RSC Advances

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View Article OnlineView Journal | View Issue

Enhanced antimi

Applied Biotechnology Research Center, Baq

Tehran, Iran

† Electronic supplementary informationused materials and equipment in the pEDX spectra, and the photos of disk coubeen given in this section. The LC-Msequence have been reported, as well. See

Cite this: RSC Adv., 2021, 11, 38961

Received 22nd October 2021Accepted 29th November 2021

DOI: 10.1039/d1ra07821f

rsc.li/rsc-advances

© 2021 The Author(s). Published by

crobial treatment by a clay-baseddrug nanocarrier conjugated to a guanidine-richcell penetrating peptide†

Mohammad Reza Khodabakhshi and Mohammad Hadi Baghersad *

In this study, a novel and efficient drug delivery system is proposed for the enhancement of antimicrobial

properties of antibiotic medications such as vancomycin (VCM) and levofloxacin (OFX). The architecture

of the designed drug carrier is based on halloysite nanotubes (HNTs) with a rolled-laminate shape,

suitable for the encapsulation of drug and further release. In order to make them capable for magnetic

direction to the target tissue, the exterior surface of the tubes is composed of iron oxide nanoparticles

(Fe3O4 NPs), via an in situ process. The main role in the antimicrobial activity enhancement is played by

a cell-penetrating peptide (CPP) sequence synthesized in the solid phase, which contains three arginine–

tryptophan blocks plus a cysteine as the terminal amino acid (C(WR)3). The drug content values for the

prepared nanocargoes named as VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3, have been

estimated at ca. 10 wt% and 12 wt%, respectively. Also, the drug release investigations have shown that

above 90% of the encapsulated drug is released in acetate buffer (pH ¼ 4.6), during a 90 minutes

process. Confocal microscopy has corroborated good adhesion and co-localization of the particles and

the stained living cells. Moreover, in vitro antimicrobial assessments (optical density, zone of inhibition,

and minimum inhibitory concentration) have revealed that the bacterial cell growth rate is significantly

inhibited by suggested nanocargoes, in comparison with the individual drugs in the same dosage. Hence,

administration of the presented nanocargoes is recommended for the clinical treatment of the infected

target organ.

1. Introduction

One of the permanent challenges in the administration ofantibiotic compounds is resistance of bacteria to the anti-infection drugs.1 For instance, an acquired bacterial resis-tance to antibiotic vancomycin (called vancomycin-resistantenterococci) is a well-known type in Enterococcus bacteriumcaused by alteration of the peptidoglycan synthetic pathway.2 Asanother example, bacterial resistance to beta-lactam antibioticssuch as penicillin and cephalosporin via secretion of metallo-beta lactamase enzymes can be mentioned.3 As a traditionalresolution for this issue, co-administration of the antibioticdrug with an assistant has been suggested (like co-amoxiclave).4

Today, with the advent of nanotechnology, the therapeuticeffects of the useless medications have been revived. Briey,tiny size drug carriers, surface-functionalization with the

iyatallah University of Medical Sciences,

(ESI) available: The tables listing theresented work. Moreover, DLS curves,nter experiments on the products haveS results of the synthesized peptideDOI: 10.1039/d1ra07821f

the Royal Society of Chemistry

various protein facilitators, and enhanced internalization intothe cells led to reuse the abandoned drugs.5–7 In fact, viaencapsulation of the aimed drug into the nanostructures,destructive inuences of the bacteria on the antibiotic drugsthat lead to deactivation, is decreased.8,9 All of the mentionedproperties of the nanoscale drug delivery systems help to over-come the bacterial resistance, as well. In this regard, Huh andKwon have published a review on the advances in treatment ofthe bacterial infections by the nanomaterials as a new paradigmknown as “nanoantibiotics”.10 Moreover, Cardoso and co-workers have reported a nanoscale therapeutic containingtetracycline antibiotic into silica nanoparticles to overcome thebacterial resistances.11

In addition to bacterial resistance, the use of nanomaterialsalso affects drug release, cell internalization, and dose controlstrategies.12 Briey, using the porous nanomaterials or poly-meric matrices in the composite form provides this greatopportunity to control the drug release process in the targetcells; for example extended release by the layered structures,13

and controlled release in a specic condition through using pH/temperature responsive polymers.14 Among different types ofthe nanomaterials, halloysite which is a clay-based species withthe rolled tubular structure has shown interesting properties inthe recent therapeutic substances.15,16 As the rst advantage,

RSC Adv., 2021, 11, 38961–38976 | 38961

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a layer-by-layer paradigm can be executed for the loading andsubsequent release process of the aimed drug.17 Through usingthe halloysite nanotubes (HNTs), a determined dosage of thedrug is wrapped and delivered to the target cells.18 Moreover,there is a strong hydrogen binding (H-bind) network betweenthe layers of the HNTs resulting in the formation of the stablecages for encapsulation of the drugs. This H-bind networkorigins from the silanol and aluminol groups onto the exteriorand interior surfaces of the rolled wall, respectively.19 Also, thehydroxyl (–OH) groups located onto the surfaces of the HNTs actas the suitable sites for chemical conjugations.20

As another useful species of the nanomaterials, iron oxidenanoparticles (Fe3O4 NPs) has been widely used in differentscopes such as drug delivery,21,22 catalysis,23,24 diagnosis,25

medical imaging,26 sensing,27 removal of the pollutants,28 andetc.29,30 The rst and foremost excellence of the Fe3O4 NPs is itsimpressive magnetic property which leads to extensive use indifferent applications.31,32 In the eld of drug delivery, magneticbehavior of the nano-carriers provides high convenience in bothpreparation and application processes. The conduction of themagnetic cargoes to the target tissue through applying anexternal magnetic eld has been introduced as an efficientstrategy for the enhancement of the targeted delivery.33 More-over, Fe3O4 NPs with the mean size of 35 nm provide an extremesurface area due to having a huge surface to volume ratio.34,35

Also, the surface of the Fe3O4 NPs could be chemically func-tionalized due to the presence of the –OH groups.36,37 Magne-tization of the different species through combination with theFe3O4 NPs via incorporation into the pores or H-bind interac-tions, is a common practice.38–40

Another potential resolution for the bacterial resistance inthe antimicrobial therapy is submitted based on the use ofantimicrobial peptides (AMPs).41,42 For instance, peptide nucleicacids (PNAs) as synthetic analogs of DNA with 2-([2-aminoethyl]amino)acetic acid backbone have been used due to theirinhibitory effect on transcription and translation processes. ThePNAs are able to complementary bind to DNA/RNA with highselectivity. Therefore, they have been widely used in diagnosisand treatment of various diseases.43,44 As another species ofAMPs, cell-penetrating peptides (CPPs) have been recognized asthe active structures in cellular uptake process. Concisely,through electrostatic interactions between the positive-chargedamino acids and negative-charged ingredients in the cellmembrane, cell adhesion and subsequent penetration areenhanced by the AMPs.45 Typically, arginine (Arg) is used forthis purpose due to containing a guanidine functional group atthe end of the side chain.46 As a reasonable justication, sincethe guanidine group includes more positive-charged nitrogencenters in its chemical structure (HNC(NH2)2), the electrostaticinteractions with the phosphate and sulphate ingredients(present in cell membrane), are enhanced.47 So far, so manyreports have been published about the AMP–drug conjugations,in which an antimicrobial drug is covalently linked to an AMP,resulted in the enhanced anti-infection properties.48–50

Based on the advantages described above, herein, we suggesta novel tiny-sized system constructed of the HNTs decoratedwith the Fe3O4 NPs onto the surfaces, utilizable for magnetic

38962 | RSC Adv., 2021, 11, 38961–38976

drug delivery in the antimicrobial treatments. To overcome thebacterial resistance, the antibiotic drugs are encapsulatedinside and between the rolled layers of the tubes, and then wellwrapped via freeze drying. For this purpose, vancomycin (VCM)and ooxacin (OFX) as two mostly administrated medicationsfor the bacterial infections (specically diarrhea), have beenused. In order to demonstrate the synergistic therapeutic effectsbetween the encapsulated drugs and the AMPs, the surfaces ofthe VCM@Fe3O4/HNT and OFX@Fe3O4/HNT particles arecovalently conjugated to a guanidine-rich peptide chain,synthesized on a solid resin. The structure of the target peptidechain includes an initial cysteine (C) plus three repeated blocksof tryptophan–arginine (WR). Finally, the antimicrobial prop-erties of the prepared VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 therapeutics have been evaluated bythe optical density experiment at the wavelength of 600 nm(OD600), zone of inhibition (ZOI), and disk counter experimentson Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)as Gram-positive and Gram-negative bacteria cell lines,respectively. Briey, it has been observed that the growth rate ofthe bacterial cells is inhibited for above 70% by VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes,during a 90 minutes process. Also, minimum inhibitoryconcentration (MIC) and minimum bactericidal concentration(MBC) values have been estimated for the prepared nano-cargoes and compared with the individual VCM and OFX. Theobtained results have exhibited signicant enhancement of theantimicrobial effect through incorporation of the low dosages ofVCM and OFX drugs into the Fe3O4/HNT–C(WR)3 nano-carrier.

2. Experimental2.1 Materials and equipment

All chemical and instruments used in this project are listed inTables S1 and S2, in ESI le.†

2.2 Preparation and synthetic methods

2.2.1 Preparation of the activated HNTs. HNTs powder (5.0g) was ground via ball-milling (50 Hz) for 30 min, using zirconiagrinding bowls and balls of 0.1 mm in diameter. Then, thecalcination process was performed on the ground particles viaa programmed heating up to 600 �C, during 4 h in the furnace.Aer cooling down to room temperature, the obtained neatpowder of the HNTs was transferred into a sterilized glass ask,then N2 gas was purged and the ask was well sealed. Further,2.0 g of the neat HNTs was placed into a round-bottom ask(100mL) and 50mL of hydrochloric acid (HCl, 1.0 M) was addedinto the ask. To obtain a ne dispersion of the HNTs in theacid medium, ultrasound bath (50 Hz, 100 W L�1) was used, for30 min at room temperature. Aerward, the mixture was stirredfor 24 h, at 80 �C under reux conditions. Next, the activatedHNTs were collected via centrifugation (4000 rpm, 15 min), andrinsed with deionized water for three times. Finally, the ob-tained powder was dried in the vacuum oven and stored ina sterilized glass ask, under N2 gas.51

© 2021 The Author(s). Published by the Royal Society of Chemistry

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2.2.2 Preparation of Fe3O4 NPs. In a round-bottom ask(100 mL), FeCl2$4H2O (1.0 mmol) and FeCl3$6H2O (1.3 mmol)were dissolved in deionized water (30 mL) and stirred at 80 �C,under N2 atmosphere for 2 h. Then, a solution of NH3$H2O (15mL, 25 wt%) was dropwise added into the ask during thestirring. Then the reaction mixture was cooled down to roomtemperature and the obtained magnetic NPs were magneticallycollected and washed for several times with deionized water.Finally, they were dried in oven at 60 �C.52

2.2.3 In situ composition of Fe3O4 NPs with HNTs. For thispurpose, the same procedure as the above section was per-formed with this difference that the neat HNTs (0.5 g) weredispersed in the solution of the iron salts, before addition of theNH3$H2O solution.53

2.2.4 Functionalization of Fe3O4/HNTs nanocompositewith mercaptopropyl silane (MPS). In a double-necked round-bottom ask (50 mL), Fe3O4/HNTs particles (0.5 g) weredispersed in toluene (10 mL) via ultrasonication. Then, themixture was stirred under N2 atmosphere at room temperature,and 3-mercaptopropyl trimethoxysilane (MPTMS) (7.0 mL) wasadded to the ask drop-by-drop using a glass syringe tted inseptum. Next, the mixture was stirred for 12 h under N2 atmo-sphere, under reux conditions (105 �C). Aerward, the ob-tained Fe3O4/HNT–MPS particles were magnetically collectedand washed with hot ethanol (10 mL) for several times. Ulti-mately, the particles were dried in oven at 60 �C.53,54

2.2.5 Incorporation of the VCM and OFX in nano-carriers.In four separate conical tubes (15 mL, Falcon Centrifuge Tubes,polypropylene), the aqueous solutions of VCM and OFX (5.0 mL,20 mg mL�1) were placed and then Fe3O4/HNT–MPS particles(0.1 g) were dispersed in the tubes via ultrasonication (50 Hz, 5min), at room temperature. Next, the tubes were sealed andcovered by the foil, and the content of the tubes were shaken for24 h. Aer completion of the process, the obtained nanocargoes(VCM@Fe3O4/HNT–MPS and OFX@Fe3O4/HNT–MPS) wereseparated via centrifugation (4000 rpm, 15min), and washed fortwo times with deionized water, via vortex mixing and centri-fugation. Finally, the obtained particles were redispersed in thedeionized water, transferred to the glass Petri dishes, and driedby freeze drier, for 48 h.55

2.2.6 Synthesis of the C(WR)3 peptide chain. The C(WR)3peptide sequence was synthesized in the solid phase; where 2-chlorotrityl chloride (CTC, loading 0.8, 0.5 g) was well washedwith DMF (10 mL) via shaking, swollen in DCM (10 mL), anddried by the vacuum pump. Then, the rst amino acid (Fmoc–Trp(Trt)–OH, 1.0 mmol) was dissolved in DMF (10 mL) andDIEA (8.0 mmol). Then, the prepared mixture was added to theswollen CTC placed in a glass vessel, and shaken for 2 h, atroom temperature. Aerward, removal of the Fmoc protectinggroup was carried out with piperidine solution (25% in DMF).Further, next amino acid (Fmoc–Arg(Pbf)–OH) was activated bydissolving in a solution of DMF (10 mL), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrauoroborate (TBTU, 1.3mmol), and N,N-diisopropylethylamine (DIPEA, 0.8 mL). Theobtained solution was then added into the vessel and shaken for2 h, at room temperature. This process was continued for the

© 2021 The Author(s). Published by the Royal Society of Chemistry

next amino acids, and aer completion of the process (attach-ment of Fmoc–Cys(Trt)–OH), the synthesized peptide chain wasseparated from the CTC resin by a solution of TFA 96%, TES2.0% and deionized water 2.0%. Then, the CTC resin wasremoved via ltration, and TFA was evaporated by rotary evap-orator, under reduced pressure. Next, cold diethyl ether wasadded to form a white powder, and themixture was stirred in anice bath, for 1 h. Finally, the white powder was obtainedthrough paper ltration.55

2.2.7 Preparation of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes. For disulde bondconjugation, aer dispersion of 0.1 g of the VCM@Fe3O4/HNT–MPS or OFX@Fe3O4/HNT–MPS particles in 3.0 mL of colddeionized water (4 �C), H2O2 (30 wt%, 0.2 mL) and Cys-containing peptide chain (3.0 mL, 0.5 M, in ethanol) wereadded to the mixture drop-by-drop using two separate syringes,during the shaking for 1.5 h.56 Aer completion of the process,the obtained VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes were separated via centrifugation(4000 rpm, 15 min), and washed for two times with deionizedwater, via vortex mixing and centrifugation. Finally, the parti-cles were redispersed in the deionized water, transferred to theglass Petri dishes, and dried by freeze drier, for 48 h.

2.3 Confocal microscopy

Initially, all glassware were sterilized with autoclave, at 120 �C.In a glass tube with a threaded cap, 0.05 mg of VCM@Fe3O4/HNT–C(WR)3 particles was dispersed in 1.0 mL of Dulbecco'smodied Eagle's medium (DMEM), and the bacterial cells (S.aureus-ATCC 12600 or E. coli-ATCC 9637) with the density of 3�108 CFU mL�1 were added. The content of the tube was incu-bated at 37 �C, at 95% humidity. Aer 90 min incubation, cellstaining was performed using three drops of diluted crystalviolet in phosphate buffer saline (PBS, 0.1 M), and a drop ofLugol's solution. Then, 30 mL of the sample was poured ona laminated glass slide and dried.55

2.4 Antimicrobial experiments

2.4.1 Cell cultivation. For cultivation, S. aureus-ATCC 12600and E. coli-ATCC 9637 cells were incubated in the LB brothmedium with density value of 106–107 CFU mL�1. Cultivationwas done in the sterilized glassware at 37 �C and 95% humidity.Incubation was carried out in a shaking incubator with180 rpm.55

2.4.2 Optical density experiments. Cell cultures werecarried out in the presence of the VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 particles (50 mg mL�1, inDMEM), dispersed via ultrasonication (50 kHz, 100 W L�1) for2 min. A dispersion of related particles was used as blankcontrol. Also, culture at particle-free medium was used asa control. More dispersions including Fe3O4/HNTs, Fe3O4/HNT–C(WR)3, and also individual C(WR)3 peptide sequence, VCMand OFX with the same concentration as the main samples wereassessed. Three samples for each condition were prepared ina 96-well plate. Ultimately, the prepared samples were studiedby a UV-vis spectroscopy at 30, 60, and 90 min.55

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2.4.3 Zone of inhibition experiments. Initially, appropriatedispersions of the solid particle samples including VCM@Fe3-O4/HNT–C(WR)3, OFX@Fe3O4/HNT–C(WR)3, HNTs, Fe3O4/HNTs, and Fe3O4/HNT–C(WR)3 were prepared in DMEMmedium, with the concentration of 10 mg mL�1. Also, theindividual VCM, OFX, and C(WR)3 peptide sequence were dis-solved in DMEM with the same concentration as the particles,and the sterilized disks were used for the subjection to the cells.Cultivation was performed in the sterilized Petri dishes con-taining the LB broth liquid medium and the bacterial cells, viaincubation at 37 �C for 24 h. The formed zones of inhibitionwere evaluated and reported in centimeter unit. Three samplesfor each compound were prepared and studied to checkreproducibility of results.55

3. Results and discussion3.1 Preparation of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes

Initially, to prepare a uniform collection of the tubes, HNTswere ground by ball-milling. Then, to remove the unwantedllers, calcination was performed at high temperatures. Next, inorder to activate silanol and aluminol groups, treatment of thetubes with HCl was carried out.35,51 To magnetize the tubes, co-deposition of the iron(II) and (III) chloride salts in an alkalinecondition was performed through an in situ manner, resultingin F3O4/HNTs nanocomposite. For this purpose, the HNTs wereinitially well dispersed via ultrasonication.57 To functionalizethe surface of the F3O4/HNTs, mercapto silane compound wereused. Due to the presence of the propyl groups in the structureof mercapto propyl silane (MPS), the hydrophobicity of thesurfaces is a bit increased (video le no. 1), which is appropriatefor prevention of drug leaching. In this stage, the surface of theparticles are covalently functionalized through silicon–oxygenbonds.58–60 In the next stage, the silanized-Fe3O4/HNTs nano-particles were dispersed in the concentrated solutions of VCMand OFX drugs, and then shaken in the darkness. Lightprotection was preformed via insulation of the reaction askwith the pieces of foil, as ordered in the analytical papers of theVCM and OFX for preservation. On the other hand, C(WR)3peptide sequence was synthesized using solid-phase peptidesynthesis techniques, described in literature.55 For disuldebond conjugation between the F3O4/HNT–MPS particles andC(WR)3 peptide sequence, a cold solution of hydrogen peroxidewas used.56 Scheme 1 presents the successive stages of thepreparation route of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes.

The C(WR)3 peptide sequence was synthesized on CTC resin,respect to the approved procedures for solid-phase peptidesynthesis. Briey, CTC seeds were well washed with DMF, andthen swollen in DCM. Shaking and drying by the vacuum pumphave been used within the successive stages of the synthesis.Fmoc–Arg(pbf)–OH was dissolved in DMF and DIEA, and theprepared solution was added to the swollen CTC placed ina glass vessel, and shook. Aerward, removing of the Fmoc-protecting group was carried out using piperidine solution(25% in DMF). Next, Fmoc–Trp(boc)–OH was dissolved in

38964 | RSC Adv., 2021, 11, 38961–38976

a mixture of DMF, TBTU (as a coupling reagent), and N,N-dii-sopropylethylamine (DIPEA), and then added into the vesseland shook. This process was repeated for the next amino acidsuntil three successive blocks of arginine–tryptophan (RW) wasobtained. As the last amino acid, Fmoc–Cys(Trt)–OH was addedto the sequence. Finally, the synthesized peptide sequence(C(WR)3) was separated from the resin using a mixture of TFA(96%), triethyl silane (TES, 2.0%), and deionized water (2.0%).The synthesized peptide sequence was identied by liquidchromatography-mass spectrometry (LC-MS) (Fig. S1, in ESIsection†).

3.2 Characterization of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes

3.2.1 FTIR spectroscopy. As the rst method for charac-terization of the products, Fourier-transform infrared (FTIR)spectroscopy was used to investigate functionalization of theHNT cargo aer successive stages of the preparation. As isobserved in Fig. 1, successful formation of the Fe3O4 NPs isconrmed by two indicative peaks; one at ca. 580 cm�1 relatedto Fe–O bond, and another one at 3000–3500 cm�1 belonging tothe hydroxyl groups (O–H) present on the surface of the parti-cles (spectrum 1).61 HNTs are generally characterized by a sharppeak at ca. 1100 cm�1, which is related to the stretching vibra-tions of Si–O–Si bands in the silica network. The broad peak ofthe hydroxyl groups (silanol and aluminol) is also appeared at3100–3600 cm�1 (spectrum 2).62 In the same spectrum, there arealso some other peaks with weak intensity appeared at ca. 1635,1750, 2960, and 2990 cm�1, which are attributed to theunwanted materials inside the tubes. In spectrum 3 thatbelongs to the ground HNTs, this is clearly seen that the similarpeaks are appeared at the same areas, conrming well preser-vation of the silica network during the ball-milling process.Aer acid treatment of the HNTs (spectrum 4), two separatepeaks have been appeared at ca. 3624 and 3695 cm�1 that areattributed to the stretching vibrations of AlO–H bonds, presentonto the interior surface of the rolled walls of HNTs.63 Activationof these hydroxyl groups before encapsulation of drug into thetubes is of high importance, since they play a key role in theformation of the H-bond network between the walls and wrap-ping the cargo.64 In spectrum 5 that is related to the magnetizedHNTs, this is observed that a broad peak has been added in thearea of 3000–3600 cm�1, corroborating the presence of thehydroxyl groups of Fe3O4 NPs. Moreover, the sharp peak of Fe–Obond at ca. 580 cm�1 has been added to the spectrum, verifyingwell composition of Fe3O4 NPs with the HNTs. Furthermore,appearance of a weak peak at ca. 2972 cm�1 veries the pres-ence of the C–H bonds with hybridation sp3, coming frommercaptopropyl silane (MPS). Finally, in spectrum 6 belongingto VCM@Fe3O4/HNT–C(WR)3 nanocargo, a tangible increase inthe peak intensity of C–Hsp3 at ca. 2959 cm�1, and alsoappearance of the new peaks in a range of 1600–1800 cm�1

coming from carbonyl groups in the structure of VCM andC(WR)3 peptide sequence, clearly corroborate successful addi-tion of the VCM and C(WR)3 to the composite.

© 2021 The Author(s). Published by the Royal Society of Chemistry

Scheme 1 Schematic presentation of the successive stages of the preparation route of VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes.

Fig. 1 FTIR spectra of (1) Fe3O4 NPs, (2) natural HNTs, (3) groundHNTs, (4) activated HNTs, (5) Fe3O4/HNTs–MPS nanocomposite, and(6) VCM@Fe3O4/HNT–C(WR)3 nanocargo.

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3.2.2 EDX and XRD spectroscopy. One of the mostly usedmethods for investigation of the new ingredients added to thenanostructures, is energy-dispersive X-ray (EDX) analysis. Viathis method, the presence of the expected elements with therelated quantities in the samples is conrmed. Fig. S2 (in ESIsection†) illustrates the obtained EDX spectra of eight samplesincluding natural, calcinated, and acid-activated HNTs,magnetized HNTs with Fe3O4 NPs, MPS-functionalized Fe3O4/HNT, VCM@Fe3O4/HNT–MPS, VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 composite structures. As is seen, all

© 2021 The Author(s). Published by the Royal Society of Chemistry

essential elements exist in the spectra of each sample, corrob-orating successful combination of the materials. Froma comparison between the obtained spectra for the individualHNTs, this is quickly found out that a cleaner structure hasbeen resulted aer the calcination and acid-treatmentprocesses. As shown, only two peaks are observed in the EDXspectrum of activated HNTs, verifying successful removal of theunwanted materials such as calcium, sodium, and carbon.Moreover, increase in the peak intensity correlates with theabundance of the elements. As presented by quantitative resultstables, weight percentage of carbon and nitrogen atoms hasraised aer conjugation of the Fe3O4/HNTs to the C(WR)3peptide structure. Also, the peaks related to chlorine and uo-rine elements have emerged at the areas of ca. 2.6 and 0.6 keV,aer incorporation of VCM and OFX in the tubes, respectively.

For further conrmation of successful composition of Fe3O4

NPs and HNTs, X-ray diffraction (XRD) patterns were providedand compared together. As demonstrated in Fig. 2, all referencepeaks related to the structure of Fe3O4 NPs (JCPDS no. 19-0629)have been appeared in the pattern of Fe3O4/HNTs, corrobo-rating well combination of the spherical particles with thenanotubes. These indicative peaks have appeared at 2q ¼ 31.1�,35.4�, 43.3�, 53.5�, 57.3�, and 62.6�. Also, in the pattern of Fe3O4/HNTs composite, there are two peaks at 2q ¼ 20.2� and 23.8�

marked with Miller indices (0 2 0) and (0 0 2), respectively,which verify the presence of HNTs (JCPDS no. 29-1489).

3.2.3 TGA analysis. Thermogravimetric analysis (TGA) wasperformed on the samples to investigate decomposition statesof the used ingredients via thermal degradation. From a quicklook at Fig. 3a, the structural differences between Fe3O4/HNTs,VCM@Fe3O4/HNT–C(WR)3, and OFX@Fe3O4/HNT–C(WR)3composites are found out. In all three curves, a partial increasein the weight ratio has occurred at the beginning of study. Thisincrease is most likely coming from the adsorption of the

RSC Adv., 2021, 11, 38961–38976 | 38965

Fig. 2 XRD patterns of Fe3O4 NPs and Fe3O4/HNTs composite.

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moisture in the air by hot surfaces of the particles. For Fe3O4/HNTs composite, almost a gradual descending trend isobserved until 800 �C, in which ca. 8% of the weight has beenlost. This amount of weight loss is attributed to the removal ofthe water molecules from the structure at the early stages ofdegradation. At higher temperatures, the captured water by theunderlying layers (especially alumina and silica networks) areseparated.65 For the VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3-O4/HNT–C(WR)3 structures, this is clearly observed that morevalues of the weight loss occurred during thermal degradationprocess in a range of 50–800 �C. For VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 structures, ca. 4% and 3% of theweight have been lost, respectively, due to separation of the

Fig. 3 (a) TGA curves of Fe3O4/HNTs, VCM@Fe3O4/HNT–C(WR)3, and OFtemperature magnetic-hysteresis curves of Fe3O4 NPs and VCM@Fe3O4

38966 | RSC Adv., 2021, 11, 38961–38976

water molecules. Then, the weight ratios of both samplesgradually reduced to ca. 90% and 86% by increase in thetemperature to around 580 �C, for VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3, respectively. At this stage, theorganic structures including C(WR)3 and parts of the encapsu-lated VCM and OFX drugs are decomposed.66 Aerward, therolled structure of HNTs are probably dissociated and thecombined Fe3O4 NPs start to degradation. Simultaneously, theentrapped VCM and OFX inside the underlying layers of therolled tubes start to release and decomposition.

3.2.4 VSM analysis. To investigate magnetic properties ofthe formulated nanocargoes, vibrating-sample magnetometer(VSM) analysis was used. As shown in Fig. 3b, magnetization

X@Fe3O4/HNT–C(WR)3, composites, under air atmosphere. (b) Room-/HNT–C(WR)3 composite.

© 2021 The Author(s). Published by the Royal Society of Chemistry

Fig. 4 SEM images of (a) Fe3O4 NPs, (b) natural HNTs, (c) acid-treated HNTs, (d) ground HNTs, and (e and f) Fe3O4/HNTs composite. TEM imagesof (g) Fe3O4 NPs, and (h) VCM@Fe3O4/HNT–C(WR)3 composite. (i) Size-distribution diagram of acid-treated HNTs, related to the green box in theimage (c).

Fig. 5 UV-DRS curves of the neat HNTs, individual VCM, VCM@HNT,

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saturation of the neat Fe3O4 NPs occurs at 57 emu g�1, throughapplication of a 10 K Oe magnetic eld. Whereas, this valuereduced to ca. 22 emu g�1 for VCM@Fe3O4/HNT–C(WR)3composite. Obviously, since other combined components suchas HNTs, VCM, and C(WR)3 are not magnetic, the magneticproperty of the nal product is much less than the neat Fe3O4

NPs. However, this value of magnetization saturation isadequate for execution of the magnetic direction of nano-cargoes in the body.67

3.2.5 SEM/TEM imaging. To investigate size distributionand composition states of the formed particles, scanning-electron and transmission-electron microscopy (SEM andTEM) were employed. As illustrated in Fig. 4a, the neat Fe3O4

NPs with spherical morphology and average size of ca. 42 nmhave been successfully synthesized. As is seen in the preparedSEM image, agglomeration of the particles is observed in someareas that is most likely due to super-paramagnetic property ofthe particles. Next, the tubular structure of the natural HNTs isrecognized in Fig. 4b. Also, the existence of the excess materialswith the HNTs is clearly veried in the prepared SEM image (b).As described in the preparation section, to make a uniform andclean collection of the HNTs, grinding by ball-milling was per-formed. As shown in Fig. 4c, well uniformity in size and shape ofthe HNTs has been obtained aer the grinding process.Generally, estimation of the mean size of the particles is carriedout via two ways; rst, high-resolution (HR) TEM imaging andselected area (electron) diffraction (SAED) are performed on the

© 2021 The Author(s). Published by the Royal Society of Chemistry

sample.68,69 Second, a zoomed-out image is selected andimported into Digimizer soware, then the size of the particlesthat are distinguishable by eyes is estimated.70 In this work, weused the second method and selected an area for evaluation ofsize distribution of for the neat HNTs (green box in image c).

and VCM@Fe3O4/HNT composites.

RSC Adv., 2021, 11, 38961–38976 | 38967

Table 1 The obtained results from drug content study on VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 samples

Sample Aa (a. u.) Cb (mg mL�1) Drug contentc (wt%) Rel. E.d (%)

VCM@Fe3O4/HNT–C(WR)3 0.138 0.20 10.0 8.1OFX@Fe3O4/HNT–C(WR)3 0.680 0.24 12.0 9.2

a UV-vis absorbance activity of the ltered solutions aer separation of the particles, at 281 and 298 nm. b Concentration of VCM or OFX in theinitial samples containing 50 mg of particles and 25 mL of DMSO. c Drug content values were estimated via calculation methods given in theESI section. d Relative errors were obtained by considering the values for three samples for each case (n ¼ 3).

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The resulted size distribution diagram has been given in Fig. 4i,presenting the mean size of the neat HNTs around 1600–2100 nm before the grinding process. To corroborate the effi-ciency of the grinding stage, size distribution state ofa suspension of HNTs in the aqueous medium has beeninvestigated by Zetasizer analysis. Fig. S3 (in the ESI section†)demonstrates the obtained intensity-based dynamic-light scat-tering (DLS) curves related to the HNTs suspension samplesbefore and aer grinding process, conrming breakage of thetubes into smaller pieces. The average size of the HNTs has beenestimated to be ca. 400 nm, aer the grinding process. Obvi-ously, the mean size of the particles in the colloidal form is lessthan the solid state, since irradiation of the ultrasound waves isused for dispersion of the particles in the liquid medium.Fig. 4d illustrates the neat structure of the HNTs aer acidtreatment and grinding processes. Fig. 4e and f conrmssuccessful composition of the Fe3O4 NPs with the HNTs. Fig. 4gpresents the TEM image of the synthesized Fe3O4 NPs, in whichdark spots come from the spherical nanoparticles. In Fig. 4h,incorporation of the Fe3O4 NPs onto the exterior surfaces of theHNTs in the nal structure of VCM@Fe3O4/HNT–C(WR)3 isveried. In this image, the presence of the dark spots onto thesurface of the tubes well corroborate combination of the parti-cles with the tubes.

3.2.6 UV-DRS spectroscopy. Fig. 5 shows UV-vis differentialreectance spectroscopy (UV-DRS) curves related to neat HNTs,individual VCM, VCM@HNT, and VCM@Fe3O4/HNT compos-ites, prepared for comparison and investigation of drug loadinginto the tubes. All samples were well rinsed with deionizedwater and ethanol for several times to remove any excesscompounds from the surfaces. As is observed, the maximumvalues of the UV-vis reectance activity of the neat HNTs (blueline) and individual VCM (violet line) have been obtained at ca.225–325 and 235 nm, respectively. From the curve ofVCM@HNT (gray line), it is concluded that the VCM has beensuccessfully incorporated into the tubes, as it shows reectanceactivity in a wavelength range of ca. 220–330 nm. In the curverelated to VCM@Fe3O4/HNT composite (red line), this is seenthat an additional sharp shoulder has been added in the area of335–425 nm. This peak is attributed to the Fe3O4 NPs that havebeen incorporated into the composite structure.71

Fig. 6 UV-vis absorbance–concentration calibration curves of VCMand OFX standard solutions in PBS (0.1 M, pH ¼ 6.8), obtained in fivepoints. Error bars represent standard deviations for three samples ofeach concentration (n ¼ 3).

3.3 Therapeutic properties of VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 nanocargoes

3.3.1 Drug content of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes. To make an

38968 | RSC Adv., 2021, 11, 38961–38976

approximate estimation of drug content loaded into the Fe3O4/HNT–C(WR)3 nanocarrier, UV-vis absorbance spectroscopy wasused. For this purpose, two calibration curves (absorption byconcentration) were drawn using ve standard solutions ofVCM and OFX in phosphate buffer saline (PBS, 0.1 M, pH¼ 6.8),with the concentrations of 5, 10, 15, 20, and 25 ppm. From theobtained line equations, concentration of the unknown solu-tions (in ppm) can be the obtained through putting the UV-visabsorbance values (y) in the equations (Fig. 6).72 For prepara-tion of the samples, 50 mg of VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 particles were put in two separateasks, then ultrasonicated in 25 mL of dimethyl sulfoxide(DMSO), for an hour at room temperature. Simultaneously, thesame procedure was carried out for a sample of Fe3O4/HNT–C(WR)3 particles to provide an appropriate solution for the baseline. Aer completion of the process, the particles werecollected by an external magnet, and separated. The excessparticles were removed by centrifugation (4K rpm) and paperltration, until a clear solution was obtained. Then, dilution ofthe obtained solutions with the PBS medium was performedthrough transfer of 1.0 mL of the sample solution to 25 mL ofthe PBS. Finally, the obtained solutions were studied by UV-visspectroscopy at 281 and 298 nm for VCM and OFX, respectively.As summarized in Table 1, drug content values for VCM@Fe3O4/

© 2021 The Author(s). Published by the Royal Society of Chemistry

Table 2 The obtained results from drug release study on VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 samples

Sample Conditiona Ab (a. u.) Time (min) Releasec (%) Rel. E.d (%)

VCM@Fe3O4/HNT–C(WR)3 PBS (6.8) <0.01 60 Partial 10.9PBS (6.8) 0.033 90 23.9 11.1PBS (9.0) 0.058 60 42.0 10.2PBS (9.0) 0.092 90 66.7 7.4AcB (4.6) 0.108 60 78.2 5.5AcB (4.6) 0.126 90 91.3 5.2

OFX@Fe3O4/HNT–C(WR)3 PBS (6.8) 0.061 60 8.8 9.3PBS (6.8) 0.109 90 16.0 8.2PBS (9.0) 0.309 60 45.4 4.7PBS (9.0) 0.412 90 60.6 5.8AcB (4.6) 0.566 60 83.2 4.2AcB (4.6) 0.632 90 92.9 3.6

a All samples were prepared in 2.0 mgmL�1 concentration, and stirred at 37 �C. Ultrasonication (50 kHz, 100W L�1) has been applied for 2minutes,to make a ne dispersion of the particles. b UV-vis absorbance activity of the ltered solutions were recorded at 281 and 298 nm for VCM and OFX,respectively. All absorbance digits were compared with the maximum values (A0 ¼ 0.138 and 0.680) obtained in drug content experiment. c Thepercentage of the released amounts have been calculated via the following equation, A/A0 � 100. d Relative errors were obtained by consideringthe values for three samples for each case (n ¼ 3).

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HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes havebeen estimated (10.0 � 0.8) wt% and (12.0 � 1.1) wt%,respectively.

3.3.2 Drug release behavior of VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 nanocargoes. To do investiga-tions on drug release process from VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 nanocargoes, similar portions ofthe particles were well dispersed in three buffered media, andstirred at 37 �C. For this purpose, PBS (0.1 M) with the pH valuesof 6.8 and 9.0, and acetate buffer (AcB, 0.1 M) with pH ¼ 4.6were used. This is an important point for the prepared cargoesto release majority of the encapsulated drug in the acidicmedium, since the cargoes will experience a similar conditioninto the cytoplasm environment aer the internalizationprocess.73,74 According to literature, there are hydrogen bond (H-bond) interactions between the silanol and aluminol groups inthe layered structure of HNTs, which provides a suitablesubstrate for drug encapsulation. In the acidic conditions, thisH-bond network is dissociated and consequently the loaded

Fig. 7 Confocal microscopy images of the co-localized VCM@Fe3O4/Hcoli (yellow color) bacteria cells, during 90 minutes incubation (tempercrystal violet (emission at 590 nm).

© 2021 The Author(s). Published by the Royal Society of Chemistry

drug is released.51 This structural feature of the HNTs results inthe huge prevention of drug leaching in the pH-neutral envi-ronments. The release amount of VCM and OFX were moni-tored by UV-vis absorbance spectroscopy, via the sameprocedure explained in the section of drug content estimation.Also, the calibration curves and line equations presented inFig. 6 were used for this purpose. Table 2 reports the obtainedresults from drug release screening experiments. According tothe table, as expected, the major release; (91.3 � 4.7)% for VCMand (92.9 � 3.3)% for OFX, occurs in the acidic conditionsprovided by AcB, during a 90 minutes process.

3.3.3 Cell attachment and uptake of the nanocargoes. Toinvestigate cellular uptake process for the prepared therapeuticnanocargoes, VCM@Fe3O4/HNT–C(WR)3 particles were studiedon two cell lines; S. aureus, as a Gram-positive and E. coli, asa Gram-negative bacterial strain. To make a meaningfulcomparison, individual VCM was also subjected to the cells asa control. For this purpose, a 90 minutes process was executedat 37 �C and 90% humidity. In order to make the target cells

NT–C(WR)3 nanoparticles (blue color) with S. aureus (red color) and E.ature: 37 �C, humidity: 90%). Cell staining has been carried out using

RSC Adv., 2021, 11, 38961–38976 | 38969

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visible in confocal microscopy, cell-membrane staining wascarried out using crystal violet, which shows the photo-luminescence emission activity at ca. 590 nm.75 Generally, themerge panels reveal cell-attachment and co-localization ratios,where a compilation of two different colors appears. Also, theintensity of the merged colors origins from the compilationratio.76 Fig. 7 illustrates the obtained images from confocalmicroscopy aer incubation of the compounds with the cells. Asis observed, for the S. aureus cells, formation of more amountsof purple color with high intensity in the merge panel of VCM-nanocargo conrms higher degree of co-localization incomparison with the individual VCM. In the same way, for E.coli strain, formation of the white color in the merge panelcorroborates the overlap of the yellow and blue colors, origi-nating from co-localization of the VCM and the living cells. As isseen in the merge panels, the white color is observed in moreareas of the image of VCM-nanocargo, than the individual VCM.These observations well disclose that cell adhesion as the mostimportant stage of the therapeutic program is signicantlyenhanced through conjugation of the C(WR)3 peptide sequenceto the drug carriers.

3.3.4 Antimicrobial behavior of VCM@Fe3O4/HNT–C(WR)3and OFX@Fe3O4/HNT–C(WR)3 nanocargoes

3.3.4.1 Optical density experiment. In order to evaluateantimicrobial potency of the prepared VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes, opticaldensity experiments (OD600) were performed on the samples.77

For this purpose, the same concentration (50 mg mL�1) of thenanocargoes and related controls including Fe3O4/HNTs, Fe3O4/HNT–C(WR)3, and individual C(WR)3 peptide sequence, VCMand OFX were assessed over S. aureus and E. coli, as Gram-positive and Gram negative bacteria strains, respectively. TheOD activity of the samples was studied at four times; 0, 30, 60,and 90 minutes. Initially, cell colonies were cultivated in LB

Fig. 8 Bacteriostatic effects of VCM@Fe3O4/HNT–C(WR)3 and OFX@FeFe3O4/HNTs, Fe3O4/HNT–C(WR)3, and individual C(WR)3 peptide sequenhave been prepared and subjected to the cells in the same concentratfrequency and 100 W L�1 power density (2 minutes, at room temperdispersions. The error bars show relative error (%) for three samples of e

38970 | RSC Adv., 2021, 11, 38961–38976

broth medium with a cell density value of ca. 107 CFU mL�1, viaincubation for 24 hours at 37 �C and humidity of 95%. It shouldbe noted that sterilization of the tools at 120 �C before startingthe work is essential. Dulbecco's modied Eagle's medium(DMEM) was used for preparation of the dispersions of theparticles. As presented in diagrams (Fig. 8), signicant growthinhibitions occurred by VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes, compared respect tothe controls. During a 90 minutes process, (75.2 � 1.9)% and(64.8 � 1.5)% growth inhibition have been obtained bysubjection of VCM@Fe3O4/HNT–C(WR)3 particles to the S.aureus and E. coli cells, respectively. These values increased to(78.0 � 1.6)% and (70.2 � 1.4)% for the OFX@Fe3O4/HNT–C(WR)3 particles. This difference may be due to more loadingratio of OFX into the tubes than the VCM (Section 3.3.1). For theindividual VCM and OFX with the same concentration as theparticles, (60.1 � 1.9)% and (62.9 � 2.0)% on S. aureus, and(48.8 � 2.8)% and (46.0 � 1.5)% on E. coli, bacteriostatic effectshave been observed, during 90 minutes. Since, there have beenno noticeable antimicrobial effects for the neat Fe3O4/HNTs,individual C(WR)3, and Fe3O4/HNTs–C(WR)3, the observeddifference between the individual drugs and the nal products(nanocargoes) is attributed to the efficacy of the C(WR)3 peptidesequence. As is seen, there was also no remarkable growthinhibition effect for the individual C(WR)3, concluding that thiscompound is playing a role just in the cell-penetration process.Partial antimicrobial effects have been exhibited for the indi-vidual C(WR)3 and Fe3O4/HNTs–C(WR)3 on both cell lines.These amounts of growth inhibition can be ascribed to thesulfur and disulde bonds, which are able to induce the anti-microbial trait.78 Since the prepared nanocargoes will be sug-gested for the clinical use, it is essential to evaluate thecytotoxicity levels of the products on eukaryotic cells, as well.For this purpose, 3T3 broblast strain was chosen as a human

3O4/HNT–C(WR)3 nanocargoes on (a) S. aureus and (b) E. coli strains.ce, VCM and OFX have been experimented as the controls. All samplesion of 50 mg mL�1 (in DMEM). Ultrasonication in a bath with 50 kHzature) was performed on the heterogeneous samples to make fineach condition (n ¼ 3).

© 2021 The Author(s). Published by the Royal Society of Chemistry

Table 3 The obtained results from ZOI experiments on VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes incomparison with the controls

SampleZone diameteron S. aureus (cm)

Zone diameteron E. coli (cm)

VCM 3.2 � 0.1 2.3 � 0.1OFX 3.2 � 0.1 2.6 � 0.2C(WR)3 Partial PartialHNTs Partial PartialFe3O4/HNTs Partial PartialFe3O4/HNT–C(WR)3 Partial PartialVCM@Fe3O4/HNT–C(WR)3 3.7 � 0.2 3.0 � 0.2OFX@Fe3O4/HNT–C(WR)3 3.9 � 0.2 3.2 � 0.1

Fig. 9 Digital images of the inhibition zones created by VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes, in comparisonwith the controls including VCM, OFX, HNTs, Fe3O4/HNTs, Fe3O4/HNT–C(WR)3, and C(WR)3 peptide sequence, over S. aureus (a–c) and E. coli(d–f) cells. Incubation was carried out at 37 �C and humidity of 95%, for 24 hours.

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normal cell line. Briey, the same procedure as the bacterialcells was implemented at which the 3T3 cells were cultured inLB broth medium with a cell density value of �107 CFU mL�1,and the same concentration (50 mg mL�1) of the individualVCM, OFX, and both nanocargoes were subjected to the cellsaer well dispersion in DMEM by ultrasound waves. As shownin Fig. S4 (in ESI le†), no signicant growth inhibition wasobserved aer subjection of the nanocargoes during a 90 minincubation process. As is seen, aer passing 30 minutes ofincubation, the growth rate has been a bit affected by theindividual VCM and OFX. This partial inuence can be attrib-uted to the change of pH to acidic values aer dissolution ofVCM and OFX.79

3.3.4.2 Zone of inhibition and colony counter experiments. Toperform more investigations on the bacteriostatic properties ofthe prepared VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes, the zones of growth inhibition(ZOIs) in the cultivation Petri dish (containing nutrient agar)have been diametrically evaluated. For this purpose, S. aureusand E. coli cells were incubated in the presence of 10 mg of thepowder samples of the products and the controls, for 24 hours.Fig. 9 illustrates the provided images from dishes aer thecompletion of the incubation time. As is observed, for S. aurous(images a–c), VCM@Fe3O4/HNT–C(WR)3 (VCM-nanocargo) andOFX@Fe3O4/HNT–C(WR)3 (OFX-nanocargo) have created inhi-bition zones with (3.7 � 0.2) cm and (3.9 � 0.2) cm diameters,

© 2021 The Author(s). Published by the Royal Society of Chemistry

respectively. Whereas, the same dosage of the individual VCMand OFX have formed zones with (3.2� 0.2) cm diameter. Thesemeaningful differences in the antimicrobial activity betweenthe prepared nanocargoes (with only ca. 11 wt% drug content)and the individual drug compounds, well corroborateenhancement of antimicrobial properties through incorpora-tion of the drugs inside the nano-carrier and conjugation to theC(WR)3 peptide sequence. As well, this is seen that the controlssuch as HNTs, Fe3O4/HNTs, Fe3O4/HNT–C(WR)3, and C(WR)3peptide sequence have solely shown no considerable

RSC Adv., 2021, 11, 38961–38976 | 38971

Table 4 Obtained results from MIC/MBC experiments onVCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nano-cargoes, over S. aureus and E. coli cells

SampleMIC(mg mL�1)

MBC(mg mL�1)

S. aureus VCM 8 64OFX 2 2VCM@Fe3O4/HNT–C(WR)3 8 64OFX@Fe3O4/HNT–C(WR)3 2 2

E. coli VCM 32 128OFX 2 4VCM@Fe3O4/HNT–C(WR)3 16 64OFX@Fe3O4/HNT–C(WR)3 2 4

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antimicrobial effects on both cell lines. The same result isobserved for the E. coli cells (images d–f), with this differencethat the growth inhibition has been a bit reduced totally for allsamples. It may origin from higher resistance of Gram-negativebacteria against membrane penetration.80 To have a quantita-tive assessment on bacteria cell killing by the prepared nano-cargoes, disk counter experiment was performed via subjectionof VCM@Fe3O4/HNT–C(WR)3 particles to S. aureus cells. Forthis purpose, bacteria cells were cultivated in LB broth mediumfor 24 hours at 37 �C. Then, the opacity of the solution reachedto 0.4, and a suspension of VCM@Fe3O4/HNT–C(WR)3 inDMEM (5 mg mL�1) was prepared using an ultrasound bath forwell dispersion. The population of the bacteria cells in thedishes was counted over 24 hours. The obtained results haverevealed that great antibacterial treatment is executed by theprepared nanocargoes, as ca. (96.6 � 14.3)% cell death wasoccurred in the dishes (Fig. S5, in ESI section†). Table 3concisely reports the obtained results from the ZOI experimentson the samples.

3.3.4.3 Minimum inhibitory and bactericidal concentrations.As another comparative method, minimum inhibitory concen-tration (MIC) and minimum bactericidal concentration (MBC)values were evaluated for the samples of VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 nanocargoes, and theobtained results were compared with the controls. MIC and

Table 5 The weight values of recollected Fe3O4/HNTs, and corre-sponding concentrations measured by ICP analysis in the supernatantsafter circulation for 120 hours, at 37 �C (pharmacodynamics)

EntryTime ofexperiment (h)

Weighta

(mg)Rel. errorb

(%)Fec

(ppm)Si(ppm)

Al(ppm)

1 0 100 — — — —2 12 97.2 5.3 Trace Trace Trace3 24 91.9 6.6 1.08 0.72 0.364 48 68.3 8.6 4.32 3.10 1.375 72d 52.5 11.5 6.36 4.65 2.026 120 34.3 17.6 8.74 6.36 2.81

a 100 mg of Fe3O4/HNTs was dispersed in 10.0 mL of HAS. b Relativeerrors have been calculated for three samples for each condition (n ¼3). c Concentrations of the leached elements in the media, obtainedby ICP analysis. d Estimated half-life.

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MBC are dened based on the concentration of an antimicro-bial agent that inhibits the growth (for bacteriostatic agents) orkills all bacteria cells (for bactericidal agents).81 According to thestandard procedure, the bacteria cells (S. aureus and E. coli)were cultivated in agar LB broth medium at 37 �C, for 24 hours(108 CFU mL�1). A series of diluted samples of VCM@Fe3O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3 suspensions witha geometric sequence of concentration (2, 4, 8, 16, 32, 64, 128,and 526 mg mL�1) have been experimented in 2.0 mL of Muel-ler–Hinton broth (M–H broth) liquid medium followed by freshbacterial suspension with a turbidity of 0.5. Disk countermethod was used for evaluation of the growth inhibition orbactericidal effects. Table 4 summarizes the obtained resultsfrom MIC/MBC experiments. As is observed, similar MIC/MBCvalues have been obtained for the prepared nanocargoes andthe individual VCM and OFX. The point is that only 10 wt% and12 wt% of the nanocargoes belong to the VCM and OFX, whilethe same values as the pure drugs are obtained. In the case of E.coli cell line, this is seen that both MIC and MBC have beenreduced for VCM@Fe3O4/HNT–C(WR)3 than the individualVCM, meaning that the carried VCM overcomes the bacterialresistance via incorporation into the tubes and conjugation tothe C(WR)3 peptide sequence.

3.3.4.4 Physiological stability experiments. Since aggregationof the particles may negatively affect the internalization anddrug release processes, it is of high importance to investigateaggregation states of the magnetic nanocargoes in a simulatedphysiological environment. For this purpose, a circulationsystem was prepared and VCM@Fe3O4/HNT–C(WR)3 nano-cargoes were experimented in human serum albumin (HSA,25%) solution.13 Accordingly, circulation of the particles (0.4 mgmL�1) was performed for 15, 30, and 60 minutes in HSA at37 �C, and ultimately aggregation states were investigated byDLS analysis. As shown in the obtained DLS spectra (Fig. S6–S8,in the ESI section†), the average particle size has increased fromca. 513 nm to 1355 nm during a 60 min circulation, meaningthat the efficiency of the prepared nanocargoes may decreaseover the time due to the particle aggregation. This point clearlyhighlights the importance of the magnetic direction of theparticles in the body's internal environment, which expeditesthe drug delivery process. It should be also noticed that elec-trostatic repulsion between the positive-charged guanidinegroups onto the surfaces may assist dispersion of the particlesand slow down the aggregation process.

To investigate structural stability and degradation state ofthe designed drug carrier in the simulated physiologicalconditions, nano-carrier particles were experimented at 37 �C.For this purpose, four identical samples of Fe3O4/HNTsdispersion (10 mgmL�1) in HSAmediumwere stirred for 12, 24,48, 72, and 120 hours. Aer completion of each time, theparticles were magnetically separated from the mixture and wellrinsed with deionized water and ethanol, dried, and weighted.Also, the supernatant was evaluated for tracing any metal ions(Si, Al, and Fe) released from degraded Fe3O4/HNTs. For thisaim, inductively coupled plasma (ICP) analysis was employed.To have statistical evaluation, the whole assessment wasrepeated for three times. As reported in Table 5, half-life of the

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Fe3O4/HNTs in the physiological conditions has been estimatedto be ca. 72 hours, which is an appropriate time in the phar-macodynamics studies.82

4. Conclusion

Incorporation of different types of medications in the deliverysystems has shown to be a great method for the enhancement oftherapeutic effects. In this report, a novel designed drugdelivery system constructed of the natural HNTs, Fe3O4 NPs,and a conjugated peptide chain including cysteine, arginine,and tryptophan (named as C(WR)3), has been presented andsuggested for targeted delivery of VCM and OFX antibiotics. Thedesigned CPP sequence containing arginine–tryptophan blocks(synthesized on the CTC solid resin) conjugated to the particles,has enhanced cell adhesion and subsequent internalizationinto the living cells, conrmed by confocal microscopy. Thedrug release studies have shown that above 90% of the encap-sulated drug in the tubes is released in the acidic conditions.This property corroborates major release of drugs in acidicenvironment of the cells, aer the internalization process. Thedrug content of the prepared VCM@Fe3O4/HNT–C(WR)3 andOFX@Fe3O4/HNT–C(WR)3 nanocargoes has been evaluated tobe 10 wt% and 12 wt%, respectively. As the main brilliant point,it has been observed that almost similar bacteriostatic effectshave been observed for the prepared nanocargoes (VCM@Fe3-O4/HNT–C(WR)3 and OFX@Fe3O4/HNT–C(WR)3) and the indi-vidual VCM and OFX, in the same dosage. The antimicrobialexperiments have revealed that the growth rate is highlyinhibited through subjection of the nanocargoes to the Gram-positive and -negative bacteria strains, during a short time. Incomparison with the neat VCM and OFX with the same dosageas the nanocargoes, the signicant enhancement of the anti-microbial effects has been corroborated. Since, the designednanocargoes are suggested for the clinical use in the real-life,the cytotoxic effects on 3T3 broblast as a eukaryotic strainhas been investigated, as well. The results demonstrated thatthe human normal cells are not affected by the subjectednanocargoes. Moreover, the half-life of the designed nano-cargoes has been estimated around 72 hours in the simulatedphysiological environment, which is an appropriate time forbiodegradation. However, biodistribution and aggregationstates of the particles in the body's internal environment shouldbe further investigated via animal testing, because they maysignicantly affect the cellular uptake and drug releaseprocesses. Also, the release strategy could be controlled throughcoating the designed nanocargoes with different types ofmaterials like polymers. For example, to have a delayed releasein drug delivery to intestine, some polymeric structuresresponsive to alkaline conditions can be used for the coating ofthe whole cargo. Overall, via performing these studies on thepresented samples, we can get closer to the real-life utility inantimicrobial treatment.

Conflicts of interest

The authors declare no conict of interest.

© 2021 The Author(s). Published by the Royal Society of Chemistry

Acknowledgements

The authors gratefully acknowledge the partial support from theResearch Council of the Baqiyatallah University of MedicalSciences.

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