Peptide-carbon quantum dots conjugate, derived from human Retinoic Acid Receptor Responder Protein 2, against antibiotic-resistant gram positive and gram negative pathogenic bacteria

nanoma e ials A icle Pep ide-Ca bon Quan um Do s Conjuga e, De i ed om Human Re inoic Acid Recep o Responde P o ein 2, agains An ibio ic-Resis an G am Posi i e and G am Nega i e Pa hogenic Bac e ia Aninda Mazumda 1,2,* , Yazan Haddad 1,2 , Ved an Milosa lje ic 1,2 , Hana Michalko a 1, Roman Gu an 1,2 , Sukanya Bhowmick 1,2 and Ami a a Moulick 1,2,* 1Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, Zemedelska 1, CZ-613 00 B no, Czech Republic; [email p o ec ed] (Y.H.); [email p o ec ed] (V.M.); [email p o ec ed] (H.M.); [email p o ec ed] (R.G.); [email p o ec ed] (S.B.) 2Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyno a 123, CZ-612 00 B no, Czech Republic *Co espondence: [email p o ec ed] o [email p o ec ed] (A.M.); [email p o ec ed] (A.M.) Recei ed: 29 Janua y 2020; Accep ed: 11 Feb ua y 2020; Published: 14 Feb ua y 2020   Abs ac : An ibio ic- esis an bac e ial in ec ions ha e become global issues o public heal h, which inc eases he u e need o de elop al e na i es o an ibio ics. He e, he HSER (Homo sapiens e inoic acid ecep o ) pep ide was designed om e inoic acid ecep o esponde p o ein 2 o Homo sapiens, and was conjuga ed wi h syn hesized CQDs (ca bon quan um do s) o enhanced an ibac e ial ac i i y in combina ion, as indi idually hey a e no highly e ec i e. The HSER–CQDs we e cha ac e ized using spec opho ome e , HPLC coupled wi h elec osp ay-ioniza ion quad upole ime-o - ligh mass spec ome e (ESI–qTOF) mass spec ome e , ze a po en ial, ze a size, and FTIR. The ea e , he an ibac e ial ac i i y agains Vancomycin-Resis an S aphylococcus au eus (VRSA) and Esche ichia coli (ca bapenem esis an ) was s udied using g ow h cu e analysis, u he suppo ed by mic oscopic images showing he p esence o cell deb is and dead bac e ial cells. The an ibac e ial mechanism o HSER–CQDs was obse ed o be ia cell wall dis up ion and also in e ac ion wi h gDNA (genomic DNA). Finally, oxici y es agains no mal human epi helial cells showed no oxici y, con i med by mic oscopic analysis. Thus, he HSER–CQDs conjuga e, ha ing high s abili y and low oxici y wi h p ominen an ibac e ial ac i i y, can be used as a po en ial an ibac e ial agen . Keywo ds: bac e ial in ec ions; an ibio ic- esis an ; ca bon quan um do s; an ibac e ial ac i i y; oxici y 1. In oduc ion The e olu ion o bac e ia wi h e e y gene a ion o acqui e esis ance owa ds new an ibio ics ha e made i a challenge o ea pa hogenic bac e ia, as hei pa e n o esis ance di e s [ 1 ]. Thus, bac e ial in ec ions due o he apid inc ease in an ibio ic esis ance ha e become a majo h ea o global heal h. An ibio ic esis ance occu s due o incomple e cou se o an ibio ic dose and i s misuse [ 2 ], exposu e o cons an s ess, changes in genomic le el, and ho izon al gene ans e . The numbe o mul id ug- esis an pa hogenic bac e ia is inc easing e e y day, c ea ing wo se si ua ions o he clinical se ings o deal wi h i [3,4]. The Wo ld Heal h O ganiza ion in he yea 2017 lis ed he an ibio ic- esis an bac e ia in h ee di e en ca ego ies depending on he h ea le el p io i ies: c i ical, high, and medium [ 5 ]. The Vancomycin-Resis an Nanoma e ials 2020,10, 325; doi:10.3390/nano10020325 www.mdpi.com/jou nal/nanoma e ials Nanoma e ials 2020,10, 325 2 o 19 S aphylococcus au eus (VRSA) is a he highes h ea le el and he ca bapenem- esis an En e obac e iaceae (CRE) is in he c i ical le el. The las eso o he g am posi i e and nega i e bac e ia is ancomycin and ca bapenem, bu hese a e also becoming esis an , c ea ing global epidemiological and clinical bu den. The VRSA is esis an due o i s abili y o ejec he en y o ancomycin in o i s own sys em and p e en i s own al e ed au olysis, along wi h gene ansc ip ion al e a ion [ 6 ]. Whe eas, he En e obac e iaceae a e esis an due o he de elopmen o ca bapenemase [ 7 , 8 ]. An imic obial agen s like os omycin, polymyxin, colis in, and ni o u an oin ha e become ine ec i e due o plasmid-media ed esis ance in CRE. Whe eas combina o ial ea men using di e en an ibio ics can be used bu i has some oxici y, limi a ions, and side e ec s [ 9 , 10 ]. Thus, he e is an u gen need o de elop a be e ea men me hod agains he VRSA and CRE in ec ions [ 5 , 11 ]. This highligh s he eme gency o de eloping al e na i es o an ibio ics ha ha e high an imic obial ac i i y wi h less o negligible oxici y. An imic obial pep ides (AMPs) can be an in e es ing al e na i e o adi ional an ibio ics due o i s na u al occu ence in almos all species, hei amphipa hic na u e, and hei assis inna e immuni y. They ha e he abili y o p o ide highly e ec i e and non-speci ic de ensi e ac i i y agains in ading pa hogens [ 12 – 14 ]. Few AMPs ha e been es ed clinically ha showed some an imic obial ac i i y [ 15 ]. Though, he e a e ew limi a ions o hese an imic obial pep ides as hey can be uns able, in e ac non-speci ically, and ha e a oxic na u e [ 16 ]. Some o hem a e highly e ec i e agains bac e ia and ungi, and ew also impa an icance ac i i y. The p oblem wi h solubili y is mos ly negligible, and he p esence o seconda y s uc u es (like helices and be a shee s), wi h he possibili y o modi y hem, ele a e hei alue o e en be e ea men . The nanopa icles help in he ad ancemen o nanomedicine, which in u n ha e helped in a ge ed d ug deli e y, low dosage, and high ac i i y wi h he limi a ion o oxici y, solubili y, and s abili y [ 17 ]. Ca bon nanopa icles, like g aphene quan um do s, g aphene oxide, and CQDs, ha e ecei ed g ea a ac ion due o hei an imic obial p ope ies [ 18 ]. CQDs a e eme ging wi h wide applica ions in senso s, ca alysis, medicine, and bio-imaging. Thenanosized CQDsp o ide ad an agewi h hei wide su acea ea, physiochemical, andop icalp ope ies[ 19 ]. Thei pho o-induced edoxcha ac e is icsandin insicop icalp ope iescan be enhanced and exploi ed o in oduce a new pla o m o na u al/ isible ligh -ac i a ed an imic obial agen s [ 20 ]. Thus, hey can be used as an an imic obial agen wi h he scope o modi y hei su ace cha ge and unc ional g oups o be e pe o mance. They in e ac wi h he bac e ial su ace causing cell wall damage and an inc ease in endogenous eac i e oxygen species (ROS), leading o cy oplasmic leakage and cell dea h. S udies showed ha some CQDs we e biocompa ible in in i o and in i o se up [ 19 , 21 ]. Though hey ha e some limi a ions, like sho exci a ion/emission wa eleng h wi h poo pene a ion capabili y, unclea pho oluminescence, no an ibac e ial ac i i y in ew cases, and poo mul i-colo emissions. Consequen ly, CQDs can be modi ied and conjuga ed wi h pep ides o ob ain an imic obial ac i i y. Thus, an eligible al e na i e o an ibio ics a e an imic obial pep ides and nanopa icles. The o ma ion o conjuga es using pep ides wi h nanoma e ials o o he chemicals is possible and p o ides a wide ange o p oduce new pa icles wi h medical applica ions [ 17 ]. HSER (Homo sapiens e inoic acid ecep o ) is a 22-amino acid esidue pep ide de i ed om e inoic acid ecep o esponde p o ein 2 o Homo sapiens. Re inoic acid ecep o esponde p o ein 2 was known o exhibi de ensi e esponse agains g am posi i e and nega i e bac e ia [ 22 ]. The pep ide was syn hesized and he an ibac e ial ac i i y agains VRSA and E. coli was seen o be poo . Fu he mo e, he ca bon quan um do s (CQDs) we e syn hesized based on ou p e ious s udies [ 23 ]. Then, conjuga ion o he pep ide was done using CQDs o inc ease he an ibac e ial ac i i y o HSER a e he o ma ion o HSER–CQDs as a whole. The p esen expe imen is mainly based on he molecula dynamics (MD) simula ion o he designed HSER pep ide o unde s and he s uc u e o he pep ide, which was accompanied by he syn hesis and cha ac e iza ion o he HSER-CQDs conjuga e. The ea e , he an imic obial ac i i ies o HSER–CQDs we e es ed agains VRSA, and E. coli wi h he de e mina ion o hei espec i e MIC (minimum inhibi o y concen a ion). Fu he , he luo escence li e/dead cell imaging and phase Nanoma e ials 2020,10, 325 3 o 19 con as mic oscope imaging agains VRSA and E. coli was s udied. The ea e , he mechanism o he HSER–CQDs we e s udied. Finally, he oxici y es s like hemoly ic assay and MTT assay we e pe o med. Thus, he HSER–CQDs showed an ibac e ial ac i i y ha can be used as a be e ea men s a egy agains mul id ug- esis an bac e ial in ec ions and a po en ial subs i u e o an ibio ics wi h medicinal alues. 2. Expe imen al 2.1. Molecula Dynamics (MD) Simula ion The pep ide was de i ed om he e inoic acid ecep o esponde p o ein 2 o Homo sapiens due o i s de ensi e esponse agains g am posi i e and nega i e bac e ia. The s uc u e was cons uc ed di ec ly om sequence (CCVRLEFKLQQTSCRKRDWKKP) using he build s uc u e ool in UCSF Chime a 1.10.2, wi h s anda d Φ / Ψ angle alues o alpha helix ( − 57 ◦ and − 47 ◦ , espec i ely) and Dunb ack o ame lib a y o side-chains, hen hyd ogens we e added. Molecula dynamics (MD) simula ion was p epa ed and un on GPU (NVIDIA GTX1080Ti) using G omacs 2018 wi h Ambe 99SB o ce ield. The s anda d leap- og in eg a o was used o calcula ion o ajec o ies in s eps o 2 s. The Ve le scheme was used o neighbo sea ching, while he cu -o me hod was used o Van de Waals in e ac ions a 1.2 nm. Pa icle mesh Ewald (PME) was used wi h all elec os a ics calcula ions a 1.2 nm cu o . B ie ly, he pep ide was sol a ed in SPC wa e model in dodecahed on pe iodic box and neu alized wi h Cl − ions. Only 335 s eps o s eepes descen minimiza ion we e equi ed o achie e con e gence wi h maximum o ce <1000 kJ/mol/nm. NVT equilib a ion was done o du a ion o 1 ns using LINCS cons ain s o H-bonds, and modi ied Be endsen he mos a coupling (300 K) in wo g oups (p o ein and non-p o ein). NPT equilib a ion was done o du a ion o 1 ns using LINCS cons ain s o H-bonds, modi ied Be endsen he mos a coupling (300 K), and iso opic B endsen p essu e coupling (1 ba ). MD P oduc ion was pe o med in NPT o 1000 ns using LINCS cons ain s o H-bonds, modi ied Be endsen he mos a coupling (300 K), and Pa inello-Rahman iso opic p essu e coupling (1 ba ). T ajec o ies and ene gies we e sa ed e e y 10 ps and he o al numbe o ajec o y ames was 100,000. Pos MD analysis was done by cen e ing he p o einin he box, ollowed by i ing o backbone and emo al o all wa e molecules om he sys em. Time e olu ion o seconda y s uc u e was pe o med acco ding o he s anda d DSSP me hod. Visualiza ion and u he analysis was done in UCSF Chime a. Clus e ing analysis o minimal backbone in s eps o 50 ames was used o iden i y he mos s able con o ma ion (la ges clus e ). Roo mean squa e de ia ions (RMSD) o he backbone (87 a oms) and o all a oms in pep ide (191 a oms) we e calcula ed wi h e e ence o he i s ame (α-helix) and o he ep esen a i e ame o op clus e . 2.2. Chemicals and Syn hesis o he HSER-CQDs Conjuga e All he chemicals, eagen s o pep ide syn hesis, CQDs syn hesis, and s anda ds we e pu chased om Sigma-Ald ich (S . Louis, MO, USA) in ACS pu i y, unless no ed o he wise. All he expe imen s we e done main aining s e ile condi ion o ob ain he bes esul s. Acco ding o ou p e ious s udies he p epa a ion o he wa e soluble CQDs we e done and b ie ly he syn hesis o CQDs is desc ibed [24]. To he solu ion o ci ic acid (2.1 g), and Mili-Q wa e 0.8 mL o e hylenediamine was added in a 100 mL h ee-necked lask in s i ing condi ion o a couple o minu es (min). Then he olume was made o 50 mL. Then he solu ion was subjec ed o mic owa e i adia ion o 10 mins (Mul iwa e 3000, An on Paa GmbH, G az, Aus ia). The solu ion was pu i ied by dialysis agains Mili-Q wi h a D-Tube maxi dialyze o e nigh o emo e he unbound e hylenediamine [ 23 ]. Fmoc solid phase syn hesis was used o syn hesize HSER on Libe y Blue pep ide syn hesize (CEM, Ma hews, NC, USA). The sequence o HSER is CCVRLEFKLQQTSCRKRDWKKP, a s e ch o 22 amino acids. Equal p opo ion (1:1 a io) o he HSER and CQDs was added in a ial and kep in o a o Mul i Bio RS-24 (Biosan, Riga, La ia) o 24 h a a cons an o a ion and ime Nanoma e ials 2020,10, 325 4 o 19 in e al ib a ion. The solu ion was pu i ied again by dialysis agains Mili-Q wi h a D-Tube maxi dialyze o 24 h o emo e he unbound pep ide om he solu ion. 2.3. Cha ac e iza ion o he HSER-CQDs Conjuga e The HSER-CQDs we e p epa ed and isualized unde a UV ansillumina o a exci a ion wa eleng hs ( λex ) o 270 and 312 nm (T ansillumina o Mul iband TFX-35.MC(Vilbe Lou ma , Coll é gien, F ance). A mul i unc ional mic opla e eade Tecan In ini e m200 PRO (Tecan g oup L d., Männedo , Swi ze land) was used o measu e he abso bance and luo escence o CQDs, HSER, and HSER -CQDs [ 25 ]. Samples we e sepa a ed using he KNAUER PLATINblue V6900A HPLC sys em, which consis ed o wo P–1 pumps and an au o-sample AS–1 (Knaue , Be lin, Ge many). Mobile phase A and B consis ed o ace oni ile 0.1% o mic acid and wa e wi h 0.1% o mic acid. G adien mode had a low a e o 0.5 mL/minu e. Injec ed sample olume was 20 µ L. P io o injec ion, he samples we e dilu ed wi h mobile phase A o ge a inal concen a ion o 10 µ g/mL. HPLC was coupled wi h elec osp ay-ioniza ion quad upole ime-o - ligh mass spec ome e (ESI–qTOF–MS) B uke Maxis Impac (B uke Dal onik GmbH, B emen, Ge many). The ollowing pa ame e s o mass spec ome e we e used: End pla e o se po en ial, 500 V; capilla y po en ial, 4500 V; nebulize gas (N2) p essu e, 4 ba ; d ying gas (N2) low a e, 6 L/min −1 ; d ying empe a u e, 300 ◦ C. Mass ange was se om 50 o 3000 m/z. Posi i e ion mode was used. P io o HPLC-MS analysis, a mass spec ome e was calib a ed using ESI-TOF uning mix. The size dis ibu ion and he a e age size o he nanopa icles we e de e mined using Mal e n Ze asize (NANO-ZS; Mal e n Ins umen s L d., Wo ces e shi e, UK), which has quasielas ic lase ligh sca e ing. The 1.5 mL o he CQDs solu ion, HSER, and HSER–CQDs we e added o he polys y ene la ex cell and measu ed a an de ec o angle o 173 ◦ a 633 nm wa eleng h, wi h empe a u e o 25 ◦C, he e ac i eindex o 0.30and eal e ac i eindexo 1.59. Fou ie ans o m in a ed spec oscopy (FTIR) analysis was done by ilm o each eeze d ied samples using an FTIR spec opho ome e MATTSON 3000 FT-IR (Madison, WI, USA) wi h a wa enumbe ange o 400–4000 cm−1, wi h 4 cm−1 esolu ions. 2.4. Cul i a ion o Bac e ia Bac e ial s ain, ancomycin- esis an S aphylococcus au eus (VRSA) CCM 1767, was ob ained om he Czech Collec ion o Mic oo ganisms, Facul y o Science, Masa yk Uni e si y, B no, Czech Republic, and Esche ichia coli ATCC BAA 2340 was ob ained om Ame ican Type Cul u e Collec ion (ATCC), Uni ed S a es. The media used o he g ow h o he bac e ia was Muelle Hin on (MH) media o 15 mL in E lenmeye lasks and he bac e ia was inocula ed and cul u es we e kep in shake a 37 ◦ C a 130 pm o 24 h. Fu he , dilu ion o he cul u e was done using he MH b o h o ob ain 0.1 Abso bance (0.5 MacFa land s anda ds) a OD600 nm and used o consecu i e expe imen s [26]. 2.5. Minimum Inhibi o y Concen a ion (MIC) De e mina ion The s anda d b o h mic o-dilu ion me hod (Eu opean Commi ee on An imic obial Suscep ibili y Tes ing) was used o de e mine he suscep ibili y o bac e ial cul u es agains HSER-CQDs and i s indi idual componen , wi h he de ec ion done by he unaided eye. MIC was calcula ed using he solu ions om high o low concen a ion g adually. The di e en concen a ion o HSER–CQDs (250, 125, 75, 50, 25, and 10 µ g/mL) was added o he mic opla e wells and mixed wi h he bac e ial cul u es (0.5 MacFa land wi h inal dilu ion 1:100 using MH medium). Then he pla e was incuba ed a 37 ◦ C o 24 h. The well wi h lowes concen a ion o an ibac e ial agen wi h almos no bac e ial g ow h was conside ed he MIC and he con ol was bac e ia wi hou any ea men [26]. 2.6. G ow h Cu es and Viabili y Pe cen age The g ow h cu e was ob ained using he Mul iskan EX (The mo Fishe Scien i ic, Wal ham, MA, USA) bymeasu ing heabso bance ode e mine hean ibac e ialac i i y. Di e en concen a ionso heHSER–CQDs, Nanoma e ials 2020,10, 325 5 o 19 HSER, and CQDs we e used o check he an ibac e ial e ec s on g am posi i e and nega i e bac e ia. The posi i e con ol was only bac e ia wi hou any ea men . To compa e he ac i i y o HSER–CQDs, he bac e ia was ea ed wi h di e en concen a ion o HSER and CQDs, espec i ely. Fu he , he iabili y pe cen age was calcula ed om he abso bance alue a e 24 h, compa ing i wi h he abso bance alue o he posi i e con ol [27]. 2.7. Mic oscopy o An ibac e ial Agen s agains Bac e ia in Ambien Ligh and Li e/Dead Cell Assay Ini ially he samples we e incuba ed wi h bac e ia and HSER–CQDs conjuga e ( espec i e MIC) o 4 h a 37 ◦ C in shaking incuba o . The op ical Olympus BX51 luo escence mic oscope equipped wi h a 40 × phase con as lens was used o s udy he an ibac e ial ac i i y o he HSER–CQDs agains VRSA and E. coli. The numbe o cells obse ed pe sample was obse ed om 10 andomized mic oscopic g id ields. The li e/dead cell assay is based on wo luo escen dyes, namely, SYTO9, which pe mea es bo h damaged and in ac cell memb anes, and p opidium iodide (PI), o s ain only he cells wi h damaged cell memb anes [ 16 ]. A o al o 1 µ L o bo h he s ains we e added a e he ea men o hebac e ialcells o luo escencemic oscopy. Anin e edOlympusIX71S8F-3 luo escencemic oscope (Olympus Co po a ion, Tokyo, Japan) equipped wi h Olympus UIS2 se ies objec i e LUCPlanFLN 40 × (N.A. 0.6, WD 2.7–4 mm, F.N. 22), a me cu y a c lamp X-ci e 12 (120 W; Lumen Dynamics, Mississauga, ON, Canada), and a Came a Olympus DP73 was used o ob ain he mic oscopic image o s udy he ou comes o li e/dead bac e ial cells assay. The numbe o cells obse ed pe sample was obse ed om 10 andomized mic oscopic g id ields. The images we e p ocessed using he S eam Basic 1.7 So wa e. 2.8. Cell Memb ane B eak and Leakage Assay and In e ac ion wi h DNA Ini ially, VRSA was ea ed wi h HSER–CQDs (25 µ g/mL) and incuba ed a 37 ◦ C o 4 h in shaking condi ion. Fu he , he bac e ial cell memb ane damage and cell cy oplasmic leakage a e he ea men by HSER–CQDs was s udied by cen i uging he samples, and he supe na an was used as he empla e o PCR and he ampli ica ion was done using 16S RNA gene p ime s (16S Fo wa d- ACTGGGATAACTTCGGGAAAC, and 16S e e se- CAGCGCGGATCCATCTATAA), and con i ma ion was done using aga ose gel elec opho esis. The con ol was he supe na an o VRSA cul u e wi hou ea men and he 100 bp ladde was used. The gels we e isualized and documen ed unde Azu e c600 (Azu e Biosys ems Inc., USA). Simila ly, he genomic DNA (gDNA) om VRSA was isola ed and hen incuba ed wi h HSER–CQDs and kep o 1 h. Then, he samples we e used as he empla e o PCR eac ion and he ampli ica ion was done using 16S RNA gene p ime s, and con i ma ion was done using aga ose gel elec opho esis wi h espec o he 100 bp ladde . The con ol was he gDNA o VRSA cul u e wi hou ea men . The gels we e isualized and documen ed unde Azu e c600 (Azu e Biosys ems Inc., Dublin, CA, USA). 2.9. In luence and Toxici y o HSER–CQDs on Euka yo ic Cells (Cy o oxici y Assay and Hemolysis Assay) The PNT1A (p os a e epi helial cells), HBL-100 (mamma y gland epi helial cells), MDA-MB-468 (mamma y gland adenoca cinoma cells), and Du-145 (p os a e adenoca cinoma cells) human cell line we e cul u ed and used. The MTT (3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) assay was used o check he iabili y. B ie ly, in 50 µ L medium, he suspension o 5000 cells was added o each well o mic o i e pla es, ollowed by incuba ion o 24 h a 37 ◦ C wi h 5% CO 2 . A e 24 h ea men , 10 µ L o MTT (5 mg/mL in phospha e bu e ed saline (PBS)) was added o he cells and incuba ed o 4 h a 37 ◦ C. A e ha , MTT-con aining medium was eplaced by 100 µ L o 99.9% dime hyl sul oxide (DMSO) and incuba ed o 5 min, he abso bance o he samples was de e mined a 570 nm using In ini e m200 PRO (Tecan, Männedo , Swi ze land) [28]. Nanoma e ials 2020,10, 325 6 o 19 Fu he , esh human blood was collec ed om he olun ee , wi h signed in o med consen , by a enipunc u e om an an ecubi al ein. RBCs we e isola ed om blood and he suspensions we e washed wi h 150 mM NaCl solu ion h ee o i e imes. RBCs was incuba ed wi h he HSER-CQDs o 1 h a 37 ◦ C. The posi i e and he nega i e con ol was 0.1% T i on X-100 and PBS, espec i ely. The deg ee o hemolysis was de e mined by measu ing he abso bance o he supe na an a 540 nm, a e cen i uga ion, and calcula ed acco ding o he ollowing equa ion: h= A −Ac A100% −Ac ×100, (1) whe e his pe cen age o hemolysis; A c is he abso bance o he supe na an om nega i e con ol (PBS, pH 7.4); A is he abso bance o he supe na an om he samples incuba ed wi h he HSER-CQDs; and A 100% is he abso bance o he supe na an o posi i e con ol (0.1% T i on X-100), which causes comple e lysis o RBCs [26]. 2.10. Endogenous Reac i e Oxygen Species (ROS) P oduc ion Assay Bac e ial endogenous ROS p oduc ion was measu ed by using DCFDA Cellula Reac i e Oxygen Species De ec ion Ki (Abcam Ab113851) acco ding o he gi en p o ocol and ins uc ions o bac e ial cells [ 29 , 30 ]. To he cell suspension o VRSA, 10 µ M o 2 0 ,7 0 –dichlo o luo escein diace a e (DCFDA) was added and kep a 37 ◦ C o 30 min. in he da k condi ion ollowed by a wash using 1 × washing bu e . Then, VRSA cells we e ea ed wi h 75 µ g/mL o HSER, HSER–CQDs, and CQDs o 3 h. The luo escence in ensi y was measu ed by Tecan in ini e m200 p o (wa eleng h o exci a ion a 485 nm and emission a 535 nm. The ROS was de e mined by compa ing wi h he con ol, which was ea ed wi h H2O2showing posi i e ROS p oduc ion [31]. 2.11. Mic oscopic Analysis o HSER-CQDs In e ac ion agains Euka yo ic Cells The MDA-MB-468 (mamma y gland adenoca cinoma cells), Du-145 (p os a e adenoca cinoma cells), PNT1A (p os a e epi helial cells), and HBL-100 (mamma y gland epi helial cells) human cell line we e cul u ed and used. B ie ly, he suspension o 5000 cells in 300 µ L medium supplemen ed wi h g ow h ac o s was added o each well o mic o i e pla es, ollowed by incuba ion o 24 h a 37 ◦ C wi h 5% CO 2 o ensu e cell g ow h. A e 24 h ea men wi h HSER–CQDs, he cells we e obse ed unde an in e ed Olympus IX 71S8F-3 luo escence mic oscope (Olympus Co po a ion, Tokyo, Japan) equipped wi h Olympus UIS2 se ies objec i e LUCPlanFLN 40 ×(N.A. 0.6, WD 2.7–4 mm, F.N. 22), a me cu y a c lamp X-ci e 12 (120 W; Lumen Dynamics, Mississauga, ON, Canada), and a Came a Olympus DP73 o ob ain he b igh ield mic oscopic image using S eam Basic 1.7 So wa e. The numbe o cells obse ed pe sample was obse ed om 10 andomized mic oscopic g id ields. 3. Resul s and Discussion The syn hesis and cha ac e iza ion o HSER–CQDs, along wi h i s an ibac e ial e icacy agains VRSA and E. coli, was s udied and he MIC was de e mined. Fu he he oxici y es showed i has no oxici y agains euka yo ic cell lines (p os a e and mamma y gland epi helial cell lines (HBL-100, and PNT1A). The mechanism o ac ion was based on cell wall damage and leakage o cellula con en . Some in e ac ion o he HSER–CQDs wi h gDNA was obse ed, and assay showed negligible ROS gene a ion. VRSA and E. coli was used as model o ganism o es he an ibac e ial e ec o he HSER–CQDs due o i s pa hogenici y [26]. 3.1. Pep ide S uc u e The la ges p o ein con o ma ion clus e was eached a e ~550 ns and ep esen ed a nea ly con inuous du a ion o 21.85% o en i e simula ion (437 o 2000 ames) (Figu e 1A). The ep esen a i e ame (Figu e 1B) o he op clus e was 81,251 (o o al 100,000). The ideo Nanoma e ials 2020,10, 325 7 o 19 o he las 300 nanoseconds (ns) o simula ion, whe e he pep ide has s able con o ma ion, is shown in ESI (Elec onic Supplemen a y In o ma ion). The a oms a e chlo ide ions. Nanoma e ials 2020, 10, x FOR PEER REVIEW 7 o 21 300 nanoseconds (ns) o simula ion, whe e he pep ide has s able con o ma ion, is shown in ESI (Elec onic Supplemen a y In o ma ion). The a oms a e chlo ide ions. Figu e 1. Molecula dynamics simula ion o pep ide. (A) Clus e analysis o Molecula dynamics( MD) ajec o ies. Snapsho s we e aken e e y 50 ames and he inal numbe o ames was 2000. The op clus e was p edominan in he second hal o he simula ion. (B) The pep ide s uc u e a ame #81,251 is ep esen a i e o he op clus e . The backbone ibbon is shown in g een while he molecula su ace is shown in s anda d he e oa om colo . The e a e 10 in a-model hyd ogen bonds in his ame dona ed by he ollowing a oms: A g4 (HE, HH21), Gln10 (HE21, HE22), Gln11 (HE21), A g15 (H, HH11), A g17 (HE, HH21), and Asp18 (H). Only h ee o hese in ol ed he backbone. (C) Time e olu ion g aph o seconda y s uc u e change. A e he i s 550 ns o he simula ion, he pep ide displayed a s able seconda y s uc u e as ollows: 1–6 (coil), 7 (β-b idge), 8–11 (bend), 12–14 ( u n), 15 (coil), 16 (bend), 17 (β-b idge), 18–22 (coil). (D) Roo mean squa e de ia ions (RMSD) analysis o backbone and all a oms. The s uc u e de ia ed om he o iginal α-helix in he i s ame by ange o 5–11 Å. The RMSD o he backbone o he op clus e was below 4 Å, while o all a oms was below 6 Å. In bo h he a e age s uc u e o la ges clus e and he ep esen a i e ame, hyd ogen bonds we e obse ed a he in he side-chains han he backbone. Namely, Glu6 and Asp18 side-chains p o ided accep o oxygens o he amides o a ginines and glu amines (A g4, A g15, and A g17 in addi ion o Gln10 and Gln11). This obse a ion is impo an o in e p e a ion o FTIR ib a ional spec a. Addi ionally, indica ing ha possible change in p o ona ion s a es (due o pH change) will be de imen al o s able olding. The ime e olu ion g aph o he seconda y s uc u e (Figu e 1C) shows a combina ion o alpha helix ( esidues 5–10) bends and u ns in he i s hal o he simula ion. In he second hal o he simula ion, a s able con o ma ion was o med in he middle o pep ide. I was ea u ed by u ns ( esidues 12–14) su ounded by bends and inally a be a b idge connec ing esidues 7 and 17. The es o he pep ide endings we e domina ed by andom coil (Figu e 1C). RMSD analysis con i med hese obse a ions (Figu e 1D). O e all, RMSD we e highe by ~1 Å in all a oms compa ed o backbone-only a oms. Wi h e e ence o he i s ame (α-helix), he Figu e 1. Molecula dynamics simula ion o pep ide. ( A ) Clus e analysis o Molecula dynamics( MD) ajec o ies. Snapsho s we e aken e e y 50 ames and he inal numbe o ames was 2000. The op clus e was p edominan in he second hal o he simula ion. ( B ) The pep ide s uc u e a ame #81,251 is ep esen a i e o he op clus e . The backbone ibbon is shown in g een while he molecula su ace is shown in s anda d he e oa om colo . The e a e 10 in a-model hyd ogen bonds in his ame dona ed by he ollowing a oms: A g4 (HE, HH21), Gln10 (HE21, HE22), Gln11 (HE21), A g15 (H, HH11), A g17 (HE, HH21), and Asp18 (H). Only h ee o hese in ol ed he backbone. ( C ) Time e olu ion g aph o seconda y s uc u e change. A e he i s 550 ns o he simula ion, he pep ide displayed a s able seconda y s uc u e as ollows: 1–6 (coil), 7 ( β -b idge), 8–11 (bend), 12–14 ( u n), 15 (coil), 16 (bend), 17 (β-b idge), 18–22 (coil). ( D ) Roo mean squa e de ia ions (RMSD) analysis o backbone and all a oms. The s uc u e de ia ed om he o iginal α -helix in he i s ame by ange o 5–11 Å. The RMSD o he backbone o he op clus e was below 4 Å, while o all a oms was below 6 Å. In bo h he a e age s uc u e o la ges clus e and he ep esen a i e ame, hyd ogen bonds we e obse ed a he in he side-chains han he backbone. Namely, Glu6 and Asp18 side-chains p o ided accep o oxygens o he amides o a ginines and glu amines (A g4, A g15, and A g17 in addi ion o Gln10 and Gln11). This obse a ion is impo an o in e p e a ion o FTIR ib a ional spec a. Addi ionally, indica ing ha possible change in p o ona ion s a es (due o pH change) will be de imen al o s able olding. The ime e olu ion g aph o he seconda y s uc u e (Figu e 1C) shows a combina ion o alpha helix ( esidues 5–10) bends and u ns in he i s hal o he simula ion. In he second hal o he simula ion, a s able con o ma ion was o med in he middle o pep ide. I was ea u ed by u ns ( esidues 12–14) su ounded by bends and inally a be a b idge connec ing esidues 7 and 17. The es o he pep ide endings we e domina ed by andom coil (Figu e 1C). RMSD analysis con i med hese obse a ions (Figu e 1D). O e all, RMSD we e highe by ~1 Å in all a oms compa ed o backbone-only a oms. Wi h e e ence o he i s ame ( α -helix), he de ia ions in he backbone and all a oms we e in he ange o 4–11 Å du ing all simula ion and 7–11 Å in he second hal . Wi h e e ence o he ep esen a i e ame o he op clus e , RMSD d opped om 4–12 Å in he i s hal o 1–6 Å in he second hal o he simula ion o bo h he backbone and all a oms. Nanoma e ials 2020,10, 325 8 o 19 3.2. Syn hesis and Cha ac e iza ion o he HSER-CQD Conjuga e The HSER pep ide and CQDs in 1:1 a io helps in he o ma ion o he HSER–CQDs conjuga e, which was dialyzed and obse ed unde UV– ansillumina o and showed a bluish luo escence. The spec opho ome ic analysis was done and he abso bance was de ec ed in he spec al ange om 230 o 1000 nm and he maxima abso p ion peak o HSER and he HSER-CQDs conjuga e was ob ained a 280 nm (Figu e 2A). Ou expe imen al esul s clea ly indica e he absence o CQDs abso p ion peak in isible spec al ange. Howe e , he lowe abso bance peak o HSER de ec ed a 280 nm belongs o ansi ion o di used pi elec ons o he a oma ic ing o y osine o yp ophan [ 32 ]. On he con a y, he highe abso bance peak o HSER–CQDs was sligh ly shi ed and de ec ed a 270 nm. Such a peak p obably belongs o he π–π* ansi ion o a oma ic –C=C– and –C–C– bonds in he sp2 hyb idized domain o CQDs co e [ 33 ], which indica es ha he in e ac ion be ween HSER and CQDs has occu ed. The luo escence p ope ies o HSER and CQDs can be obse ed by wo luo escence peaks a 370 and 445 nm om he espec i e spec a, as shown in Figu e 2B. The CQDs emission peak was ound a 445 nm, which is asc ibed o su ace o molecule cen e o CQDs [ 34 ]. Such luo escence beha io o CQDs is common and usually is a ibu ed o he di e si y o pa icle size as well as he su ace s a e o CQDs [ 35 ]. The lowe emission peak o HSER was de ec ed a 370 nm, while he highe emission peak o he HSER–CQDs conjuga e was shi ed and shows he p esence o bo h peaks a 350 and 425 nm, con i ming he in e ac ion be ween HSER and CQDs. A peak de ec ed a 350 nm usually comes om he p esence o ca boxyl (–COOH) o amine (–NH2) g oups on CQDs su ace, which a e a ibu ed o n– π * ansi ion o –C=O, C–N o –C–OH bonds in he sp3 hyb idized domains [ 33 ]. This p obably indica es he possible in e ac ion be ween CQDs su ace wi h ca boxyl o amine g oup om HSER. Xiaohui Gao and co-wo ke s epo ed ha shi s in he emission peak con ibu e o he s ong in e ac ion be ween wa e molecules and exci ed dipole momen s in CQDs in solu ion [ 36 ]. Shi ing o peaks con i m he s uc u al change in HSER due o in e ac ion wi h CQDs [ 37 ]. The dialyzed HSER CQDs we e u he cha ac e ized using HPLC coupled wi h ESI–qTOF–MS o ob ain he indi idual peaks o HSER–CQD componen s, bu , due o he small size o CQDs, hey we e no spo ed in HPLC; howe e , sha p peaks o he p esence o HSER we e ob ained, as shown in he Figu e 2C. The FTIR analysis (Figu e 3A) o he syn hesized CQDs showed wo majo bands in he amide a ea, namely a 1550 and 1385 cm −1 , which a e cha ac e is ic o seconda y amines and ca boxyla es, espec i ely [ 38 ]. The b oad ange band nea 3000s cm −1 (Figu e 3B) indica es o ma ion o hyd ogen-bonded hyd oxy g oups (–OH) as compa ed wi h equi alen CA ib a ions [ 38 ]. The in ensi ies o hese h ee bands we e isibly dec eased ollowing he coa ing by HSER pep ide. In pep ides and p o eins, he amide I band (in he 1600s cm −1 ) is o en used as an indica o o seconda y s uc u e. This is based on he con ibu ions o C=O s e ching and C-N s e ching, which a e in luenced by he s eng hs o hyd ogen bonds in ol ing hem [ 39 ]. Acco dingly, he α -helix s uc u e is co ela ed wi h equency ange 1660–1648 cm −1 , whe eas he β -shee s uc u e is co ela ed wi h equency ange 1640–1625 cm −1 . Fo una ely, due o a ailable da a om MD simula ions, i is clea ha such in e p e a ion o s uc u e can be biased in his si ua ion. P edic ed HSER s uc u e appea ed o be s abilized by amide-based elec os a ic in e ac ions o side-chains (Hyd ogen bonds). In a mo e p ecise desc ip ion, he ib a ions o he backbone do no in ol e many hyd ogen bonds and hus hey can be assigned o he diso de ed seconda y s uc u e wi h equency ange 1657–1642 cm −1 [ 40 ]. The 1625 cm −1 in HSER–CQDs is an indica o o sho ange backbone agg ega ion, while he dec ease in 1650 cm −1 co ela es wi h loss o andom backbone and in e up ed side-chain in e ac ions in HSER–CQDs. The con ibu ion o amide bands is domina ed by he backbone pep ide bond, ne e heless, se e al side-chain ib a ions all in he amide I and II ange [ 41 ]. Thei ex ension coe icien (M −1 cm −1 ) con ibu ions in he amide I a e in he ollowing ange: A g ( ε =300–490), Gln (ε=360–380), and Lys ( ε =60–130). As o he amide II egion (in he 1500s cm −1 ) hey a e in he ollowing ange: Gln ( ε =220–240), Asp ( ε =290–380), Glu ( ε =450–470) and Lys (ε=60–130). Fu he mo e, he Cys hiol (–SH) s e ching peak a 2551 cm −1 [ 41 ] was absen in bo h HSER Nanoma e ials 2020,10, 325 9 o 19 and HSER–CQDs spec a, sugges ing ha sul ide b idges we e o med in bo h cases. O e all, FTIR ib a ional spec a con i med he coa ing o CQDs wi h HSER pep ide, mos ly ia s ong non-co alen agg ega ion. This was cha ac e ized by amide-based sho - ange hyd ogen bonds (1625 cm −1 ) be ween pep ide backbone and seconda y amines, ca boxyla es, and hyd oxyl g oups o he CQDs su ace. Nanoma e ials 2020, 10, x FOR PEER REVIEW 9 o 21 = 360–380), and Lys (ε = 60–130). As o he amide II egion (in he 1500s cm −1 ) hey a e in he ollowing ange: Gln (ε = 220–240), Asp (ε = 290–380), Glu (ε = 450–470) and Lys (ε = 60–130). Fu he mo e, he Cys hiol (–SH) s e ching peak a 2551 cm −1 [41] was absen in bo h HSER and HSER–CQDs spec a, sugges ing ha sul ide b idges we e o med in bo h cases. O e all, FTIR ib a ional spec a con i med he coa ing o CQDs wi h HSER pep ide, mos ly ia s ong non-co alen agg ega ion. This was cha ac e ized by amide-based sho - ange hyd ogen bonds (1625 cm −1 ) be ween pep ide backbone and seconda y amines, ca boxyla es, and hyd oxyl g oups o he CQDs su ace. Figu e 2. (A) The luo escence spec a we e ob ained wi h he exci a ion o 320 nm and emission a ange o 360 o 850 nm; and he bo le wi h black cap con ains Ca bon Quan um Do s(CQDs) and he b own-capped bo le con ains he HSER-CQDs conjuga e showing bluish luo escence. (B) The Abso bance spec a ob ained a a ange o 230 o 1000 nm. (C) Samples we e sepa a ed on column Zo bax eclipse AAA C18 (3.5 µm pa icles; 150 × 4.6 mm) using he KNAUER PLATINblue V6900A HPLC sys em, which consis ed o wo P 1 pumps and an au osample AS 1 (Knaue , Be lin, Ge many). Mobile phase A consis ed o wa e wi h +0.1% o mic acid. Mobile phase B consis ed o ace oni ile wi h +0.1% o mic acid. G adien mode: 0 min 3% B– > 30 min 97% B– > 40 min 97% B– > 42 min 3% B– > 50 min 3% B– > STOP. Flow a e was 0.5 mL/min. Injec ed sample olume was 20 µL. P io o injec ion he samples we e dilu ed wi h mobile phase A o ge a inal concen a ion 10 µg/mL. HPLC was coupled wi h ESI-QqTOF mass spec ome e B uke Maxis Impac (B uke Dal onik GmbH, B emen, Ge many). The ollowing pa ame e s o mass spec ome e we e used: End-pla e o se po en ial, 500 V; capilla y po en ial, 4500 V; nebulize gas (N 2 ) p essu e, 4 ba ; d ying gas (N 2 ) low a e, 6 L/min −1 ; d ying empe a u e, 300 °C. Mass ange was se om 50 o 3000 m/z. Posi i e ion mode was used. P io o HPLC-MS analysis a mass spec ome e was calib a ed using ESI-TOF uning mix. Figu e 2. ( A ) The luo escence spec a we e ob ained wi h he exci a ion o 320 nm and emission a ange o 360 o 850 nm; and he bo le wi h black cap con ains Ca bon Quan um Do s(CQDs) and he b own-capped bo le con ains he HSER-CQDs conjuga e showing bluish luo escence. (B) The Abso bance spec a ob ained a a ange o 230 o 1000 nm. ( C ) Samples we e sepa a ed on column Zo bax eclipse AAA C18(3.5 µ mpa icles;150 × 4.6mm)using heKNAUERPLATINblueV6900AHPLCsys em,whichconsis ed o wo P 1 pumps and an au osample AS 1 (Knaue , Be lin, Ge many). Mobile phase A consis ed o wa e wi h +0.1% o mic acid. Mobile phase B consis ed o ace oni ile wi h +0.1% o mic acid. G adien mode: 0 min 3% B– >30 min 97% B– >40 min 97% B– >42 min 3% B– >50 min 3% B– >STOP. Flow a e was 0.5 mL/min. Injec ed sample olume was 20 µ L. P io o injec ion he samples we e dilu ed wi h mobile phase A o ge a inal concen a ion 10 µ g/mL. HPLC was coupled wi h ESI-QqTOF mass spec ome e B uke Maxis Impac (B uke Dal onik GmbH, B emen, Ge many). The ollowing pa ame e s o mass spec ome e we e used: End-pla e o se po en ial, 500 V; capilla y po en ial, 4500 V; nebulize gas (N 2 ) p essu e, 4 ba ; d ying gas (N 2 ) low a e, 6 L/min −1 ; d ying empe a u e, 300 ◦ C. Mass ange was se om 50 o 3000 m/z. Posi i e ion mode was used. P io o HPLC-MS analysis a mass spec ome e was calib a ed using ESI-TOF uning mix. Howe e , he pa icle size dis ibu ion showing a e age pa icle size and he ze a po en ial o he p epa ed HSER–CQDs we e analyzed using DLS a e pu i ica ion, using dialysis in D-Tube maxi dialyze ubes agains Mili-Q (Figu e 3C,D). The ea e , he o ma ion o nanopa icles was also con i med by ze a size and po en ial measu ing. Ze a size esul s ob ained indica es ha he size o CQDs is 4 ± 2 nm, while he size o HSER-CQDs was ound o be 23 ± 3 nm (Figu e 3D). Fu he mo e, he alue o he ze a po en ial showed ha he lowes ze a po en ial was ound in case o CQDs a − 3 mV, and he highes ze a po en ial show HSER wi h 33 mV. Finally, he HSER-CQDs conjuga e e eal he change o ze a po en ial a e in e ac ion o be 23 mV, showing he endencies o agg ega e which can be connec ed wi h pep ide agg ega ion on he su ace o nanopa icles. Thus, a e he cha ac e iza ion o HSER–CQDs was comple ed, i was used o u he s udy he an ibac e ial ac i i y agains pa hogenic bac e ial s ains. Nanoma e ials 2020,10, 325 16 o 19 showed he p esence o a high amoun o dead bac e ial cells. The mechanism o ac ion is based on change in s uc u e o HSER a e in e ac ing wi h he CQDs, which was suppo ed by FTIR esul s showing ha he cell wall dis up ion occu ed along wi h some in e ac ion in p esence o gDNA, which s op i s ampli ica ion. Finally, he HSER–CQDs we e ound o be compa ible wi h RBCs and non oxic agains no mal human epi helial cells, wi h he help o MTT and hemoly ic assay and u he con i med by mic oscopic analysis. Thus, HSER–CQDs can be used as a subs i u e o an ibio ics. Supplemen a y Ma e ials: The ollowing a e a ailable online a h p://www.mdpi.com/2079-4991/10/2/325/s1, Figu e S1: G ow h cu e and iabili y pe cen age in p esence o HSER, Figu e S2: G ow h cu e and iabili y pe cen age in p esence o CQDs, Figu e S3: ROS, Figu e S4: Op ical image o VRSA and E. coli, Figu e S5: The cy o oxici y es . Au ho Con ibu ions: All he au ho s esea ched da a, w o e he manusc ip , and e iewed and edi ed he a icle subs an ially. A.M. (Aninda Mazumda ) pe o med mos o he expe imen s including designing pep ide, syn hesis o CQDs, mic obiological es s, and w o e he mos pa o he manusc ip . Y.H. helped in he MD simula ion and de elopmen o he mechanism o ac ion. V.M. helped in he syn hesis o he pep ide and he cha ac e iza ion o he conjuga e. H.M., R.G. and S.B. helped wi h di e en expe imen s. A.M. (Ami a a Moulick) supe ised he whole expe imen . All au ho s ha e ead and ag eed o he published e sion o he manusc ip . 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