M EDTA 0125 (vv) TEMED was mixed withquercetin solution in DMSO The absorbance was measuredat 406 nm immediately after addition of quercetin (119863

0) and

after 20min (11986320) In control phosphate buffer was used

instead of the sample and the absorbances 119888119863

0and 119888119863

20

were measured correspondinglyThe percent of inhibition ofquercetin autoxidation by SOD1 in experimental samples wascalculated by the formula [(119888

119863

0minus

119888

119863

20) minus (119863

0minus119863

20)](

119888119863

0minus

119888

119863

20) times 100 One unit of SOD1 activity was defined as the

amount of SOD1 which inhibits the quercetin autoxidationby 50 The protein content was determined using MicroBCA Protein Assay Kit (Pierce Rockford IL)

23 In Vitro Drug Release Study Onemg of SOD1 nanozymewas dispersed in 05mL of PBS transferred to 100 kDaMWCO membrane and centrifuged for 5min at 1200timesgSupernatant (about 005mL) was diluted by PBS to theinitial volume and centrifuged againThe ldquofiltration-dilutionrdquoprocedure was repeated 5 times SOD1 activity and proteinconcentration were measured in the initial nanozyme solu-tion in each filtrate and in the final supernatant In anotherseries of experiments equal amounts of freshly dissolvednanozyme in PBS were incubated at room temperature fordifferent time periods Then the solutions were filtered
















3 Results




0

5

10

15

20

25

Initi

al ac

tivity

()


(a)

0

5

10

15

20

25

Initi

al p

rote

in co

ncen

trat

ion

()


(b)






Day 5

PlaceboSODNanozyme

NS NS

NSNS

0

1

2

3

Scor

e

Day 8Day 3 Day 4

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast





NSNSNS

NS

NS

NS

PlaceboSODNanozyme

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

0

1

2

3

Scor








120572






Median

Range


NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75




2-macroglobulin and




NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results




0

5

10

15

20

25

Initi

al ac

tivity

()


(a)

0

5

10

15

20

25

Initi

al p

rote

in co

ncen

trat

ion

()


(b)






Day 5

PlaceboSODNanozyme

NS NS

NSNS

0

1

2

3

Scor

e

Day 8Day 3 Day 4

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast





NSNSNS

NS

NS

NS

PlaceboSODNanozyme

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

0

1

2

3

Scor








120572






Median

Range


NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75




2-macroglobulin and




NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

Initi

al ac

tivity

()


(a)

0

5

10

15

20

25

Initi

al p

rote

in co

ncen

trat

ion

()


(b)






Day 5

PlaceboSODNanozyme

NS NS

NSNS

0

1

2

3

Scor

e

Day 8Day 3 Day 4

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast





NSNSNS

NS

NS

NS

PlaceboSODNanozyme

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

0

1

2

3

Scor








120572






Median

Range


NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75




2-macroglobulin and




NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 5

PlaceboSODNanozyme

NS NS

NSNS

0

1

2

3

Scor

e

Day 8Day 3 Day 4

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast





NSNSNS

NS

NS

NS

PlaceboSODNanozyme

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

0

1

2

3

Scor








120572






Median

Range


NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75




2-macroglobulin and




NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















120572






Median

Range


NS

0

2

4

6

8

Tota

l sco

re

lowast

lowastlowast

25ndash75




2-macroglobulin and




NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















NS

0

2

4

6

8

10

Tota

l sco

re

Median

Range

lowastlowast

lowastlowast

25ndash75


Day 4 Day 8

NS

NS NS

NS NS

0

1000

2000

3000

Leuk

ocyt

es co

unt (

fr

om co

ntro

l)

PlaceboSODNanozyme

lowast



2-macroglobulin





(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)







4 Discussion



(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)











50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















50 termed ldquoSOD1











5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions


50block




Acknowledgments



References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article superoxide dismutase 1 nanozyme for...

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