Arabic gum plus colistin coated moxifloxacin-loaded nanoparticles for the treatment of bone infection caused by Escherichia coli

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Acta Biomaterialia

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Arabic gum plus colistin coated moxiﬂoxacin-loaded nanoparticles for the treatment of bone infection caused by Escherichia coli

J.J. Aguilera-Correa

^a^,^b^,¹

, M. Gisbert-Garzarán

^a^,^c^,¹

, A. Mediero

^d

, R.A. Carias-Cálix

^e

, C. Jiménez-Jiménez

^a^,^c

, J. Esteban

^b^,^f^,^∗

, M. Vallet-Regí

^a^,^c^,^∗

a Departamento de Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid, Instituto de Investigación Sanitaria Hospital 12 de Octubre i+12, Plaza Ramón y Cajal s/n, Madrid 28040, Spain

b Networking Research Centre on Infectious Diseases (CIBER-ID), 28029 Madrid, Spain

c Networking Research Centre, Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid 28029, Spain

d Bone and Joint Unit, IIS- Fundación Jimenez Diaz, UAM, Avenida Reyes Católicos, 2, Madrid 28037, Spain

e Pathology Department, Fundacion Jimenez Diaz University Hospital, UAM, Madrid, Spain

f Clinical Microbiology Department, IIS-Fundación Jiménez Diaz, UAM, Avenida Reyes Católicos, 2, Madrid 28037, Spain

a rt i c l e i nf o

Article history:

Received 3 August 2021 Revised 20 September 2021 Accepted 7 October 2021 Available online 13 October 2021 Keywords:

Osteomyelitis Escherichia coli Bioﬁlm Nanoparticles Arabic gum Colistin Moxiﬂoxacin

a b s t r a c t

Osteomyelitisisaninﬂammatoryprocessofboneandbonemarrowthatmayevenleadtopatientdeath.

EventhoughthisdiseaseismainlycausedbyGram-positiveorganisms,theproportionofboneinfections causedbyGram-negativebacteria,suchasEscherichiacoli,hassignificantlyincreasedinrecentyears.In thiswork,mesoporoussilicananoparticleshavebeenemployedasplatformtoengineerananomedicine abletoeradicateE.coli-relatedboneinfections.Forthatpurpose,thenanoparticleshave beenloaded withmoxifloxacinand further functionalizedwithArabicgum and colistin(AG+CO-coated MX-loaded MSNs).ThenanosystemdemonstratedhighaffinitytowardE.colibiofilm matrix,thankstoAGcoating, andmarkedantibacterialeffectbecauseofthebactericidaleffectofmoxifloxacinandthedisaggregating effectofcolistin.AG+CO-coatedMX-loadedMSNs wereabletoeradicatethe infectiondevelopedona trabecularboneinvitro and showedpronouncedantibacterial efficacyinvivoagainst anosteomyelitis provoked byE. coli.Furthermore, AG+CO-coated MX-loadedMSNs wereshowntobe essentially non- cytotoxicwithonlyslighteffectoncellproliferationandmildhepatotoxicity,whichmightbeattributed tothenature ofbothantibiotics. Inviewoftheseresults,thesenanoparticles maybeconsideredas a promisingtreatmentforboneinfectionscausedbyenterobacteria,suchasE.coli,andintroduceageneral strategyagainstboneinfectionsbasedontheimplementationofantibioticswithdifferentbutcomple- mentaryactivityintoasinglenanocarrier.

Statementofsigniﬁcance

Inthiswork,weproposeamethodologytoaddressE.coliboneinfectionsbyusingmoxifloxacin-loaded mesoporoussilicananoparticlescoatedwithArabicgum containingcolistin(AG+CO-coatedMX-loaded MSNs).TheinvitroevaluationofthisnanosystemdemonstratedhighaffinitytowardE.colibiofilmmatrix thankstotheArabicgumcoating,adisaggregatingandantibacterialeffectofcolistin,andaremarkable antibiofilmactionbecauseofthebactericidalabilityofmoxifloxacinandcolistin.Thisanti-E.colicapacity

Abbreviations: AG, arabic gum; AG + CO, arabic gum containing colistin; APTES, 3-(aminopropyl)triethoxysilane; BHI, brain-heart infusion; CAMHB, cation adjusted Müller- Hinton broth; CFU, colony-forming unit; CO-FITC, colistin labeled with ﬂuorescein isothiocyanate; CO, colistin; CTAB, cetyltrimethylammonium bromide; Cyp3A, cytochrome P450 3A4; DLS, dynamic Light Scattering; DMSO, dimethyl sulfoxide; FTIR, Fourier transformed Infrared; GFP, green ﬂuorescent protein; HBSS, Hanks’ balanced saline solution;

Hs, hepatocytes; IL-6, interleukine 6; KCs, Kupffer cells; MBC, minimum bactericidal concentration; MBEC, minimum biofilm eradication concentration; MBIC, minimum biofilm inhibitory concentration; MIC, minimum inhibitory concentration; MSNs, mesoporous silica nanoparticles; MX, RhB moxifloxacin; NPCs, non-parenchymal cells; PBS, phosphate buffer saline; PI, propidium iodide; RANKL, receptor activator for nuclear factor κB Ligand; RhB, Rhodamine B isothiocyanate; SEM, scanning electron microscopy;

TEOS, tetraethyl orthosilicate; TGA, thermogravimetric Analysis; TNF- α, tumour necrosis factor α; TVX, trovaﬂoxacin.

∗Corresponding authors at: Departamento de Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, Madrid 28040, Spain.

E-mail addresses: john_j2a@hotmail.com (J.J. Aguilera-Correa), migisber@ucm.es (M. Gisbert-Garzarán), aranzazu.mediero@quironsalud.es (A. Mediero), rafael.carias@quironsalud.es (R.A. Carias-Cálix),carlaj05@ucm.es (C. Jiménez-Jiménez),jestebanmoreno@gmail.com (J. Esteban),vallet@ucm.es (M. Vallet-Regí).

1 These authors contributed equally to this work.

https://doi.org/10.1016/j.actbio.2021.10.014

1742-7061/© 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

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ofAG+CO-coated MX-loadedMSNswas broughtoutinaninvivorabbitmodel ofosteomyelitiswhere thenanosystemwasabletoeradicatemorethan90%ofthebacterialloadwithintheinfectedbone.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Osteomyelitis is an inﬂammatory process of bone and bone marrow caused by at least one microorganism, which causes lo- calbonedestruction,necrosis,andappositionofnewboneandcan compromiseboneorjointinfection[1].Despiteitsincidenceisal- most 22 cases per 100,000 person-years [2], the implications of thisdiseasearebeyondthesenumbers,includingmortality[3].

Althoughosteomyelitiscanbevirtuallycausedbyanymicroor- ganism, boneinfections are mainlycaused byGram-positive bacteria,suchasStaphylococcusaureus,whichisresponsibleforupto 90% ofthe casesofpyogenicosteomyelitis [1].However,the pro- portion of bone infections caused by Gram-negativebacteria has signiﬁcantly increasedoverthelast fewyears [4,5].Among them, enterobacteria,like Escherichiacoli,haveattractedmuch attention owingtotheirabilitytoreachantibioticmulti-resistance[6,7].Be- sides,thebacterialpathogenyofthisinfectioninvolvesthebioﬁlm development, which is a growthformincreases the resistanceto multiple adverse situations, includingphagocytosis by phagocytic cellsfromimmunesystems,aswellasantibiotics[8].Forthisrea- son, themostfrequenttreatmentsforosteomyelitisincludeintra- venousantibioticadministrationtogetherwithsurgicalremovalof portionsofinfectedordeadbone[9,10].

In the last few decades, the applicationof nanotechnology to medicine, so-called nanomedicine, has attracted much attention andit isexpectedtorevolutionizethebiotechnologicalandphar- maceutical industries shortly. In thisregard, nanoparticles acting as drug delivery vehicles account for 75% of the market share of approved nanomedicines [11]. Among the different types of nanocarriers, mesoporous silica nanoparticles (MSNs) are consid- eredaspromisingcandidates fordrugdeliveryowing tofeatures, such as large surface areas (ca. 1,000 m²/g) and pore volumes

Scheme 1. Mesoporous silica nanoparticles (green) loaded with moxiﬂoxacin (yel- low) (a) and coated with Arabic gum (purple) plus colistin (blue).

(ca. 1 cm³/g), tunable pore size and morphologies, ease of sur- facemodiﬁcation,andbiocompatibility[12–15].As aresult,MSNs havebeenwidelyappliedtotreatseveraldiseases,includingcom- plexbonediseases,suchasosteoporosis,bonecancerandbonein- fection [16]. Regarding the latter, the suitability ofloading MSNs withlow-molecular-weightantibioticsforeliminatingE. coli bone infectionshasbeenreported[17–19].Asidefromtreatingbonein- fections,endowing thenanoparticleswithabilityto recognizein- fectionis ofmajorimportance. Inthisregard, some authorshave reportedtheuseofantibodies,aptamers,dendrimers,orproteins, amongothers,totargetboneinfection[20].

Inthiswork, wehaveemployed MSNstoengineer a drugde- livery nanovehicle forthe potential treatment of bone infections causedbyE.coli(Scheme1).Becauseofthepotentialtranslationof thisnanomaterial, two clinicallyrelevant antibiotics,moxifloxacin (MX)andcolistin(CO),wereselectedaspayloads.MXisafourth- generationfluoroquinolone antibacterialagentwithabroadspec- trum ofactivity, encompassing Gram-negative andGram-positive bacteria[21],that hasbeenused inmonotherapyinbone-related infection[22–24].COisapolymyxinagent(polymyxinE)thathas proven to be effectiveagainst Gram-negativeinfections [25],and wasselectedbecauseofitsanti-E.coliabilityvouchedbyprevious invitroandinvivostudiesofprostheticjointinfection[26].Given thepivotalroleofbiofilmformation duringthistypeofinfection, we aimed to endow MSNs withselectivity forthe E. coli biofilm modeltoenhancetheefficacyofthetreatment.Forthatpurpose, thesurface ofnanoparticleswasmodifiedwithArabic gum(AG), abranched-chain,complexpolysaccharidecomposed of1,3-linked beta-D-galactopyranosyl monomers connected to the main chain through1,6-linkages[27],whosedegradationbysecretedbacterial enzymeswasobserved to improvethe retentionof MSNson the biofilm.Furthermore,itwasobservedthattheuseoftheAGcoat- ingimprovedtheadsorptionofCOonthesurfaceofMSNs.Finally, thebactericidaleffectofthisbiocompatiblenanocarrierwasexten- sivelyanalysed invitroandinvivo,showingpromisingresults.To the best of our knowledge, this is the first time that MSNs are engineered to carry low molecular weight anda high molecular weightantibiotic atthe sametime, insteadofjustone antibiotic, whileachievingsignificanttargetingcapacityforE.colibiofilmand substantialefficacyinvivo.

2. Materialsandmethods

2.1. SynthesisofMCM-41mesoporoussilicananoparticles

The following compounds were purchased from Sigma- Aldrich(USA):Tetraethylorthosilicate(TEOS);Ammoniumnitrate;

Cetyltrimethylammonium bromide (CTAB); Rhodamine B isothiocyanate(RhB);3-(Aminopropyl)triethoxysilane(APTES).

Mesoporous silica nanoparticles were synthesized through a modiﬁcation of the Stöber method [28]. For that purpose, H₂O (480 mL), NaOH(2 M, 3.5mL) andCTAB(2.74 mmol, 1g), were mixedina1-Lﬂaskandheatedto80°C.Then,TEOS(22.39mmol, 5 mL) was added dropwise (0.33 mL/min) over 15 min and the whole mixture wasthen stirred at80 °Cfor 2 h. After that, the nanoparticleswere collectedby centrifugation andwashed twice withwaterandonce withethanol. The organictemplate wasre- movedbyionicexchange,usingasolutionofNH₄NO₃ (10mg/mL)

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inethanol(95%).Forthatpurpose,nanoparticlesweredispersedin 350 mLof such solution,reﬂuxed for3h and subsequentlycen- trifuged and washed withwaterand ethanol. The whole process wasrepeatedtwotimes,andtheﬁnalsurfactant-freenanoparticles werestoredinabsoluteethanol.

The biological experiments were performed using Rhodamine B-labeled MSNs.Forthisend, RhB(0.002 mmol,1.07mg)wasre- acted with APTES (0.009 mmol, 2.2 μL) in 40 μL of ethanol for 2h.Then,thismixturewasgentlymixedwithTEOS(22.39mmol, 5 mL) and the synthesis of MSNs was carried out as described above.

ThenanoparticleswerecharacterizedintermsofFourierTrans- formed Infrared (FTIR) spectroscopy, Thermogravimetric Analysis (TGA), DynamicLightScattering (DLS), Zetapotential, andN₂ ad- sorption analysis. FTIR spectra were collectedin a Nicolet Nexus (Thermo FisherScientiﬁc)equippedwithaGoldengateattenuated total reﬂectance device, averaging 64 scans in the range 4,000–

400 cm⁻¹ (resolution 1 cm⁻¹). TGA measurements were carried out in a Perkin Elmer Pyris Diamond TG/DTA analyser, applying 5°C/minheatingrampsfromrt to600°C.DLSandZetapotential measurementswereperformedinaZetasizerNanoZS(MalvernIn- struments)equippedwitha633nmlaser.Samplesweredispersed indistilledwaterwithsonicationandplacedinaDTS1070dispos- able folded capillary cell (Malvern instruments) for data acquisi- tion.N₂adsorptionanalysis(adsorptionanddesorptionisotherms) were obtained in a Micromeritics ASAP 2020. Samples were de- gassedundervacuumfor24handanalyzedat77K.Poresizewas estimatedfromthemaximumoftheporesizedistributionplot.

2.2. Arabicgumcoatingofnanoparticlesandantibioticreleasefrom coatednanoparticles

Arabic gum coating was performed by adapting a methodol- ogypreviously described[29].TheuseofArabicgumisbasedon the enterobacterialabilityto degradeanduseitascarbonsource [30,31].MSNswereinitiallyfunctionalizedwithAGbeforethean- tibioticsincorporationtofindouttheconditionsthatprovidedsig- nificant AGdeposition onto thesurface. Briefly, Arabicgum (10%, w/v) was preparedby dissolving 4 g of Arabic gum from acacia treepowder(SigmaAldrich,USA)in20mLdistilledwater.Theso- lution was stirred withlow heat (40–45 °C) for60 min using a hotplatemagneticstirrerandwaslefttocooldowntoroomtem- perature.Then, 1mLofArabicgum10% wasdepositedina4mL glass recipientundervigorous agitationatroom temperatureand mixed with1 mL of water with12 mg/mL of MSNsfor 10 min.

Then, the nanoparticleswere centrifuged andwashed twice with distilledwater,leadingtoAG-coatedMSNs.

For the synthesis of Arabic gum plus colistin-coated MSNs, 50 mgof colistin sodium methanesulfonate(Sigma Aldrich,USA) were mixed with1 mL of Arabic gum 10% and the coating was carriedoutasdescribedabovetoyieldAG+CO-coatedMSNs.

ThecoatednanoparticleswerecharacterizedintermsofFourier TransformedInfrared(FTIR)spectroscopy,ThermogravimetricAnal- ysis(TGA),TransmissionElectronMicroscopy(TEM),DynamicLight Scattering (DLS) andZetapotential. TEMimageswere takenona JEOL JEM 1400. Samples were dispersed in distilled water under sonicationandthenfewdropsweredepositedontocarbon-coated coppergrids.

2.3. Antibioticloadingintothenanoparticleandantibioticrelease

One millilitre of a 5 mg/mL solution of moxiﬂoxacin (Sigma Aldrich,USA)(MX)inHEPES buffer[32] (Lonza, Switzerland)was addedto12mgofMSNs.TheseMSNswereloadedat500rpmand 5 °C for 24 h [33]. After loading, MSNs were washed two times withHEPESbuffer(MX-loadedMSNs).

To determine the moxifloxacin release from loaded MSNs, 12 mg of MSNs were suspended in 1 mL of phosphate buffer saline(PBS) (pH=7.4)(Lonza, Switzerland).This suspensionwas placedwithanothermillilitreofPBSintothelowerchamberfrom aTranswell® 6-wellplate(Corning,USA). Then,the uppercham- ber of the Transwell® 6-well platewas placed and1 mL of PBS wasadded. This buffer wasselected because is one of the most usedbuffer forantibiotic release[34–38].Thefinal concentration ofnanoparticleswas4mg/mLperwell(n= 4).Theplatewasin- cubatedat 37 °C and5% CO₂.Periodically,300 μL ofeach upper chamberfromwellwere sampledandreplacedby 300μLofnew PBS. These300 μL were used todetermine moxifloxacin concentration by measuring the fluorescence using an excitation wave- lengthof294nmandanemissionwavelengthof503nm[39],and acalibration curve madewithaconcentration rangefrom125 to 0.122μg/mL.Thisexperimentwasperformedfourtimes.

TodeterminethecolistinreleasefromArabicgumpluscolistin coatedMSNs,12mgofMSNswerecoatedwith50mgofcolistin inpresence orabsence of1 mLof Arabic gum20%. Colistinpre- viously labeled withﬂuoresceinisothiocianate (CO-FITC) to mon- itor the release. For that reason, 50 mg of colistin and 0.1 mg ofFITCwere dissolved in DMSOandstirredovernight atRT. The mixturewasthen precipitatedincoldether/acetone (90:10),centrifuged andwashed withethanoluntil no FITCwasobserved in thesupernatant.CO-FITCMSNswere suspendedin1mLofphos- phate buffer saline(PBS) (Lonza, Switzerland) to evaluatethe re- leasekinetics.Thismillilitrewasplaced withanother millilitreof PBSintothelowerchamberfromaTranswell® 6-wellplate(Corn- ing,USA).Then,theupperchamberoftheTranswell® 6-wellplate wasplacedand1mLofPBSwasadded.Theﬁnalconcentrationof nanoparticleswas4mg/mLperwell (n= 3).Theplatewasincu- batedat37°Cand5%CO₂.Regularly,300μLofeachuppercham- berfromwell weresampledandreplacedby 300μLofnewPBS.

These300 μLwere used to determinecolistin-FITCconcentration by measuring the ﬂuorescence usingan excitation wavelength of 490nmandanemissionwavelengthof525nm,andacalibration curvemadewithaconcentrationrangefrom500to0.244μg/mL.

2.4. Microbiologicalstudies

E. coli ATCC 25922-GFP strain was used in all microbiological studies. This strain can produce a green ﬂuorescent protein (GFP).Thestrainwaskeptfrozenat–80°Cuntilexperimentswere performed. Accordingto therecommendationsof thecommercial house, this strain was grown in tryptic soy broth (BioMérieux, France) supplementedwith100μg/mLofampicillin (Merck,USA) at37 °Cin5% CO₂.Thepurityof itsaxenic culture wascorrobo- ratedevery dayby inoculating each broth ona blood tryptic-soy agar(BioMérieux,France).

2.4.1. Bacteria-nanoparticleinteraction

TheE.coli-nanoparticleinteractionwasevaluatedbyusingfour differentexperiments, (1)useof Arabic gumcoating asa carbon source by E. coli, (2) E. coli bioﬁlm-nanoparticles interaction, (3) bactericidal ability of Arabic gum plus colistin coating against E.

coli and, (4) E. coli effect on the release of drugs loaded in the nanoparticles.

Toevaluate theuse ofArabicgum coating asa carbonsource by E. coli, 500 μL of PBS with 10 mg/mL (1% p/v) of each type ofnanoparticle (MSNsandAG-coated MSNs)were depositedin a well from a 24-well plate. Then, 10 μL of a bacterialsuspension with3.18× 10⁶ CFU/mL were added.PBS supplementedwith1%

of Arabicgum wasused as positive control. Then, the platewas incubatedstaticallyat37°Cand5%CO₂for24h.Afterincubation, 300 μL of each well were placed in a 96-well ﬂat-bottom plate 220

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(Thermo Fisher Scientiﬁc, USA) and their ﬂuorescence was mea- suredusingan excitationwavelengthof488nm andanemission wavelength of510nm. Thisexperimentwasperformedby dupli- cateandfourtimes(n=8percondition).

Todescribe theE. colieffecton thereleaseofdrugsloaded in AG-coatedMSNs,12mgofMSNswereloadedwith500μLofpro- pidium iodide (PI) in water(500 μg/mL) (SigmaAldrich, USA) at 750rpmand5°Cfor24h.Afterloading,thesenanoparticleswere rinsed one time with 1 mL of distilled water. After this, 12 mg of each PI-loaded MSNswere suspended in 1 mL of brain-heart infusion (BHI) (BD, USA) to determine the PI release from AG- coatedPI-loadedandnoncoated PI-loadedMSNs.This suspension wasmixed1:1withanothersuspension(10⁸CFU/mLofbacteriain BHI)intothelowerchamberfromaTranswell® 6-wellplate.Then, the upper chamber of the Transwell® 6-well plate was inserted and1mLofBHIwasadded.Theﬁnalconcentrationofnanoparti- cleswas4mg/mLperwell. Theplatewasincubatedat37°Cand 5%CO₂.Periodically,300μLofeachupperchamberfromwellwere sampledandreplacedby300μLofsterileBHI.These300μLwere used todeterminethePIconcentration bymeasuring theﬂuores- cence usinganexcitation wavelengthof493nm andanemission wavelengthof636nm, anda calibrationcurve madewitha con- centrationrangefrom250to0.122μg/mL.Thebacterialconcentra- tion wasestimatedby using awell from a6-well platewiththe same bacterial concentration butwithout MSNs. This experiment wasperformedbytriplicate.

To study E. coli biofilm-nanoparticles interaction, 100 μL of saline 0.9% NaCl (B. Braun, Germany) with 3.23 × 10⁸ CFU/mL were placedineachwellofa96-well flat-bottomplateandincu- batedstaticallyat37°Cand5%CO₂for1.5h.Thesupernatantwas then removed, andeach well wasrinsed two timeswith 150 μL ofsaline.Then,200μLofwound-likemediumwereaddedtoeach well, andthe platewasincubatedstatically at37 °C and5% CO₂ for48h.Wound-likemediumiscomposedby5mLofBoltonbroth (SigmaAldrich,USA),4.5mLofbovineadultserum(SigmaAldrich, USA), and0.5 mLof lakedhorse blood(Oxoid, USA)[40,41].The useofwound-likemediumhadnootherpurposebutmimickingin vitrothe nutritionalconditionsthat a bacteriumcausingbone in- fectioncouldfindinvivo.Afterincubation,thesupernatantwasre- moved,andeachwellwasrinsedtwotimeswith200μLofsaline.

Then, 150μLofsaline with2mg/mLofeach typeofMSN (MSN, AG-coatedMSN,andAG+CO-coatedMSNs)wereaddedtothecor- respondingwellsandincubatedat100rpm,37°C,and5%CO₂ for 30minand3h.Afterthat,eachwellwasrinsedagainwith200μL of saline and stained with 1% of safranin, according to a previously reportedmethodology [42].The experimentwasperformed bytriplicate(n=24percondition).

TodemonstratethebactericidalabilityofArabicgumpluscol- istincoating againstE.coli,500μLofsalinewith1× 10⁸CFU/mL plus 500 μLofsaline with4mg/mLofeach type ofnanoparticle (MSNs,AG-coatedMSNs,andAG+CO-coatedMSNs)wereplacedin a 2mL tube(finalconcentration of2mg/mL). Thisconcentration ofbacteria waschosen tobringto lighttheantibacterial effectof MSNs without needing the bacterial growth.Each tube wasagi- tatedat1.400rpmand37°Cfor30min.Afterthis,150μLofeach tube were mixed with150 μLof tryptic-soy brothsupplemented with20%alamarBlue(BIO-RAD,USA)[43]inawellfroma96-well flat-bottom plate, andwere incubated at 100 rpm and 37 °C for 2h.Thefluorescencefromeachwell wasthenmeasuredusingan excitation wavelength of560 nm andan emission wavelength of 590 nm. This experiment was performed four times (n = 4 per condition).Tosupportvisuallythenumericalresults,theprevious experiment wasanalysed using transmissionelectron microscopy (TEM). The protocolforTEM hasbeen described previously [44]. Semithinsections(0.6 μm)forlight microscopyandthinsections (60 nm) forTEMof resin-included bacteria were cut usinga Le-

icaUltracutultramicrotomeUC7(Leica).Sectionswerecollectedon 200meshnickelgridsandexaminedusingaJeol JEM1400trans- missionelectronmicroscope(JeolLtd,Tokyo,Japan).

2.4.2. Minimalinhibitoryconcentrationandminimalbactericidal concentration

Minimuminhibitoryconcentrations(MIC)weredeterminedus- ingthepreviouslydescribedbrothmicrodilutionmethod[45]with onemodification.TheMICistheminimumconcentrationrequired toinhibitthebacterialvisiblegrowth.Themainmodificationcon- sisted of supplementing all the broth used with 100 μg/mL of ampicillin.Inbrief,aseriesofnanoparticleconcentrationsstarting from2,000to1.953μg/mLwithatwo-folddilutionwereaddedto cationadjustedMüller-Hintonbroth(SigmaAldrich,USA)(CAMHB) toa final volume of100 μLper well. Onehundred microlitresof bacterialsuspensioninCAMHBcontainingapproximately1.6× 10⁶ colony-formingunitspermillilitre(CFU/mL)wasaddedtoaCostar 96-wellround-bottompolypropyleneplate(CorningInc.,USA)fol- lowedbystaticincubationat37 °Cand5%CO₂ foratleast20h.

Afterincubation,MICwasdeterminedmeasuringfluorescenceus- ing an excitation wavelength of 488 nm and an emission wave- lengthof510nm.Minimumbactericidalconcentration(MBC)were determined using the flash microbiocide method previously described[46]. TheMBCis definedastheminimum concentration requiredto kill a certainbacterial concentration. Briefly,10 μLof each wellwere mixedafter24hincubation with190 μLoftryp- ticsoy brothinanew96-wellplate,whichwasfurtherincubated statically at 37 °C and 5% CO₂ for 24 h. After incubation, MBC wasdeterminedbymeasuringthefluorescence,usinganexcitation wavelengthof488nmandanemissionwavelengthof510nm.The experimentswereperformedbytriplicate.

2.4.3. Minimalbioﬁlminhibitoryconcentrationandminimalbioﬁlm eradicationconcentration

Minimal biofilm inhibitory concentrations (MBIC) and mini- malbiofilmeradicationconcentrationsweredeterminedusingthe methodologypreviouslydescribed[47].TheMBICistheminimum concentration requiredtoinhibitthevisiblegrowth ofabacterial biofilm.ForMBIC,biofilmformationonpegsfromtheCalgaryde- vicewasinduced by inoculating200 μLoftryptic-soy brothcon- taining10⁶ CFU/mL ofbacteria per well in a 96-well flat-bottom plate(ThermoFisherScientific, Massachusetts,UnitedStates).The lid(ThermoFisherScientific)oftheCalgarydevicewasthenplaced andthe platewasincubated inturmoil at37 °C and5% CO₂ for 24h.Afterincubation,thepegsfromthelidwererinsedtwotimes inwellscontaining200μLofsaline.Afterwards,thelidwasplaced inaplatewithdifferentMSNconcentrationsstartingfrom2,000to 1.953 μg/mLwitha two-folddilutionwereaddedto CAMHBto a finalvolumeof200μLperwellandwasincubatedbystaticincu- bationat37°Cand5%CO₂foratleast20h.Afterincubation,MBIC wasdeterminedbymeasuringthefluorescence,usinganexcitation wavelengthof488nmandanemissionwavelengthof510nm.The MBEC is the minimum concentration required to kill a bacterial biofilm.ForMBEC,thelidfromtheMBICwasrinsedtwotimesin aplatewithwellscontaining200μLofsaline0.9%NaCl,placedin aplatewith200μLoftryptic-soybroth,andincubatedstaticallyat 37°Cand5%CO₂for24h.Afterincubation,MBECwasdetermined by measuringthe fluorescence,usingan excitation wavelengthof 488nmandanemission wavelengthof510nm. Theexperiments wereperformedbytriplicate.

2.4.4. Anti-biofilmefficacyofAG+CO-coatedMX-loadedMSNs A four-mL flat-bottom sterile tube with25 mg of bovine tra- becularbone(Bio-OssSpongiosafrom0.25to1mm;Inibsa,Spain) was rinsed with 1 mL of saline. Then, 500 μL of saline with 3.15× 10⁸ CFU/mLwere addedto eachtube andwere incubated

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staticallyat37°Cand5%CO₂ for1.5h.Thesupernatantwasthen removed,andeachtubewasrinsedtwotimeswith1mLofsaline.

Afterward, 1 mL of wound-like medium were placed into each tube,whichwerefurtherincubatedstaticallyat37°Cand5%CO₂ for48h. Thewound-like mediumisa biofilmmodelthat resem- bles the type of biofilm that is usually found inclinical practice [48]. The rationale forusing this medium relies on its composi- tion.Amongothers,thiswound-likemediumcontainserythrocytes and serum, which are components that the nanoparticles would havetodealwithinaninfectioninahumanbody.Hence,thepur- poseofusingbovinetrabecularboneandwound-likemediumwas to mimic in vitro ina very realistic way the conditionswhere a bacterium causingosteomyelitiswouldgrowinvivo.After biofilm formation,each tubewasrinsedtwo timeswith1mLsaline and treated inpresence or absence of1 mL of two effectiveconcen- trations of nanoparticles,which were further incubated statically at 37 °C and 5% CO₂ for 24 h. Such concentrations, 31.25 and 62.5μg/mL, wereestimatedaccordingtothe MBECobtained, and consideringpreviousstudiesthatstablish4× MICasagoodther- apeutic approach [32,49]. After incubation,each tubewas rinsed two timeswith1mLofsaline andalltrabecular bonefromeach tube weretransferred to a5mL round-bottomtube containing1 mL of saline. All tubes were sonicated at room temperature for 5 min[50].Then, thenumberofbacteriawasdeterminedasCFU per gram of bone by using the drop platemethod [51]in Mac- Conkeyagarplates(BioMérieux,France).Theplateswereincubated at37°Cand5%CO₂ atleast24h.Theexperimentwasperformed fivetimes.

Tosupport visuallythe numericalresults,theprevious experiment wasanalysedusing laserconfocal microscopyandscanning electron microscopy(SEM).Forlaser confocalmicroscopy,the ex- perimentwasperformedina4× 2glass-bottomplate(ibidi,Ger- many)wherethetubeswerereplacedbywells,andalltheabove- mentionedvolumeswerereplacedby300μLofeachmedium.Af- ter24h,eachwellwasdirectlyanalysedinaLeicaDMIRBconfo- cal laser-scanningmicroscope (Leica,Germany)without removing thesupernatant.ForSEMstudy,thesamesampleswereﬁxedwith 2.5% glutaraldehydein 0.1M sodiumcacodylate buffer atpH7 at 4 °C for 90 min. Samples were then dehydratedwith increasing concentrations ofethanol(30, 50,70, 90, and100%) at22°C for 10min.MicrographswereobtainedusingaﬁeldemissiongunJEOL JSM6400scanningelectronmicroscope(JeolLtd,Tokyo,Japan).

2.5. Cellstudies

MC3T3-E1 cells were inoculated in a concentration of 10,000 cells/cm² on 96-well plates with

α

^-minimum ^essen-

tial medium with 10% foetal bovine serum and 1% penicillin- streptomycin (

α

^MEM, Invitrogen, Thermo Fisher Scientiﬁc).

RAW264.7cellswereseededinaconcentration of5000cells/cm² on 96-well plates with

α

^-minimum ^essential ^medium ^with ^10%

foetal bovine serum and 1% penicillin-streptomycin (

α

^MEM, ^In-

vitrogen, ThermoFisher Scientiﬁc Inc. USA).After celladherence, MC3T3-E1 cells medium wasreplaced by

α

^MEM ^with⁵⁰ ^mg/mL

ascorbicacid(Sigma-Aldrich,USA),10mMß-glycerol-2-phosphate (Sigma-Aldrich, USA), and part of the RAW264.7 cells was incubated in the presence of 50 ng/mL of Receptor Activator for Nuclear Factor

κ

^B ^Ligand ^(RANKL) ^(R&D ^Systems, Bio-Techne, Madrid, Spain) to promote osteoclast differentiation. All types of cells (MC3T3-E1,RAW264.7 andRAW264.7 osteoclastprecursors) cells were treated with 31.25 and 62.5 μg/mL of AG+CO-coated MX-loaded MSNs (n=8 per concentration). Non-treated cells incubated only with growth medium were considered as control (n = 8). All growth media were refreshed every 48 h. These MSNs concentrations were chosen based on the microbiological susceptibility results. Cytotoxicity was tested by CytoTox 96®

NonRadioactive Cytotoxicity Assay (Promega, USA) after 48 h of incubation, according to previously published methodology [52]. Cell proliferation was determined by addition of alamarBlue®

solution(BIO-RAD,USA) at10% (v/v)tothecellculture at14and 21daysofcultureforMC3T3-E1[53]cellsor4daysforRAWcells [54,55]. The 14-daysand21-days incubations of MC3T3-E1 allow to emulate how the cell proliferation ofthis type of cellswould be affected upon incubation with the nanosystem as it would theoretically takeplace in vivo.Data wererepresented asrelative proliferation of each treatment compared to the control, which wasconsideredas100%ofcellproliferation.

2.6. Hepatotoxicitymodel

Seven 7-week-old male RjHan:SD - Sprague Dawley rats(Jan- vierLabs,LeGenest-Saint-Isle,France)wereusedforprimaryhep- atocytes and Kupffercells (KCs)isolation aspreviously described [56,57].First,theliverwasperfusedinsituwith200mLofHanks’

balanced salinesolution (HBSS)withoutCa²⁺ andMg²⁺ (Thermo Fisher Scientiﬁc) at 10 ml/min and 37 °C without recirculation.

The organ was then excised and inserted into a sterile plastic bag where ex situperfusion was continued with 60 mL of 0.2%

pronase(Merck,New Jersey, UnitedStates) inHBSS accompanied by225mLof0.01%collagenase(Boehringer-Mannheim,Ingelheim, Germany)inHBSSunderthesameconditions.Theliver wasthen separatedfromtheperfusiondevice,thecapsulewastakenoff and thetissue wasdivided intovery smallpieces.Tissue wasdissoci- ated in a mixture containing 0.03% pronaseand 0.01% DNAse in 100 mLofHBSSandwasincubatedat37 °C withconstantshak- ing for 30 min. Subsequently, a 125 μm nylon mesh ﬁlter was used to ﬁlterthe liver homogenate removing the undigested tissue.Theobtainedcellsuspensionwascentrifugedat70× gat4°C for4min.Maturehepatocytes(H)were foundinthepelletwhile non-parenchymal cells (NPCs) were detected in the supernatant.

Thecentrifugationswererepeatedtwomoretimes,andallthesu- pernatants werecollectedforKCsisolation. Topurify thehepato- cyte population, the pellets were treated with a Percoll density- gradientcentrifugation(GEHealthcare, Marlborough,MA).ForKCs isolation,supernatantsweretreatedwith100mLofHBSSsupple- mentedwith100μg/mLDNAseand1%BSA,achievingtheirdisso- ciationby gently shakingfor 5minutes. Thecell suspension was thenﬁlteredusinga297μmnylonmeshﬁlterandcentrifugedat 500 × gfor6 minat4 °C.Toseparate the KCsfromother NPCs cells,thesuspensionwassubjectedtoanOptiprepdensitygradient (Sigma-Aldrich,Missouri,UnitedStates)andcentrifugedat1400g for17minat4°C,thusisolatingtheKCs.

Thesecellco-cultureswereseededata2:1ratio(H:KC)on48- well Type-I collagen-coatedplates.First, 375,000hepatocytecells were plated into each well and allowed to adhere for approximately 1h at37 °C / 5% CO₂ withgentle shaking every 20 min.

Themediumwaschangedafterhepatocytesattachmenttoremove unbound cells. Then, 187,500 KCs from the same donor-matched were added toeach well and againtheKCs were allowed to adhere for 1 h, with manual gentle shaking every 20 min. After- ward,themediumofthewellswaschanged formaintenanceand later use.Thecellswere incubatedwith twodifferent concentrations of AG+CO-coated MX-loadedMSNs (32.25and62.5 μg/mL) for1h.Cellsincubatedwith50μMoftrovaﬂaxacin(TVX)(Sigma Aldrich, Missouri,United States) plus 1 μg/mL lipopolysaccharide (LPS)(Sigma Aldrich, Missouri,UnitedStates) were used asposi- tivecontrol.Thesupernatantofeachconditionwasthencollected andstoredat-80°Cuntilthetime ofanalysis.Thelevels ofeach cytokine were measured using rat TNF-

α

ând ÎL-6ÊLISA ^kits âc-

cordingtothemanufacturer’srecommendations(LifeTechnologies, ThermoFisher Scientiﬁc,Massachustts,United States)andquanti- ﬁedusingaSynergy^TMHTXMulti-ModeMicroplateReader(Biotek, 222

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Fig. 1. Surgical model. Skin and muscles were sectioned until the lateral epicondyle was reached ( Fig. 1 a and b). A hole 3.2 mm in diameter was drilled and cylindrical Ti- 6Al-4V implant infected with E. coli ATCC25922GFP was placed ( Fig. 1 c). The infected implant was placed in the bone marrow; the hole was closed with bone wax ( Fig. 1 d).

The wound was closed with a continuous cross suture ( Fig. 1 e). The correct location of the implant was corroborated through dorsoventral ( Fig. 1 f) and lateral ( Fig. 1 g) ﬂuoroscopy of each animal. The white bars represent approximately 2 cm.

Fig. 2. AG + CO-coated MX-loaded MSNs intraosseous treatment. Under general anaesthesia, the femur of each rabbit from the treated group was drilled with a with a 1.5 cm needle (a). The vacuum was made on the needle using drops of sterile serum (b). Finally, 4 mL of 62.5 μg/mL of AG + CO-coated MX-loaded MSNs were injected.

Vermont, USA). CYP3A activity was measured directly in cells in each wellby usingtheP450-Glo^TM CYP3A4assaywithLuciferin- IPA (Promega, Wisconsin, United States) according to the indica- tions for cultured cells, andusinga luciferin standard curve (Lu- ciferinBeetle,Promega).SampleswereanalyzedusingaSynergy^TM HTXMulti-ModeMicroplateReader.

2.7. Invivomodel

ThisstudywasapprovedbytheInstitutodeInvestigaciónSan- itaria of Fundación Jiménez Díaz (IIS-FJD) Animal Care and Use Committee,whichincludesadhocmembersforethicalissues.An- imal careandmaintenance compliedwithinstitutionalguidelines asdeﬁnedinnationalandinternationallawsandpolicies(Spanish RoyalDecree53/2013, authorizationreferencePROEX109.7/21July 18,2021,bytheMinistryoftheEnvironment,LocalAdministration and TerritorialPlanning of theCommunity of Madridand, Direc- tive 2010/63/EUoftheEuropeanParliamentandoftheCouncil of September22,2010).

SpeciﬁcpathogenfreeNewZealandwhitemalerabbits(Granja SanBernardo, Navarra,Spain)ofbetween2.5and3Kgofweight were used. All animals were housed in individual cages in an air-conditioned room at 22 ± 2 °C and light-darkness cycles of 12:12h.

2.7.1. Evaluationofsystemicadministration

Three rabbits were treatedunder generalanaesthesia withan intravenousinjectionthrough themarginalvein ofthe leftear of 62.5μg ofAG+CO-coatedMX-loadedMSNspermillilitreofrabbit blood,considering that each animal possesses 66.33mLof blood perkilogram[58].Twodaysaftertheintravenoustreatment,each animalwas euthanizedundergeneralanaesthesia by intracardiac overdoseofsodiumthiobarbital.Therabbitspleen,liverandakid- neywererecoveredthroughsterilepreparation,surgicalfieldisola- tion.Allorganswere fixed,paraffin-infiltrated,andhaematoxylin- eosinstained.

2.7.2. Osteomyelitismodel

TheE.coli ATCC25923-GFPstrainwasemployed forthisinvivo model.Eachanimalwasplacedinthesupinepositionundergen- eral anaesthesia, its righthind leg wasimmobilised and isolated in a sterile ﬁeld. Skin and muscleswere sectioned until the lateral epicondyle was reached (Fig. 1a and b). A hole 3.2 mm in diameter and 1 cm deep was drilled. A 5 mm-long and 3 mm- diameter cylindrical Ti-6Al-4V implant infected with E. coli was placed(Fig.1c).Eachimplantwasincubatedwith2mLofa3Mc- FarlandsuspensionofE.coliinsaline(≈2.58× 10⁸colony-forming unitspermillilitre) inawellfroma12-wellplatefor2hat37°C and5% CO₂ forimplant infection. After incubation,each implant

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waswashedwith2mLofsaline(B.Braun,Germany).Afterlodging theinfectedimplantinthebonemarrow,theholewasclosedwith Ethiconbonewax(Johnson&Johnson,UnitedStates)(Fig.1d).The entireareawasdisinfectedwith6-volumehydrogenperoxide.The woundwasclosedwithacontinuouscrosssutureusinga3/0Pro- lene suture (Johnson& Johnson, UnitedStates)(Fig.1e).The correct location ofthe implant was corroborated through dorsoventral (Fig. 1f) andlateral (Fig. 1g) ﬂuoroscopy ofeach animal.The behavior, temperatureandweightofeachanimalweremonitored every24hthroughouttheexperimentalprocedure.

The infected animals were randomly assigned to two groups, namelycontrolgroup(n=3)andAG+CO-coatedMX-loadedMSNs treatedgroup(n=3).ThesamplesizewasestimatedbyWilcoxon Mann-Whitneytestandana-prioritypeofpoweranalysisconsid- ering d = 4.00,

α

= 0.05, (1-

β

⁾ = 0.95, allocation ratio = 1 by usingG^∗Power3.1.9.7software[59].Thedparameterassumesthat AG+CO-coatedMX-loadedMSNstreatment can reduce thebacte- rial concentration by atleast 99% per gram ofbone when com- paredtotheuncoatedimplantgroup. Thestatisticalpowerofthe samplewas0.983.

Three days after the surgery, each animal was anaesthetized and treatedwitha 4 mL intraosseous injectionof 62.5μg/mL of AG+CO-coatedMX-loadedMSNsbyusinga1.5cmneedleandAr- row® EZ-IO® Intraosseous Vascular Access System (Teleplex, Ire- land)(Fig.2).The controlgroup receivedno treatment.Twodays aftertheintraosseous treatment,allanimals wereeuthanizedun- dergeneralanesthesiabyintracardiacoverdoseofsodiumthiobar- bital.Therabbitfemur,liverandakidneywererecoveredthrough sterilepreparation.

Formicrobiological studies, each femur withthe implant was smashed with a hammer. This smash was immersed in sterile saline and sonicated using an Ultrasons-H 3000840 low-power bathsonicator (J.P. Selecta,Barcelona,Spain) at22 °Cfor5 min [50].Theresultingsonicate wasdilutedina10-folddilutionbank and seeded on blood-chocolate agar (Biomérieux, Marcy-l’Étoile, France) usingthespread platemethod[60,61]. The concentration ofbacteriawasestimatedasCFU/gofboneandadnexa.Theliver and one kidney of each animal were intended for pathological studies. Histological sections were fixed, paraffin-infiltrated, and hematoxylin-eosinstained.

2.8. Statisticalanalysis

Statisticalanalyses were performedusingStata StatisticalSoft- ware, Release 11 (StataCorp 2009). Data were evaluated using a one-sided Wilcoxon nonparametric test to compare two groups.

Statistical signiﬁcance was set atp-values ≤ 0.05. All results are representedasmedianandinterquartilerange.

3. Results

3.1. Nanoparticlescharacterization

3.1.1. Synthesisandfunctionalizationofmesoporoussilica nanoparticles

ThesuccessfulcoatingwithAGandCOwasconﬁrmedthrough differentcharacterizationtechniques(Figs.3andS2).TGA(Fig.3a) showedadifferenceinaweightlossof8% and14%forAG-coated

Fig. 3. Physico-chemical characterization of the different nanoparticles. (a) Thermogravimetric analysis, (b) FTIR spectroscopy, (c) TEM image of phosphotungstic acid-stained AG + CO-coated MSNs, (d) MX release experiment from MSNs (black), AG-coated MSNs (green) and AG + CO-coated MSNs (red), (e) CO-FITC release experiment from CO-coated MSNs (black) and AG + CO-coated MSNs (red) (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

224

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MSNsandAG+CO-coatedMSNs,respectively,comparedtopristine MSNs. Hence, it could be concluded that the amount of CO ad- sorbed onto the surfacewas ca.6%. The presence of AGand CO wasfurtherconﬁrmedthroughFTIRspectroscopy(Fig.3b).Inthis regard, theappearance ofadeep vibrationband atca.3400 cm⁻¹ (

ν

OH) as well as a subtle one at ca.3,000 cm⁻¹ (

ν

CH) in the AG-coated MSNs spectrum were ascribed to the presence of AG.

This was in agreement with the vibration bands observed for AG alone (Fig. S3). Finally, the presence of vibrations bands at 1650 and 1,540 cm⁻¹ that were ascribed to the amide bonds of COundoubtedly conﬁrmedits successfuladsorptiononto thesurface. Besides, AG+CO-coated MSNs were stained with phosphotungstic acidandobservedundertheTEM, wheretheblurry sur- facewasindicativeoforganicmatterdeposition(Fig.3c).Zetapo- tential measurements onAG+CO-coatedMSNs yieldeda value of -21.6mV,whichwaslessnegativethanwhatobservedforMSNs(-

34.8mV), in agreement withthe presence of free amino groups throughout the structure of CO. Finally, the colloidal stability of differentnanoparticles wasanalysed through DLS measurements, yielding a size distribution centred at 190 nm (Fig. S2). In this sense,thestabilityofnanoparticlesremainedunaffectedbydiffer- entfunctionalizations,highlighting theirsuitability forbiomedical applications.

3.1.2. AntibioticreleasefromMSNs

MX releasefromthedifferent nanomaterialswasevaluated in vial. As shown in Fig. 3d, modifying the surface with a macro- molecule with affinity for the biofilm, AG, and a high molecular weight antibiotic, CO, led to release kinetics similar to that ob- servedforMSNs. ThedifferentreleasedatashowninFig.3dwere fittedtoa first-orderkineticmodelwithan empiricalnonideality

Fig. 4. E. coli -AG-coated MSNs interaction. (a) Use of AG-coated MSNs by E. coli as a carbon source. (b) Propidium iodide release from MSNs (red) and AG-coated-MSNs (purple) in presence of E. coli that is actively replicating (green). (c) E. coli bioﬁlm- AG-coated MSNs interaction. (d) E. coli planktonic cells-AG-coated MSNs interaction. FI:

ﬂuorescence intensity. ^∗: p -value < 0.05, ^∗∗∗∗: p -value < 0.0 0 01 for Wilcoxon test (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

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factor(

δ

⁾⁽^Eq.⁽¹⁾⁾^[⁶²^,⁶³^]:

Y=A

1− e^−kt

δ

(1) WhereYisthepercentageofMXreleasedattimet,Athemax- imum amountof MX released(in percentage),andk,the release rateconstant.TheobtainedvaluesaresummarizedinFig.3d.

On the other hand, the release proﬁle of CO-FITC+AG-coated MSNs (Fig. 3e) demonstrated that the AG coating enhanced the amount ofCOloadedonto thesurface, whichcould be beneﬁcial forthebiologicalstudies,asitwouldreducetheneedtousehigh dosesofthisnanomedicine.

3.2. Microbiologicalstudies

3.2.1. Bacteria-nanoparticleinteraction

AG-coated MSNs were incubated with E. coli to evaluate whetherthe polymericcoating could be effectivelydegradedinto its monomers by such enzymes and be employed as a carbon source by the bacteria. As shownin Fig.4a, E. coli concentration showedmorethantwo-foldincreaseinthepresenceofAG-coated MSNs compared to pristine MSNs(p-value < 0.0001).The enzy- matic degradationofAGwasfurtherconfirmedthrougha release experiment. For that purpose, MSNs and AG-coated MSNs were placed with bacteria to evaluate whether the enzyme-mediated degradation of AG affected the release profile (Fig. 4b). In con- trast to Fig. 1d, the PI release wasalmost equal fromMSNs and AG-coated MSNsinpresenceofE.coli.Thedifferentnanoparticles werethenfacedtoanE.colimaturebiofilmandlefttointeractfor 30 min.Asobserved inFig.4c,thehighestretentionvalueswere observedforbothAG-andAG+CO-coatedMSNs, showingthelat- ter slightlymore accumulation(p< 0.0307). However, theanaly- sis of the interaction at 3 h demonstrated that AG-coated MSNs maintained its highretentioncapability, whereasthat of AG+CO- coatedMSNssignificantlydecreased,equallingtheresultsobtained forpristineMSNs.

AG+CO-coated MSNs signiﬁcantly reduced the E. coli viability(p-value= 0.0143)compared toAG-coated MSNsandpristine MSNs(by42%and41%, respectively)(Fig.4d).Theseresultswere conﬁrmedbyusingTEM(Fig.5),whichallowedtoinspectvisually thephysicalchangesinducedbythedrug-loadednanoparticleson thebacteria.Bacteriafacedtopristine MSNsandAG-coatedMSNs showedanormalappearance,withanintactoutermembraneand bacterial wall intimately linked to the cytoplasm (Fig. 5a–d). In some cases,AG-coated MSNswereintimatelyin contactwiththe outer membrane of some bacteria (Fig. 5d). The interaction between E. coli andAG+CO-coated MSNsgave rise to thepresence of vacuoles inside the bacteria (Fig.5e andf) resulting from the detachmentofthecytoplasmicmembranefromthecellwallright wherethe bacteriumhadinteractedwithan AG+CO-coatedMSN, whichwasunequivocallyascribedtobacterialdeath(Fig.5f).

3.2.2. Minimalinhibitoryconcentration(MIC),minimalbactericidal concentration(MBC),minimalbioﬁlminhibitoryconcentration (MBIC),andminimalbioﬁlmeradicationconcentration(MBEC)

The antibacterial effectof the nanoparticleswas evaluated by studying the MIC andMBC. Forthat purpose, differentnanoparticles werefaced against planktonic E.coli atdifferentconcentra- tions. The MIC andMBC of MX against E. coli were found to be

< 0.0625μg/mLforboth. TheMIC andMBCofCOagainstE. coli were found to be 2 μg/mL for both. The MIC and MBC of MX- loaded MSNs against E. coli were found to be <1.953 μg/mL for both. The MIC and MBCof CO-coated MSNs against E. coli were found to be 15.625 μg/mLfor both. Finally,the MIC andMBC of AG+CO-coatedMX-loaded MSNsagainst E. coli were found to be 1.953and3.906μg/mL,respectively.

Fig. 5. TEM images of E. coli planktonic cells faced to MSNs (a and b), AG-coated MSNs (c and d), and AG + CO-coated MSNs (e and f). C: cytoplasm. OM: outer membrane. ROM: ruptured outer membrane. V: vacuole.

Theantibioﬁlmeffectofnanoparticleswasevaluatedbystudy- ing the MBIC and MBEC. For that, different nanoparticles were faced against an E. coli bioﬁlm at different concentrations. The MBIC and MBEC of MX against E. coli wasfound to be <0.0625 and 1 μg/mL, respectively. The MBIC andMBEC of COagainst E.

coli were found to be 64 and128 μg/mL, respectively. The MBIC and MBEC of MX-loaded MSNs against E. coli were found to be 1.953 and 15.625 μg/mL, respectively. The MBIC and MBEC of AG+CO-coated MSNs against E. coli were found to be 2,000and

>2,000μg/mL,respectively.Finally,theMBICandMBECofAG+CO- coatedMX-loadedMSNsagainstE.coliwerefoundtobe1.953and 7.813

μ

^g/mL,respectively.

Bearing in mind the above-described results, and an in vitro modelofinfectedbonewasdevelopedforthesubsequentprepara- tionofinvivoexperiment.Forthatpurpose,twoconcentrationsof AG+CO-coatedMX-loadedMSNs,31.25(4× MBEC)and62.5μg/mL (8 × MBEC), were chosen to be used against E. coli. Both concentrations of AG+CO-coated MX-loaded MSNswere able to de- creasemorethan99.9%theviabilityofbiofilm grownonthe trabecular bone compared to non-treated biofilm (p-value < 0.01) (Fig. 6a). Furthermore, the62.5 μg/mLconcentration significantly reduced98.9% more bacteria than 31.25 μg/mLconcentration (p- value = 0.0238). To support these numerical results, representative images from each treatment were taken (Fig. 6b–i). Non- treatedE. coli biofilm showed manydifferent smallviable aggre- 226

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Fig. 6. Bacterial quantity per gram of trabecular bone after 24 h treatment with each AG + CO-coated MX-coated MSNs (a). Representative confocal images from the different conditions: (b and c) positive control, (d, e and f) 31.25 μg/mL, and (g, h and i) 62.5 μg/mL of AG + CO-coated MX-coated MSNs. Green represents the E. coli viable bacteria, red represent the AG + CO-coated MX-coated MSNs, and grey represent the trabecular bone surface. ^∗: p -value < 0.05, ^∗∗: p -value < 0.01 for Wilcoxon test. Blue, pink and white bars represent 250, 25, and 7.5 μm, respectively (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

gatesofbacteriaormicrocoloniesmainlyadheredonthebonesur- face (Fig. 6b and c), an aspect that is reminiscent of the micro- scopic appearance of certain biofilms isolated fromclinical samples [64].When E. coli biofilm wasexposed to 31.25 μg/mL, the number ofaggregates wasslightlylower (Fig.6d–f). In thiscase, AG+CO-coated MX-loaded MSNs adhered on microcolonies. This MSNs attachmentwouldbe responsible forthe absenceofviable bacteria (absence of green fluorescence) in the periphery of microcolonies (Fig. 6d–f). The best resultswere obtainedwhen the E.coli biofilmwastreatedwith62.5μg/mLofAG+CO-coatedMX- loaded MSNs(Fig.6gandh).As showninFig.4g,thequantity of

viablebacteriaintothemicrocoloniesadheredonthebonesurface was extremely scarce. Likewise, AG+CO-coated MX-loaded MSNs adhered on those few aggregates, but the viable bacteria inside ofthemwere fewerthan thoseinthemicrocolonies treatedwith 31.25μg/mL(Fig.6handi).Atmicroscopiclevel(Fig.7),untreated E. coli bioﬁlm grown on bone showed bacterial clusters adhered onboneandwasembeddedinexopolymericsubstances(Fig.7a–

c), in opposition to the E. coli cells observed on the bone surface treated with 32.5 and 61.25 μg/mL (Fig. 7d–f) which were found in the form of aggregates, mainly 32.5 μg/mL, or simply as individualized cells completely covered by the nanoparticles

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Fig. 7. SEM images of E. coli bioﬁlm grown on trabecular bone after 24 h treatment with each AG + CO-coated MX-coated MSNs: (a, b andc) positive control, (d, e andf) 31.25 μg/mL, and (g, h, and i) 62.5 μg/mL of AG + CO-coated MX-coated MSNs.

Fig. 8. Cytotoxicity of AG + CO-coated MX-coated MSNs concentration on MC3T3-E1 osteoblasts (a), RAW264.7 osteoclasts (b), and RAW264.7 macrophages (c). ^∗∗: p - value < 0.01, ^∗∗∗∗: p -value < 0.0 0 01 for Wilcoxon test.

anddisplayingimportantmembranedamages,mainly61.25μg/mL (Fig.7g-i).

3.3. Cellstudies

The biocompatibility of AG+CO-coated MX-loaded MSNs was evaluated onbone-relatedcells(Fig.8).AG+CO-coatedMX-loaded were found to be non-cytotoxic for osteoclasts, and only re-

duced cytotoxicity was observed at the highest concentration (62.5μg/mL)forosteoblastsandmacrophages.Moreover,AG+CO- coated MX-loaded MSNshad an impact on the cell proliferation of osteoblast and macrophages (Fig. 9a, c), since these cells decreased their proliferation in presence of nanoparticles. Interest- ingly,osteoblastshowed a dose-dependenteffecton proliferation at14days,butthesecellsshowedbetterproliferationinpresence of62.5μg/mLthan31.25μg/mLat21days(Fig.9a).

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Fig. 9. Cell proliferation of MC3T3-E1 osteoblasts (a), RAW264.7 osteoclasts (b), and RAW264.7 macrophages (c) in presence of each AG + CO-coated MX-loaded MSNs con- centration. ^∗: p -value < 0.05, ^∗∗: p -value < 0.01, ^∗∗∗: p -value < 0.001 for Wilcoxon test.

3.4. Hepatotoxicitymodel

The resultsderived from the hepatotoxicity modelare shown in Fig.10.Tumor necrosis factor-

α

^(TNF-

α

⁾ ^increasedproportion- ally with the concentration of AG+CO-loaded MX-loaded MSNs (Fig. 10a), whereas interleukin-6 (IL-6) showed opposite behavior (Fig. 10b). In addition, cytochrome P450 metabolic capacity (CYP3A) decreased proportionally with the concentration of nanoparticles(Fig.10c).

Invivostudies

3.4.1. Evaluationofsystemicadministration

Thethreeliversshowedapreservedhepaticparenchymaarchi- tecture withabsence ofhepatocytelesion.In one oftheanimals, mild intraepithelial lymphocytosis in the ducts and presence of chronic central perivenular inflammatory aggregates were identi- fied.Intheremainingtwo,mildsignsofductulitiswere observed, with signs of lymphocytosis in bile ducts (Fig. 11a and b). The three kidneys showedarenal parenchymawithnormalmorphol- ogy and appearance without glomerular or tubular lesions, and absence of inflammation or necrosis (Fig. 11c and d). The three spleensshowedasplenicpulpwithoutrelevantmicroscopicalter- ations(Fig.11eandf).

3.4.2. Osteomyelitismodel

AG+CO-coated MX-loaded MSNscompletely eradicated E. coli infection intwo out of thethree treatedfemurs andreducedup to 99.4% in the remaining femur, compared to the control group (Fig.12).

The three livers of control (untreated) group showed liver parenchymawithmild-moderatecentralperivenousportalinflam- mation and minimal lobular hepatitis (Fig. 13a and b). The inflammatory infiltrate consisted of lymphocytes, plasma cells and eosinophils (Fig. 13c). The livers of AG+CO-coated MX-loaded MSNs-treated groupshowedliver parenchymawithpreservedcy- toarchitecture without hepatocytelesions (Fig.13d). Minimal foci ofductulitiswithsignsoflymphocytesinbileductswereobserved inallthreetreatedanimals(Fig.13eandf).Focalsignsofcholesta- sis were detected in one of the treated rabbits. All the kidneys from both control group and AG+CO-coated MX-loaded MSNs- treatedshowedrenalparenchyma ofnormalmorphologyandap- pearancewithoutglomerularortubularlesions,andabsenceofin- flammationornecrosis.

4. Discussion

Inthis study,we demonstratethe feasibilityof usingAG+CO- coated MX-loaded nanoparticles to treat bone infections caused by E. coli, showing no cytotoxicity on osteoblast, osteoclast and macrophagesin vitro, andabsenceoforgan damage in vivo upon intraosseousadministration.

Given the bacterialorigin of this kindof infection, two clini- callyrelevantantibiotics,MXandCO,were selectedascargos.Be- causeofitslowmolecularweight(401g/mol),MXwouldbeeasily loaded within themesoporous of nanoparticles,aspreviously re- ported[65,66]. Conversely,COwouldbe likelyadsorbed ontothe surfaceofnanoparticles,aspreviously statedforotherpolymixins andMSNs[32,67,68].

TheMXreleasewasmonitoredandﬁttedtoaﬁrst-orderkinetic model(Fig.1d).Inthismodel,thevaluesfor

δ

^are^comprised^be-

tween0,formaterialsthat releasethedrugattheverybeginning oftheexperiment, and1,formaterials that followtheﬁrst-order kinetics.According to Fig.1d, the

δ

^values^estimated^for ^the^dif-

ferentnanoparticleswerecloserto0,inagreementwiththepro- nouncedbursteffectobserved duringtheinitial hour. Thiskinet- icshas also been observed forother antibiotics loaded inmeso- porous silica-based nanoparticles [69]. Also,

δ

MSNs wascompara- tively lower than those of coated samples,which would account for the slight differences observed among them during release experiments. Considering these results, the AG+CO-coated MSNs would be able to release large amounts of MX in a short time, thereby achieving high local concentration of antibiotics nearby bacteria from a low quantity of nanoparticles, which might re- ducethepotentialsideeffectsassociatedwithotheradministration routes.

Eventhough COmighthavebeensimplyadsorbedonthesur- faceofparticlesanddirectlyusedagainstbacteria,ourresultsin- dicatethatincorporatingCOalongwiththeAGcoatingwasbene- ficialto enhance the final amount of this antibiotic on nanoparticles (Fig. 1e). We hypothesize that the reason behind this re- sult would be that AG can act asa polymeric mesh that would favour the retention of CO on nanoparticles during the washing steps,resultingina significantlyhigherfinalamount ofantibiotic loaded.Hence,althoughthesynergeticeffectoftheuseofpolimix- insplusanotherantibiotic hasbeenpreviouslyreported[32],this work demonstrates that theuse ofArabic gumimproves the ad- sorptioncapacityofnanoparticles,potentiallydiminishingthefinal nanoparticle dosethat wouldbe requiredduringthe treatmentif COalonewastobeused.

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Fig. 10. Tumor necrosis factor α(TNF- α) (a), interleukin 6 (IL-6) (b), and cytochrome 3A metabolic capacity (c) from the rat hepatocyte-Kupffer cells co-cultive. ^∗: p - value < 0.05, ^∗∗: p -value < 0.01 for Wilcoxon test.

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Fig. 11. Histological liver (a, b), kidney (c,d) and spleen (e, f) images with hematoxylin-eosin stain from a rabbit that received systemically one AG + CO-coated MX-loaded MSNs dose. Black, green, and red bars represent 20 0, 10 0, and 50 μm, respectively (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

Fig. 12. Quantity of E. coli in bone and adnexa. ∗p -value < 0.05 for Wilcoxon test.

AG,abranched-chain,complexpolysaccharidecomposedof1,3- linked

β

-D-galactopyranosyl monomers connected to the main chainthrough1,6-linkages[27],canbedegradedbytheenzymatic batteryofenterobacteria[70–72].Inthissense,ourresults(Fig.2a) confirmed that AG coating can be degraded by E. coli enzymes intogalactopyranose monomers that areused asa carbonsource [30,31]. Thisfinding pointsout that AGlacks antibacterial capacity per se, unlike other gums such asIranian gum do [73]. That enzymaticdegradation ofAG wasfurtherconfirmedthrough aPI releaseexperiment(Fig.2b),in whichalmost equalamountof PI was released fromMSNs and AG-coated MSNsin presence of E.

coli, demonstrating that E. coli metabolically active cells can de- gradethe AGcoating while the bacteriumgrows. The interaction betweenE.colimaturebioﬁlmanddifferentnanoparticles(Fig.2c) broughtout that AG-coated MSNsshoweda higheraccumulation thatnon-coatedonesatshorttime(30min).Interestingly,AG+CO- coatedMSNsshowedaslightbutsigniﬁcantlyhigheraccumulation than AG-coated MSNs. However, setting the incubationperiod at

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Fig. 13. Histological liver images with hematoxylin-eosin stain of untreated rabbit (a–c) and AG + CO MX-loaded MSNs-treated rabbit (d–f). Black and red bars represent 100 μm, and 50 μm, respectively (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

3 h revealed that AG-coated MSNs maintained its highaccumu- lation capabilityto bioﬁlm, whereas that ofAG+CO-coated MSNs matched that of pristine MSNs. Thus, our results would indicate that functionalizing the surface of MSNs with AG increased the targetingabilityofnanoparticlesforE.coliinfectiousfoci.Thisen- hancedaccumulationmightbetheresultofthebiomatrixaccumu- lationof enzymes[74]able tocleavethepolymeric chains ofAG, e.g.

β

-galactosidase[75].Thisenzymaticcleavagewouldfavourthe retentionofnanoparticlestobiofilmmatrixatveryshorttimesand wouldmadetheAGcoating aspecificcoatingforE.coliandother enterobacteriathatsecretetheseenzymes[76].Afterthisaccumu- lation, the colistin carried by the AG-coated MSNswould be re- leasedandexertadestabilisingeffectontheE.coli biofilmmatrix structure[77].Tothebestofourknowledge,thisisthefirststudy that uses a gumof naturaloriginasa targeting agentfora spe- cificbacterialpathogen.Overall,gumsofdifferentoriginareworth beingexplored asthereare alreadyfewexamples oftheir poten- tialinnanomedicine.Forinstance,ithasbeenreportedthat Guar gum can be used as targeting agent for colorectal cancer treatment [78–81].Similarly, Iranian gumhas been shownto present antimicrobialpropertiesperseandhasbeenusedforwoundheal- ing applications [73]. Besides,these gumshave beenrecently in- corporatedintohydrogelsthatshow uniqueadvantagescompared to other polymeric materials, and findapplication asperiodontal materials,drugcarriers,bonematrices[82–85],andartificialblood vesselsfabricatedby3Dprinting[86].Someofthosehydrogels,as those composedofArabicgum,gelatinplus polyurethane[87],or thosecontainingN–O–carboxymethylchitosan[88]havebeenalso usedforwoundhealingapplicationsandwoundinfectionpreven- tion.

ItisknownthatCOkillsGram-negativebacteriathroughoutﬁve mechanisms:(1)disruptionofthebacterialouterandinnermem- branes, (2) vesicle-vesicle contact pathway, (3) hydroxyl radical deathpathway,(4)inhibitionofrespiratoryenzymes,and(5)anti- endotoxincolistinactivity[89].Anyofthesemechanismswouldbe responsible forthe bactericidal effect observed in AG+CO-coated MSNs at short term (30 min) against the planktonic state of E.

coli. These results were further supported by TEM study, where the presence of vacuoles resulting from the detachment of the cytoplasmic membrane from the cell wall and outer membrane [90] was only detected inthe bacteriaexposed to AG+CO-coated MSNs.

The antibacterialeffect ofnanoparticleswasevaluated byMIC andMBC.Accordingtotheresultsobtained,E.coliATCC25922-GFP issusceptibletoMX(<1μg/mL)[91]andCO(≤2μg/mL)[92].In consequence, the combined use of both antibiotics seems highly appropriate for achieving outstanding therapeutic effect against

E. coli ATCC25922GFP. On the other hand, the antibiofilm effect ofnanoparticleswasevaluated by MBIC andMBEC.For thatpur- pose,differentnanoparticleswerefacedagainstanE.colibiofilmat differentconcentrations,resultingtheMBICandMBECofAG+CO- coatedMX-loadedMSNsagainstE.coli1.953 and7.813μg/mL,respectively.Inlightoftheseresults,MXwouldbeapossibletreat- mentforE.colibiofilm,asrecommendedbyotherauthors[93,94].

Despite COwas shown to be ineffectivein inhibiting and eradi- catingthe E. coli bioﬁlm, the actual role of thisantibiotic in the potentialtreatmentofosteomyelitiswillbediscussedlater.

Given the promising results, the antibacterial eﬃcacy of AG+CO-coated MX-loadedMSNs ina more realistic scenariowas studied by using an in vitromodel of bacterialbioﬁlm grown in wound-likemedium [40,41] onbovinetrabecular bone.Thehigh- estconcentration ofAG+CO-coatedMX-loadedMSNs(62.5μg/mL;

8 × MBEC) was the most effective against E. coli biofilm. The main differences between two concentrations were (1) the bacterial viability and (2) the quantity of microcolonies adhered on the bone (Fig. S4). The bacterial viabilityreduction was a consequence oftheMXreleasedfromloadednanoparticles,asthisan- tibioticisafourth-generationbroad-spectrumfluoroquinolonethat ishighlyeffectiveagainst biofilm-relatedinfection[95–97].Inad- dition,thisantibiotic is arecent fluoquinoloneandhasbeen sel- dom used in the antibiotic-loaded nanoparticle field, where the mainfluoroquinoloneusedislevofloxacin[98–101].The reduction ofthequantity ofmicrocoloniesadheredonthebonesurfacewas directlyrelatedtotheCOcontainedinAGcoatingofthesurfaceof nanoparticles,since thisantibiotic hasa destabilisingeffecton E.

colibioﬁlmmatrixstructureanditcanleadtothereleaseofplank- toniccells, whichare moresusceptibleto antibiotics[77]. Hence, theuseofAG+CO-coated MX-loadedMSNsshoweda cooperative effectbetweenbothantibiotics,conﬁrmingthesuitabilityofusing thisnanocarrierforthe treatmentofboneinfectionscausedby E.

coli.

Bone infections are linked with progressive inﬂammatory tissue destruction and can induce marked local bone resorption at sitesofinfectionandproximal abnormalbone formation.Overall, three types of cells are responsible for this process, namely osteoblasts/osteocytes,osteoclastsandmacrophages,althoughothers canbeinvolved[102].Nevertheless,thelowestcytotoxicitymedian value wasranged between93.5% for31.25μg/mLand 94.54%for 62.5μg/mL,which canbe consideredaslow foralltypesofcells used accordingto previously publishedstudies [103]. The reduc- tionofcellproliferationwouldbeaconsequenceoftwoantibiotics used,COandMX,asobservedinbothosteoblasts(at14days)and macrophages.COcaninhibitcellproliferationinadose-dependent andtime-dependentmanner [104],whilst MXhas shownan an- 232