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International Journal of Biological Macromolecules 72 (2015) 740–750

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

evelopment of biopolymer nanocomposite for silver nanoparticlesnd Ciprofloxacin controlled release

erman A. Islana, Arup Mukherjeeb, Guillermo R. Castroa,∗

Nanobiomaterials Laboratory, Institute of Applied Biotechnology CINDEFI (UNLP-CONICET, CCT La Plata), Dept of Chemistry, School of Sciences,niversidad Nacional de La Plata, Calle 47y 115, La Plata 1900, ArgentinaDepartment of Chemical Technology, University of Calcutta, 92 A.P.C. Road, Kolkata 700 009, India

r t i c l e i n f o

rticle history:eceived 4 January 2014eceived in revised form 12 August 2014ccepted 7 September 2014vailable online 23 September 2014

eywords:uar gum alkyl amineilver nanoparticlesiprofloxacinlginate

a b s t r a c t

Screening of biopolymeric gel beads containing Silver NanoParticles (Ag-NPs) stabilized in Guar GumAlkyl Amine (GGAA) and Ciprofloxacin (Cip) was carried out in order to obtain a novel nanocompositewith controlled release profile of both antimicrobians. The selected matrix composed of Alginate/HighMethoxyl Pectin (HMP)/GGAA (4:4:1) was able to co-incorporate Ag-NPs and Cip with encapsulationefficiency higher than 70%. SEM images revealed good cohesivity and compatibility between the biopoly-mers and the cargos. Beads provided protection against Ag-NPs degradation at acidic pHs and HMP wouldplayed a key role providing hydrophobic regions. While Cip release profile showed a pH independent dif-fusional process, Ag-NPs release was restricted to matrix erodability. Calcium quelating agents and/oralginate degrading enzymes could modulate the release profile. The bactericidal activity of beads wastested in liquid medium, showing cooperative effects between the antimicrobials against Pseudomonas

ectinntimicrobial matrices

aeruginosa, Escherichia coli, Bacillus cereus and Staphylococcus aureus. TEM images confirmed interactionof Ag-NPs with bacterial surfaces followed by membrane damage. Results suggested the nanocompositematrix as a promising system for oral treatment of intestinal infectious diseases caused by multidrugresistant and unknown microorganisms, since both Cip and Ag-NPs would be able to reach intestine inthe active form.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Intestinal infections become sometimes a difficult pathology toreat because of different bacterial survival strategies such as mul-idrug resistant and/or biofilms formation [1]. For example in theystic fibrosis disease, the bacteria colonize the intestine makingiofilms by mucoid polymers protecting them against antibiotics.oreover, the nutrient absorption of the patients is strongly com-

romised and followed by a slow death if is not properly treated.lso, the treatment of these pathologies generally requires the usef several doses of high antibiotic concentrations, which are asso-iated to undesirable side effects including drug accumulation inon-targeted organs, hypersensitivity, immune-suppression, andllergic reactions [2].

In the recent years, silver nanoparticles (Ag-NPs) have emergeds a powerful tool to treat microbial infections including multi-esistant pathogens. Ag-NPs inactivate a panel of drug-resistant and

∗ Corresponding author. Tel.: +54 221 4833794x132; fax: +54 221 4833794x103.E-mail address: grcastro@gmail.com (G.R. Castro).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.09.020141-8130/© 2014 Elsevier B.V. All rights reserved.

drug-susceptible bacteria (Gram positive and Gram negative), exerttheir antibacterial activity through a bactericidal rather than bacte-riostatic mechanism, and inhibiting the bacterial growth rate fromthe time of first contact with the bacteria [3]. Hence, Ag-NPs havebeen applied in diverse medical fields such as topical ointmentsand creams containing silver to prevent infection of burns and openwounds or in the development of medical devices and implants pre-pared with silver-impregnated polymers [3]. In addition to theirdirect bactericidal activity, Ag-NPs are also known to be capableof interact and disrupt biofilm formation [4,5]. However, high con-centrations of Ag-NPs were reported as toxic in living organismswhich are remarking the need of a carefully adjusted dose as keyinformation on potential adverse side effects toward establishing atreatment on humans [6]. Besides, human cells were found to have ahigh resistance to the toxic effects of silver nanoparticles comparedto other organisms. Even, silver nanoparticles present a lower tox-icity compared with silver ions and nanoparticle encapsulation also

lowered its toxicity [7]. In particular, oral delivery of Ag-NPs wasrecently reported and found that were not observable clinically rel-evant toxicity markers in an exposed time of 14 days in humans [8].However, chemically synthesized Ag-NPs are requiring protection

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y coating to prevent phenomena like aggregation, oxidation andestabilization, parameters that determine their toxicity [9,10].

Metal nanoparticles-embedded hydrogels, generally describeds polymer matrix-nanocomposites, have been postulated as anmergent new generation of antimicrobials [3,11]. Three dimen-ional, matrices based on various natural hydrophilic polymersike starch, gelatin, cellulose, chitosan, sodium alginate, and theirerivatives such as carboxymethyl cellulose or alkyl amine guarum, are known to extend effective stabilization of the nanopar-icles for a range of applications in catalysis, biomedicine, opticsnd pharmaceuticals [12,13]. Additionally, in recent work the rel-vance of Ag-NPs co-administration with an antibiotic to produce

synergic antibacterial activity was reported [14].Among antibiotics, Ciprofloxacin (Cip) is the fifth largest generic

ntibiotic produced in the world keeping 24% of the therapeuticrescription market (about US$ 2.3 million dollars/year). Cip belongo the fluoroquinolone family, a wide class of antibiotics withroad antibacterial spectrum currently used in many infections15]. However, ciprofloxacin administration is currently associatedith many undesirable and diverse toxic side effects that involveany organs and behavior [16]. Many of the reported problems

ould be associated to a poor Cip solubility and biodisponibilitynder physiological media [17]. In addition, Cip stacking phe-omena can surpass drug solubility and becoming highly toxic toumans [18].

Guar Gum (GG) is a biopolymer synthesized by the seed legumeyamopsis tetragonolobus commonly found in the Indian subcon-inent. GG is a galactomannan composed by a linear chain of �,4-D-mannopyranoses in where D-galactopyranoses residues are-1,6 linked at every second mannosyl residue forming short side-ranches (2:1 ratio), and average molecular weight of 220 kDa. Guarum has approximately 8-times the water-thickening potency oforn starch, and can be used in multi-phase formulations as anmulsifier preventing oil droplets from coalescing, and/or as a sta-ilizer in order to prevent molecular aggregation and particlesrom settling, and it can form gels in presence of calcium ions19]. However, GG has a low solubility in aqueous solutions (about.0 mg/ml). A common strategy to increase the solubility of GG is by

ntroducing polar groups in the main structure of polymer. Alkyl-tion of GG with ethylamine increases polymer solubility and itas been proposed as a matrix for drug controlled release and as atabilizer of silver nanoparticles [12,20].

Alginates (ALG) are linear anionic polysaccharides widely usedn food and pharmaceutical industries. ALG are made of �-

anuronic acid (M units) and �-guluronic acid (G units) linkedy 1-4 bounds. Their properties of being non-toxic, showing no

mmunologic response, high biocompatibility and gelation in pres-nce of divalent ions by making “egg box” structure made themery suitable gels for many therapeutic applications [21]. ALGels can be enzymatically hydrolyzed by alginate lyase (AL). AL isble to hydrolyze alginate polysaccharides (tetramers or higher)n an endo-type manner at the �-1,4-glycosidic linkage via �-limination reaction to produce mono-, di-, and trisaccharideroducts [22]. Its importance in the release of cargo molecules withigh molecular weight or a size higher than the matrix porosityecomes an interesting tool to overcome the diffusional limita-ions of encapsulated silver nanoparticles from a biopolymeric geltructure based on alginate.

Pectins are water-soluble polysaccharides present in the plantell wall. The use of pectins as matrix in oral drug delivery devicesere proposed [23]. Pectins are composed of linear polysaccha-

ides of partially metoxylated poly �-(1,4)-D-galacturonic acids.

he esterification degree (DE) of pectins has strong influence oniopolymer properties. Pectins are grouped into Low MethoxylatedLM) with DE below of 40%, Medium Methoxylated (MM) with DEange between 40 and 60%, and High Methoxylated pectins (HM)

ical Macromolecules 72 (2015) 740–750 741

with DE higher than 60%. LM and MM pectins can be gelled bymultivalent cations, meanwhile HM only by acid pH [24].

Matrices composed of alginate, pectin and their coacervatesmodulating the Ciprofloxacin release profile and providing pro-tection of labile molecules against harsh environments werepreviously reported by our laboratory [25,26].

The aim of the present work is the co-encapsulation ofsilver nanoparticles stabilized into Alkyl Amine Guar Gum (Ag-NPs(AAGG)) and Ciprofloxacin into biopolymeric gel beads toprovide a dual-component drug delivery system for the treatmentof intestinal infections and prevention of chronic colonization ofpathogens. The release profile of both antimicrobials was analyzedat different pHs and the diffusional mechanism was established.Also, stability of the Ag-NPs incorporated into the matrix wasevaluated against simulated physiological environments (gastricand intestinal) to validate their potential application in oral deliv-ery. Characterization of the matrices was carried out by statisticalanalysis of scanning electron microscopy (SEM) pictures. Finally,microbiocidal properties against Gram positive and Gram negativestrains as model recurrent opportunistic pathogens were testedand correlated by TEM images.

2. Experimental

2.1. Materials

Ciprofloxacin (Cip, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-ox-o-7-(1-piperazinyl)-3-quinoline carboxylic acid) was purchased fromSigma-Aldrich (Buenos Aires, Argentina). Low viscosity sodiumAlginate (ALG) (average M� 1.0 × 105 Da) was obtained fromBiochem SA (Buenos Aires, Argentina). Low Methoxylated (LMP,ED 33%, average M� 1.56 × 105 Da) and High Methoxylated (HMP,ED 74%, average M� 1.60 × 105 Da) pectins were kindly providedby C.P. Kelco (Buenos Aires, Argentina). All other reagents usedwere of analytical grade purchased from Sigma (St. Louis, MO)or Merck (Darmstadt, Germany). Guar Gum (GG, average M�2.20 ± 0.20 × 105 Da) was a gift from Hindustan Gums & Chemi-cals (India). The Guar Gum (GG) alkyl derivatization procedure wasrepeated following the protocol described by Das et al. [20].

2.2. Silver nanoparticles synthesis

Silver nanoparticles (Ag-NPs) stabilized in Alkyl Amine guargum (AAGG), named as Ag-NPs(AAGG), were developed followingin situ ionic silver reduction in alkaline dextrose [12]. Briefly, 10 mlof different concentrations of AgNO3 solution (2–40 mM) wereadded to 10 ml of AAGG solution (0.5%, w/v), stirred vigorously andtransferred to a pre-heated water bath at 60 ◦C. D-dextrose (2.0 mL,25 mM) and sodium carbonate (2.0 mL, 4 mM) were mixed andadded immediately into this solution dropwise. The final solutionwas heated at 60 ◦C for 10 min more. The resultant conjugates wereseparated by added 15.0 mL acetone under stirring. The productre-dissolved in HPLC grade water and precipitated with 50% (v/v)ethanol in water. The obtained powdered silver nanocompositeswere preserved in desiccators. The diameter of silver nanoparti-cles was 140 ± 10 nm determined by dynamic light scattering (Zetasizer, Malvern, UK). The Ag-NPs plasmon response was observed at405 nm [12].

2.3. Determination of Ag-NPs(AAGG) interactions withCiprofloxacin

Interaction between both antimicrobials at different pHs wasestablished by co-precipitation of both agents [25]. Briefly, ten mil-ligrams of each powder silver nanocomposites (from 0 to 40 mM ofsilver) was dissolved separately in 1.0 ml of 50 mM buffer at pH 4.0

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Acetate/Acetic acid), 7.0 (phosphate) or 9.0 (borate), respectively.n aliquot of each solution (410 �l) was mixed with 90 �l of Cipolution (100 �g/ml) and stirred at room temperature for 1 h. Later,.0 ml of cold ethanol (96.0%) was added to precipitate the Cip/Ag-Ps(AAGG) complex. The resulting suspension was centrifuged at0,000 × g. for 10 min. The soluble Cip remnant in the supernatantas determined spectrophotometrically at 277 nm. Additionally,

he spectra of Cip solutions was scanned at 0.5 nm/s from 200 to00 nm to determine the maximum wavelengths under each exper-

mental condition using an UV-Vis spectrophotometer (BeckmanU640).

.4. Stability in time of free Ag-NPS(AAGG) incubated at differenthysiological pHs

Ten milligrams of AAGG nanocomposite powder (40 mM of sil-er) were dissolved in 1.0 ml solution of 25 mM buffers at pH.2 (KCl7HCl) and 7.4 (phosphate), to simulate the gastric andhe intestinal pHs, respectively. All solutions having NPs wererotected from light along the procedures with aluminum foil. Sta-ility of Ag-NPs(AAGG) was traced by measuring the absorbance at05 nm at different time point intervals. In addition, images werebtained by Transmission Electron Microscopy (TEM).

.5. Synthesis of biopolymeric beads containing Ciprofloxacin andg-NPs(AAGG)

Biopolymeric solutions were prepared in 25 mM acetate bufferpH = 4.0) at final concentrations of 2.0% (w/v), dissolving low-iscosity sodium alginate (ALG), Low Methoxylated Pectin (LMP)r blends of both biopolymers (1:1): ALG-LMP or ALG-HMP,espectively. Later on the homogeneous biopolymer solution,iprofloxacin and AAGG nanocomposite powder (containing0 mM of Ag-NPs) were added to obtain final concentrations of00 �g/ml and 0.5% (w/v), respectively. Beads were prepared by

et spray technique, dropping each biopolymer solution into ance cooled 500 mM calcium chloride solution dissolved into 1,2-ropylene glycol-water (1:1, v/v) [26]. Beads were washed after0 min with ultrapure water, dried at room temperature for 10 minnd stored at 5 ◦C for further experiments.

.6. Encapsulation of Cip and Ag-NPs(AAGG) into biopolymericatrixes

Ciprofloxacin in 1,2-propylene glycol-water (1:1) solutionas spectrophotometrically quantified at 280 nm (maximum

bsorption peak), and Ag-NPs(AAGG) were detected by thelasmon absorbance at 405 nm. Encapsulated ciprofloxacin and Ag-Ps(AAGG) were determined by the difference between total and

he remaining concentration in the supernatant after beads forma-ion, and calculated as follows:

ncaps (%) = (Q0 − (Cr × V)) × 100Q0

(1)

In where, Q0 = initial amount of the compound (�g),r = concentration of the compound in the filtrated solution�g/ml), V = volume of filtrated solution (ml).

Additionally, the cargo of wet beads was evaluated by weighting00 mg incubated in 2.0 ml of 100 mM phosphate buffer (pH = 7.40)or 1 h (until total bead dissolution). The total Cip and/or Ag-NPs

ontent in the supernatant was assayed spectrophotometricallyfter centrifugation (3000 × g, 10 min).

Size distribution of wet and dried (after freeze dryer process)eads was determined by optical microscopy.

ical Macromolecules 72 (2015) 740–750

2.7. Morphology and structural analysis of the differentbiopolymeric matrices by Scanning Electron Microscopy (SEM)

Beads were freeze-dried during 72 h. Furthermore, sampleswere prepared by sputtering the surface with gold using a BalzersSCD 030 metalizer obtaining layer thickness between 15 and 20 nm.Beads surfaces and morphologies were observed using Philips SEM505 model (Rochester, USA), and processed by an image digitalizerprogram (Soft Imaging System ADDA II (SIS)).

SEM images were analyzed by ImageJ software (NIH, USA). Theroughness of the surface was reflected by the standard variationof the gray values of all the pixels on the image [26]. The less thestandard variation value is, the smoother the surface is. Histogramswere performed by duplicate of SEM images at 710× magnification.

2.8. Stability of Ag-NPs(AAGG) into biopolymeric matrices afterincubation at different pHs: protective analysis

Beads containing Ag-NPs(AAGG) were incubated in buffers ofpHs 1.2 and 7.4 at 37 ◦C and 100 rpm for 4 h. Later, beads weredissolved in 100 mM phosphate buffer and remnant Ag-NPs weredetermined spectrophotometrically at 405 nm in the supernatant.Time zero controls were performed at each pH.

2.9. Release from biopolymeric beads at simulatedgastrointestinal conditions

2.9.1. Release of Ag-NPs(AAGG)50.0 mg of wet beads were weighted and suspended in 1.5 ml

buffer solution at gastric and intestinal pHs: 1.2 (25 mM KCl-HCl)and at 7.4 (10 mM phosphate), respectively, and incubated at 37 ◦Cagitated at 100 rpm. Samples were taken at different time point andthe cumulative Ag-NPs(AAGG) release was determined by measur-ing the absorbance at 405 nm.

Furthermore, to elucidate the diffusion mechanism of Ag-NPs(AAGG) from the beads, kinetic release was evaluatedincubating beads of the Alg-HMP-AAGG (4:4:1) formulation at pH7.4 in 10 mM Tris/HCl buffer (which is not having quelating activityover the calcium-alginate crosslinking) in presence of 20 Units/mlof Alginate Lyase and incubated at 37 ◦C and 100 rpm. Controlexperiments were performed without the enzyme.

2.9.2. Release of CipThe same experimental conditions described previously were

carried out for Ciprofloxacin release from beads. Samples weretaken at different point times and Cip was measured at the max-imum absorbance wavelength in each buffer (� = 270–277 nm). Inorder to keep a constant vial volume 1.0 ml of fresh media wasrefilled at each sample point. Cumulative release of Cip was calcu-lated as mentioned before.

2.10. Antibacterial study

2.10.1. Ciprofloxacin antimicrobial activity in presence ofAg-NPs(AAGG) in agar plates

Inhibition zones against Pseudomonas aeruginosa were deter-mined by using modified disk diffusion method according toCLSI/NCCLS, replacing disks for sterile glass cylinders. The glasscylinders were further placed on the inoculated agar plate surface.

Solutions containing 10 �g/ml of Cip, 40 mM of silver NPs and themixture of both were tested. Each solution (25 �l) was placed insidethe cylinders on the plates and incubated at 37 ◦C for 24 h. Then,inhibition zones were determined.

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.10.2. Inhibition growth at liquid media by antimicrobialseleased from beads

Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC5922, Bacillus cereus ATCC 10876 and Staphylococcus aureus ATCC538 were cultured in nutrient broth medium and incubated at7 ◦C and 100 rpm for 12 h. An aliquot (1.0 ml) of each batch wasdded to 50 ml of fresh nutrient broth medium and incubated at7 ◦C until exponential growth phase was reached (3–4 h). Later,.5 ml of each bacterial solution was added simultaneously with0 mg of wet beads to a 1 ml cuvette (previously sterilized with UV-adiation for 40 min). Finally, cuvettes were incubated at 37 ◦C andacterial inhibition growth was traced by optical density at 600 nm.ontrols of bacterial growth without beads were performed.

.11. TEM studies

For TEM grid preparation, the bacterial solution was ten timesiluted in physiological solution and then mixed at room temper-ture with a volume of a Ag-NP(AAGG) (40 mM) solution. Afterncubation, an aliquot of the mixture was removed and applied to

ig. 1. (a) Effect of pH on ciprofloxacin binding to AAGG. (b) UV-vis scanning spectra of solates of Cip, Ag-NPs and both against Pseudomonas aeruginosa.

ical Macromolecules 72 (2015) 740–750 743

a collodion-coated Cu grid (400-mesh). Liquid excess was drainedwith filter paper. Images were obtained for bacteria exposed to Ag-NPs(AAGG) for 5 min and 12 h, in a Jeol-1200 EX II-TEM microscope(Jeol USA, Ma.).

2.12. Statistical analysis

All experiments were carried out at least in triplicates. The meanvalues were analyzed by the one way analysis of variance (ANOVA)with a significance level of 5.0% (p < 0.05) followed by Fisher’s leastsignificant difference test (p < 0.05).

3. Result and discussion

3.1. Determination of Ag-NPs(AAGG) interactions with Cip

In order to establish a comprehensive analysis of the experi-mental conditions for the nanocomposite formulation, interactionsbetween the cargo components were under study. By co-precipitation method, an interaction between Cip and AAGG of

lutions containing Cip, Ag-NPs(AAGG) and both at pH 4.0. (c) Inhibition halo in agar

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44 G.A. Islan et al. / International Journal of

bout 50% was found. Also, the interaction was independent ofg-NPs content, even when NPs concentration was increased 20

imes (p ≤ 0.05) (Fig. 1S, Supporting Material). This result is indicat-ng the specific interaction of the Cip with the modified guar gum,

hich is the coated layer on Ag-NPs surface as previously reported12]. As the pH of the mixture was raised, the Cip interactions withhe AAGG were decreasing (Fig. 1a). The maximum interaction wasound at pH 4.0 because most of the Cip molecules are positiveharged (pKa1 = 6.1) with both ionic groups (carboxyl and amine)n the protonated form. Positively charged Cip is capable to interact

ith the AAGG mainly through hydrogen bonds and Van der Waalsnteractions as previously reported with other biopolymers [25].he alkyl amines are the most basic amines with a pKa value around.0 due to the nitrogen lone pair localized on the nitrogen withoutesonance. At pH 4.0 is expected that AAGG is positive charged,eason why the chances of electrostatic attraction with Cip weremprobable. At pH 7.0 Cip molecules changed to the zwitterionictate, in which their solubility is reduced and tend to stack amonghemselves [18], becoming less available for interaction with AAGG.n the other side, the ionization of the protonated amine groups

n AAGG chains is raised up to pH 9.0 in where the polymer haso charge and precipitates, decreasing interaction with Cip. Taking

nto account these considerations, the nanocomposite biopolymeratrix will be synthesized at pH 4.0.

ig. 2. (a) Kinetic stability of silver nanoparticles measured at 405 nm after incubation att 0, 2 and 4 h (from left to right) under acidic solution (pH < 2.0) at 37 ◦C.

ical Macromolecules 72 (2015) 740–750

To corroborate the observed results, the solutions at pH 4.0 werespectrophotometrically scanned at UV–vis range. The maximumwavelength of ciprofloxacin was shifted from 277 to 275 nm inpresence of Ag-NPs(AAGG), probably due to interaction betweenCip and the AAGG biopolymer (Fig. 1b). However, no shift on Ag-NPplasmon maximum wavelength, 405 nm, was detected in the pres-ence of Cip. This result is confirming the main interaction betweenCip and AAGG polymer. Besides, the AAGG coating layer on Ag-NPssurface is capable to adsorb Cip molecules and retard their diffusion.This fact was found because of 25% reduction in the inhibition halo(about 5 mm) against Pseudomonas aeruginosa in agar plates whenCip plus Ag-NPs coated with AAGG were co-inoculated (Fig. 1c).Meanwhile, “free” Cip or Ag-NPs alone were showing a regular bac-tericidal activity against the tested pathogen. This result, remarksthe possibility of co-immobilization of both antimicrobials as theycan act as independent agents, but also suggest that the antimicro-bial capacity should be tested in liquid medium to avoid diffusionalbarriers.

3.2. Ag-NPs(AAGG) stability at different physiological pHs

In general, most of the Ag-NPs synthesis methods were focusedon the stability of the nanoparticle preparations, control the crys-tal growth and prevention of particle aggregation. Briefly, the

pHs: 1.2 (�) and 7.4 (�). (b) TEM images of silver NPs (AAGG coated) degradation

G.A. Islan et al. / International Journal of Biological Macromolecules 72 (2015) 740–750 745

te-LM

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Fig. 3. SEM images analysis of matrices: (a) Alginate, (b) LM Pectin, (c) Algina

ethods of long-live silver nanoparticle synthesis in aqueous solu-ion as well as silver powders, were based on the development ofoating process on Ag-NP surface after being synthesized to pre-ent its premature oxidation. In the present work, AAGG coatingf Ag-NPs is providing a stabilizing method against air oxidation12]. However, Ag-NPs(AAGG) were unstable when were exposedo aqueous environment under acidic pHs (Fig. 2a). No absorptionf the plasmon (405 nm) was observed after the beads incubatednder simulated gastric environment (pH 1.2 at 37 ◦C) for 4 h, sug-esting total degradation of the Ag-NPs(AAGG). On the contrary,he absorbance of Ag-NPs(AAGG) was unchanged when they werencubated at 7.4 and 37 ◦C for the same period of time. Accordingo these results it can be concluded that Ag-NPs are not capable toe used for oral delivery in the present state, as disintegration ofhe particle occurs in the stomach environment prior to reach thenfected intestine. In addition to disintegration of the particles byxidation, a pH decrease causes appreciable particle aggregation.he reduction in OH− ions concentration reduces the electrostaticepulsions leading to precipitation and sedimentations of the Ag-

Ps [27].

TEM images are allowing to trace the evolution of Ag-Ps(AAGG) after incubation at liquid medium at pH below 2.0

Fig. 2b). In a first step, robust Ag-NPs were observed as solid black

Pectin, (d) Alginate-HM Pectin bead’s surface. Insets shown ImageJ analysis.

dots and the AAGG coating was clearly visible around them, form-ing a core-shell structure. The formed AAGG network enhancesthe stability of the Ag-NPs through electrostatic and steric effectsdue to the presence of hydroxyl groups from the polymeric chains[13]. However, when AAGG began to dissolve in liquid medium, thedegradation of the whole particle surface was observed. The exposi-tion of Ag-NPs to H+ was mainly the responsible of Ag-NP oxidation,observed by a reduction of Ag-NPs size concomitantly with thedecrease of the plasmon signal at 405 nm. These results lead to con-clude that another protective layer additionally to AAGG should beadd to protect the Ag-NP surface, in order to extend the NP life dur-ing long incubation times. Hence, a Nanoparticle-in-MicroparticleDelivery System (NiMDS) could be a feasible alternative as itwas previously proposed [28]. In the present work, the designedNiMDS should contain the Ag-NPs(AAGG) and Cip encapsulatedinto biopolymer beads for enhanced pH stability and to obtain apredictable controlled release kinetic of both antimicrobials.

3.3. Co-encapsulation of Cip and Ag-NPs(AAGG) into

biopolymeric matrixes

Different biopolymer nanocomposite beads were preparedby dropping the biopolymeric solutions containing Cip and

746 G.A. Islan et al. / International Journal of Biological Macromolecules 72 (2015) 740–750

Fig. 4. Alg-HMP-GGAA beads (a) SEM

Table 1Encapsulation of ciprofloxacin and silver nanoparticles in different gel matrices.

Matrices Encapsulation (%)

Components Composition Ciprofloxacin Silver NPs

Alginate-AAGG 4:1 65.6 ± 3.6 100 ± 0.0LM Pectin-AAGG 4:1 85.4 ± 2.9 100 ± 0.0

AgosNLs

biopolymeric matrices by SEM

Alg-LM Pectin AAGG 2:2:1 70.7 ± 4.3 100 ± 0.0Alg-HM Pectin-AAGG 2:2:1 73.8 ± 2.8 100 ± 0.0

g-NPs(AAGG) on calcium chloride dissolved on 1,2-propylenelycol-water to get high encapsulation of the antibiotic, as previ-usly reported by our group [26]. The encapsulation efficiency washown in Table 1. Alginate matrix containing 0.5% AAGG with Ag-

Ps showed Cip encapsulation percentage around 65%, whereasM Pectin formulation encapsulated 85%. Both biopolymers wereelected for their crosslinking properties in presence of calcium

image and (b) size distribution.

ions. While alginate provides a very hydrophilic structure, thepectin shows methoxylated regions with hydrophobic character,in where the Cip molecules can be located [24]. For this reason,the Cip encapsulation is higher in the case of pectin comparedto the alginate ones. When blends of alginate-pectin (Low andHigh methoxylated) were designed, an intermediate Cip encapsu-lation of around 70% was obtained. Meanwhile, the Ag-NPs(AAGG)were totally encapsulated in all tested formulations. The finalAg-NPs concentration reached 4.0 mM per microbead in our formu-lations which is in the 0.3–5.0 mM therapeutic range as previouslyreported for Ag-NPs encapsulated on alginate microbeads [29].

3.4. Morphology and structural analysis of the different

Analysis of SEM images performed by the ImageJ softwarerevealed surface differences and elucidated optimal patterns of

G.A. Islan et al. / International Journal of Biological Macromolecules 72 (2015) 740–750 747

Table 2Remnant and release of silver nanoparticles from the different matrices under pH1.2 and 7.4 at 37 ◦C and after 4 h incubation.

Matrix based pH

1.2 7.4

Remnant Released Remnant Released

Free ND – 93.0 ± 1.2 –ALG-AAGG ND ND 55.9 ± 5.0 44.1 ± 5.0LMP-AAGG ND ND 96.3 ± 5.3 3.7 ± 5.3ALG-LMP-AAGG 25.2 ± 0.5 ND 45.3 ± 3.3 54.7 ± 3.3

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ALG-HMP-AAGG 35.9 ± 1.3 ND 48.0 ± 3.3 49.8 ± 5.9

D: not detectable.

he biopolymeric nanocomposites (Fig. 3). Alginate based matrixhowed a spheroid shape, due to a fast biopolymer gelation processn presence of the high calcium concentration (Fig. 2S, Supporting

aterial). Surface analysis revealed a rough surface associated tohe high standard deviation of 32, but it is showing good compatibil-ty after Ag-NPs(AAGG) incorporation (Fig. 3a). On the other hand,rosslinking process for LM pectin based matrix was less strongnd requires more time to acquire the final shape, but the resultingicrobeads are showing high spherical morphology. However, theicrobeads displayed some cracks on their surface, possibly due

o the aggressive dehydration after freeze drying procedure, andndicative of a more labile structure (Fig. 3b). Similar to alginateased matrix surface, the ALG-LMP blend showed a rough surfaceith an standard deviation of 37, besides the bead morphology was

mproved and tend to be more spherical (Fig. 3Sc and Fig. 2S, Sup-orting Material). Besides, the ALG-HM Pectin blend showed theest results displaying more regular morphology, and suggesting aood cohesivity between the biopolymers during the ionic gelation.lso, the roughness parameter was low showing a standard devi-tion of around 10 and the histogram of the grey scale displaying

narrow distribution (Fig. 3d). A good compatibility between theLG-HM Pectin biopolymers was previously reported [25,26]. Theg-NPs embedded into the microbead structure seem to be quiteell distributed. The presence of polar–OH groups of the biopoly-ers are playing a key role in coordinating silver ions and strongly

tabilize Ag-NPs, thus preventing aggregation and agglomeration13]. The developed Ag-NPs nanocomposite displayed a uniformistribution of the components and no agglomerates or irregularlyhapes were observed as previously reported in other formulations30].

.5. Protective effect and release of Ag-NPs(AAGG) fromiopolymeric matrices

Ag-NPs(AAGG) were proposed as a broad spectrum antimicro-ial for oral delivery treatment of intestinal infections. However,he degradation of free silver nanoparticles under the extremecid stomach environment in humans (pH ≈ 1.2) represents a seri-us limitation for oral delivery (Fig. 2). In order to evaluate therotective effect of the NiMDS nanocomposite beads, the stabilitynd release of Ag-NPs(AAGG) from different biopolymeric formu-ations were analyzed at pH 1.2 and 7.4 (at 37 ◦C) (Table 2). Nolasmon was detected after exposing Ag-NPs(AAGG) encapsulated

nto Alginate and LM Pectin matrices at pH 1.2 for 4 h, likewisesing free Ag-NPs(AAGG) which are indicating total NPs degra-ation. Besides, 25% and a 36% of the initial Ag-NPs encapsulatedemained intact in the Alg-LM Pectin and Alg-HM Pectin matrices,espectively, evidencing some protective effect against acidity. The

ncreased protection provided by the incorporation of HM Pectinnto the formulation was expected considering the high quanti-ies of hydrophobic regions made by methoxylated regions of theiopolymer, which probably retard H+ diffusion into the matrix

Fig. 5. Silver nanoparticles release from Alginate-High Metoxylated Pectinmicrobeads in: (a) buffers at pH = 1.2 (�), 5.0 (©) and 7.4 (�); and (b) Tris-HCl buffer(10 mM, pH = 7.4) in the absence (�) or presence (�) of Alginate Lyase (20 Units/ml).

[31]. At simulated intestinal pH (7.4), the formulations were moresusceptible to erosion by phosphate ions and matrices releasedaround 50% of nanoparticles, except for LMP-AAGG which releasedless than 5%. Based on pH stability, release profiles and SEM imageanalysis results the Alg-HM pectin matrix was selected for furtherassays.

3.6. Size distribution of ALG-HMP-AAGG nanocomposite beads

A desirable spherical shape beads of the Alg-HM Pectin was pro-duced during synthesis (Fig. 4a), revealing the good compatibilitybetween the components and the cargos. A narrow size distributionaround 2.1 mm for wet beads was observed, which directly dependon the size of the drop during crosslinking. After freeze dryer pro-cess, their size was reduced to only 800 �m, about one third of theoriginal diameter (Fig. 4b), since the unbound water was excludefrom the matrix.

3.7. Kinetic release of Ag-NPs(AAGG) from beads

The release profile of Ag-NPs(AAGG) from the Alg-HMP matrixwas evaluated at different pHs (Fig. 5a). Silver NPs released at pH1.2 were no detectable, due to the fast oxidation of the particle.At pH 5.0, practically no release from the gel was observed in the

first 3 h. After that, a linear release profile was observed until reachthe 100% at 12 h of incubation. A similar behavior was observed atpH = 7.4, but the Ag-NPs release began at 30 min of incubation. Inthis sense, the Ag-NPs release from the Alg-HM Pectin matrix was

7 Biological Macromolecules 72 (2015) 740–750

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48 G.A. Islan et al. / International Journal of

ore modulated by an erosion process of the matrix than only aiffusion one. As the bead was destabilized by the presence of cal-ium quelating agents (like phosphate ions), the matrix increasedts porosity and the output of the nanoparticles was favored.

As a mechanistic study, Ag-NPs(AAGG) release was evaluatedt pH 7.4 in presence of a non-quelating buffer (Tris-HCl). As aesult, no release was produced because of the porosity of theatrix did not allow the diffusion of the Ag-NPs (Fig. 5b). How-

ver, addition of a matrix-degrading enzyme, Alginate Lyase, in theedium facilitated the diffusion of the Ag-NPs from the beads to

he media. The Ag-NPs were fully released from the beads in theresence of 20 units/ml of enzyme in 4 h. Based on these facts, theg-NPs release profile can be modulated not only by the formu-

ation content, presence of calcium quelating agents, but also byhe presence of the enzyme at different concentrations (data nothown), due to a progressive disruption of the alginate gel structurehich change the crosslinking degree of the network and the pore

ize [25,32].

.8. Release of Ciprofloxacin from nanocomposite beads

The Cip release from ALG-HM Pectin nanocomposite showed aimilar behavior at different pHs as seems to be independent ofhis parameter. During the first 30 min almost the 10% of Cip waseleased, reaching around 50% at 4 h incubation in all tested pHs

Fig. 6). The results at pH 1.2 were similar as previously reported25,26], but the release at pH 7.4 was slowed down. This factould be possibly because of the Ag-NPs(AAGG) are stabilizinghe gel matrix and additionally the AAGG polymer is specifically

time (h ours)

0 2 4 6 8

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ig. 7. Effect of beads on growth inhibition against Gram negative bacteria: (a) Pseudtaphylococcus aureus. Control (�), beads containing: AgNPs (�), Cip (©) and both antimi

Fig. 6. Kinetic release of ciprofloxacin from beads blends made of Alginate-HMPectin-AAGG (Ag-NPs) (2:2:1) at pHs: 1.2 (�), 5.0 (©), and 7.4 (�).

interacting with Cip molecules (Fig. 1). On the other side, analyz-ing the Cip release from the previously developed nanocomposites(Alg, LMP and Alg-LMP) was possible to elucidate that alginate ismore prone to erosion by phosphate ions, while pectin gel is lesssusceptible to disruption at slight alkaline pHs (Fig. 3S, SupportingMaterial). The difference can be explained in terms of the egg boxjunction model between the biopolymeric chains and calcium ion,

since the pectin the network is less organized with more hydropho-bic regions and consequently more difficult to destabilize thanalginate [24].

time (h ours)

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omonas aeruginosa, (b) Escherichia coli, and Gram positive (c) Bacillus cereus, (d)crobials (�).

G.A. Islan et al. / International Journal of Biological Macromolecules 72 (2015) 740–750 749

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ig. 8. TEM images of Pseudomonas aeruginosa: untreated (a); treated with Ag-NPshe Ag-NPs with bacterial membrane.

.9. Antibacterial study against Gram positive and Gram negativetrains

The antimicrobial activities of Ag-NPs(AAGG) and Ciprofloxacineleased from the Alg-HM Pectin nanocomposite beads were testedgainst different microorganisms commonly associated with recur-ent pathologies. A strong decrease in bacterial growth assayedn liquid medium was observed for Cip, Ag-NPs(AAGG) and bothntimicrobians encapsulated in Alg-HM Pectin beads (Fig. 7).

Cip or Ag-NPs showed some bacteriostatic and bactericide activ-ty depending on the bacteria regardless of Gram characteristics.

eanwhile the combined release of Cip and Ag-NPs showed syn-rgisms between both antimicrobial activities on the four testedtrains.

From Gram-negative microorganisms like P. aeruginosa, the cellrowth was partially inhibited by both antimicrobials alone, buthe combination of Cip-AgNPs antimicrobial showed an enhancedctivity antimicrobial activity, which results in an O.D. decreaseelow the initial inoculum. This result can be produced first by bac-erial growth inhibition followed by lytic process (Fig. 7a). In thisase, we demonstrated that even larger Ag-NPs of 150 nm in size areapable to inhibit more than 90% of Pseudomonas aeruginosa growthnd prevent pathogenesis by avoiding the biofilm formation [33].

In the case of E. coli, a more pronounced inhibitory effect wasbserved with the same NiMDS (Fig. 7b). The presence of AgNPsn the media decreased the E. coli growth linearly (r2 = 0.98) along

h incubation. The inhibition profile of E. coli in presence of beadsontaining only Cip and beads with Cip-AgNPs was very similar,ndicating no additive effect between the antimicrobials, possiblyttributed to the faster release of Cip rather than Ag-NPs effect.

Gram positive B. cereus growth inhibition was 66% and 54% inresence of beads containing Cip and Ag-NPs, respectively after 8 hf culture. However, the simultaneous presence of Cip and Ag-NPsn the beads inhibited 78% the microbial growth of B. cereus underhe same experimental conditions, displaying a synergic effectFig. 7c). Similar results were observed on Staphylococcus aureusultures showing an inhibition of 74% in presence of Cip-AgNPsompared with the 58% found for each encapsulated antimicrobialslone (Fig. 7d).

These studies shows that silver nanoparticles and Ciprofloxacinave great promise as antimicrobial agents against common Gramositive and Gram negative human pathogen such as B. cereus,. coli, P. aeruginosa and S. aureus. The co-immobilization of bothgents at high encapsulation percentages and the controlled release

rofile from microbeads enhance their importance compared withrevious antimicrobial drug delivery systems reported [34,35].

Finally, inhibition mechanism of the Ag-NPs(AAGG) was elu-idated using Pseudomonas aeruginosa as model of opportunistic

G): for 10 min (b) and after 12 h (c). Squares are indicating the interaction zone of

pathogens (Fig. 8). TEM images are showing the bind ofAg-NPs(AAGG) onto bacterial membrane only after 10 min ofincubation (Fig. 8b). However, presence of AAGG generates an inter-ference with TEM images, and the bacterial morphology cannotbe well defined shape, but localization of Ag-NPs was clearly vis-ible. Binding of the NPs to bacteria was mainly on surface and nointernalization was observed, at least at the experimental time ofinteraction tested by the TEM images. Squares in Fig. 8b, displayedthe interaction zones of Ag-NPs with the microbial membrane. Itis generally recognized that Ag-NPs bind to the cell wall [36], thusdisturbing cell-wall permeability and cellular respiration by dissi-pating the chemiosmotic gradient. The Ag-NPs may also penetrateinside the cell causing damage by interacting with phosphorusand sulfur containing compounds such as DNA and protein, butin the case of the Ag-NPs(AAGG) was not detectable. Another pos-sible contribution to the bactericidal properties of Ag-NPs could berelated to the release of silver ions from NPs [37]. Finally, the Pseu-domonas aeruginosa membrane was damaged and bacterial lysiswas observed in presence of Ag nanoparticles after 12 h incubation(Fig. 8c).

4. Conclusions

Novel biopolymer nanocomposite microbeads were developedas a matrix for controlled release of silver nanoparticles andCiprofloxacin. The co-encapsulation of both antimicrobials is rel-evant in the field since multi-resistant opportunist pathogens arereported more often and novel strategies of delivering drugs basedon the use of green synthesis and non-toxic materials are required.The present study reports a facile preparation of a NiMDS composedof Alginate/HM Pectin made by ionic gelation process in presenceof calcium ion and 1,2-propylene glycol. The matrix was able to co-incorporate Ciprofloxacin and silver NPs (stabilized with a AAGGcoating) with high encapsulation yield, reaching therapeutic con-centration ranges. The good cohesivity and compatibility betweenthe biopolymers and the cargo molecules was corroborated by SEMimages and stability assays. The Ag-NPs seemed to be quite welldistributed and no aggregates or irregular bodies were observedwhich is relevant for the therapeutic purposes.

The proposed nanocomposite matrix is able to provide highprotection against silver NPs degradation at acidic pHs of humanstomach compared to previous reports [38]. The presence of HMPectin into the formulation is the main responsible to provide

hydrophobic regions that retard H+ diffusion inside the matrix. Inthis sense, the nanocomposite beads are potentially applicable ascarriers for the oral delivery of silver nanoparticles, preventing theiroxidation when are exposed to the gastric environment.

7 Biolog

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[36] S.C. Hayden, G. Zhao, K. Saha, R.L. Phillips, X. Li, O.R. Miranda, V.M. Rotello, M.A.

50 G.A. Islan et al. / International Journal of

In addition, the release mechanism of both antimicrobials estab-ishes an interesting dual profile. Ciprofloxacin is released by only

diffusional mechanism, meanwhile release of silver nanoparti-les is restricted to the matrix erosion. The mechanism is relevantince in the case of ciprofloxacin-resistant bacteria remaining in thentestinal tract will be killed by the Ag-NPs. In addition, the pres-nce of alginate degrading enzymes can regulate the rate of Ag-NPselease, as well as the presence of calcium quelating agents.

A synergic bactericidal activity of both antimicrobials releasedrom beads was found against pathogens commonly associatedo several pathologies such as Bacillus cereus, Escherichia coli,seudomonas aeruginosa and Staphylococcus aureus. TEM imagesonfirmed interaction of silver NPs on bacterial surface and induc-ion of membrane damage.

The results suggested the biopolymer nanocomposite as aromising system for the treatment of chronic infections caused byultiresistant and unknown microorganisms. Even though more

tudies in living organisms are necessary to carefully adjust theoses and determine the Ag-NPs cytotoxicity, the matrix propertiesake it potentially applicable for severe intestinal infections, since

ilver nanoparticles can reach intestine as an active antimicrobial.

cknowledgements

The present work was supported by grants from Fundaciónrgentina de Nanotecnología, Consejo Nacional de Investigacionesientíficas y Técnicas (CONICET; PIP 0498, Argentina), Universidadacional de La Plata of Argentina (11/X545) and Agencia Nacionale Promoción Científica y Técnica (PICT 2011-2116, Argentina). Theoctoral fellowship to GAI from the UNLP was gratefully acknowl-dged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.ijbiomac.014.09.020.

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