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Journal of Materials ScienceFull Set - Includes `Journal of MaterialsScience Letters' ISSN 0022-2461Volume 51Number 18 J Mater Sci (2016) 51:8593-8609DOI 10.1007/s10853-016-0119-x

Electrospinning and electrosprayingtechniques for designing novel antibacterialpoly(3-hydroxybutyrate)/zinc oxidenanofibrous composites

Heriberto Rodríguez-Tobías, GracielaMorales, Antonio Ledezma, JorgeRomero, Rubén Saldívar, ValerieLanglois, Estelle Renard, et al.

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Electrospinning and electrospraying techniques

for designing novel antibacterial poly

(3-hydroxybutyrate)/zinc oxide nanofibrous composites

Heriberto Rodrıguez-Tobıas1, Graciela Morales1,*, Antonio Ledezma1, Jorge Romero1,Ruben Saldıvar1, Valerie Langlois2, Estelle Renard2, and Daniel Grande2,*

1Centro de Investigación en Química Aplicada, Blvd. Enrique Reyna No. 140, Saltillo, Coahuila C.P. 25294, Mexico2 Institut de Chimie et des Matériaux Paris-Est, UMR 7182 CNRS-Université Paris-Est Créteil, 2, Rue Henri Dunant, 94320 Thiais,

France

Received: 4 March 2016

Accepted: 4 June 2016

Published online:

10 June 2016

� Springer Science+Business

Media New York 2016

ABSTRACT

This investigation concerns the design of poly(3-hydroxybutyrate) (PHB)-based

nanofibrous hybrid materials containing zinc oxide nanoparticles (nano-ZnO)

by means of two electro-hydrodynamic techniques, i.e., electrospinning of

polymer/nano-ZnO solutions and the combination of electrospinning of poly-

mer solutions with electrospraying of nano-ZnO dispersions. The analysis of the

physical properties associated with precursory solutions was performed in

order to understand the final morphology of the corresponding nanofibers. The

obtained PHB/nano-ZnO mats showed uniform fiber morphology with an

average porosity ca. 85 % with enhanced thermal stability compared to that of

pristine PHB. Differential scanning calorimetry was also used to determine the

influence of ZnO nanoparticles in the phase transitions of as-spun PHB nano-

fibers. Furthermore, the antibacterial performance against E. coli and S. aureus

proved to be dependent on the elaboration technique, thus permitting the

design of novel bacteriostatic or bactericidal PHB/nano-ZnO nanofibrous

composites.

Introduction

Tissue engineering is one of the main applications of

fibrous polymeric materials obtained by electro-hy-

drodynamic techniques, such as electrospinning and

electrospraying. Electrospun nanofibrous scaffolds

exhibit highly porous structures with fiber diameters

suitable to mimic the natural extracellular matrix,

thus promoting cell attachment and proliferation

[1–4]. Nonetheless, these morphological features

entail a huge inconvenient, namely, the adhesion of

pathogenic microorganisms [5–7]. Several strategies

have been developed in order to overcome this

problematic issue, being the most prominent the

incorporation of organic compounds [8], and more

Address correspondence to E-mail: graciela.morales@ciqa.edu.mx; grande@icmpe.cnrs.fr

DOI 10.1007/s10853-016-0119-x

J Mater Sci (2016) 51:8593–8609Author's personal copy

recently, metallic nanoparticles [9–12] with well-rec-

ognized antimicrobial properties. Electrospun poly-

mer composites containing these specific

nanoparticles can exhibit several advantages com-

pared to typical organic compound-loaded polymers,

such as higher thermal stability, enhanced mechani-

cal performance or biocompatibility, depending on

the chemical nature of nanoparticles [1, 2, 12–15].

Among the main nanoparticles investigated, zinc

oxide (ZnO) has recently attracted considerable

attention, due to its facile and cost-effective synthesis,

controlled morphology by adjustment of reaction

conditions, and strong antimicrobial activity against a

broad range of microorganisms [16–20]. Concerning

the polymer matrix, biocompatible and/or

biodegradable polymers have been the preferred

materials in order to prevent injuries or inflammation

resulting from removal or rejection of electrospun

scaffolds. In this regard, Augustine et al. [9, 21]

designed fibrous materials based on polycaprolac-

tone (PCL) and ZnO nanoparticles (ca. 60 nm in size),

which exhibited excellent wound healing perfor-

mance at low concentration (\2 wt%) and modest

antimicrobial activity against Escherichia coli and

Staphylococcus aureus only at relatively high concen-

tration ([5 wt%). Besides, the mechanical perfor-

mance of PCL-based mats was not favored by the

presence of nanoparticles. Furthermore, the authors

perceived some variations in fiber diameter with ZnO

content higher than 2 wt% which were attributed to

polymer solution viscosity; however, rheological

studies were not presented. It is noteworthy that such

PCL/ZnO fibers exhibited cytotoxicity against

fibroblasts for ZnO concentrations higher than 5 wt%,

thus limiting their application as wound dressings.

Similar results related to antibacterial activity and

tissue proliferation were obtained by Shalumon et al.

[22] for electrospun biocomposites derived from

solutions consisting of poly(vinyl alcohol) (PVA),

sodium alginate, and dispersed ZnO particles

(160 nm in size). Alternately, Wang et al. [23]

designed a hybrid mat based on chitosan which has

an inherent antibacterial activity with PVA and ZnO

nanoparticles. The formation of a chitosan–Zn com-

plex drastically diminished the proliferation of both

Candida albicans and E. coli.

Electrospraying in combination with electrospin-

ning displays two main advantages compared to

other deposition methodologies, such as immersion

of mats in nanoparticle-containing dispersion or

atomic layer deposition [24, 25]. Indeed, it consists of

a one-step process for obtaining inorganic particle-

coated polymeric fibers with the possibility of con-

trolling the size of particles/aggregates deposited

onto the polymeric fibers by variation of nanoparti-

cles dispersion parameters (concentration, solvent,

etc.), thus tuning the final properties of obtained

mats. However, the electrospinning/electrospraying

tandem technique has scarcely been explored as an

approach for designing biocompatible and/or

biodegradable mats with antibacterial properties.

Parallel investigations were carried out by Rodrı-

guez-Tobıas et al. [12] and Virovska et al. [26] in

which the influence of the fabrication technique, i.e.,

electrospinning or electrospinning/electrospraying,

on the antibacterial activity of polylactide/ZnO fibers

was investigated. The former authors reported a high

inhibition growth (up to 94 % after 24 h of incuba-

tion) for S. aureus, when the electrospinning/elec-

trospraying tandem approach was used for the

fabrication of PLA fibers with 1 wt% electrosprayed

ZnO nanoparticles. On the other hand, the latter

authors determined that after 8 h of incubation, the

growth inhibition of the same bacteria was only equal

to 30 % with a ZnO concentration of 45 wt%.

Poly(3-hydroxybutyrate) (PHB) is a semicrystalline

thermoplastic biopolyester and represents the simplest

member of the poly(3-hydroxyalkanoate) family.

These naturally occurring polyesters have attracted

much interest in biomedical applications, due to their

renewability, biodegradability, and higher biocom-

patibility compared to other biodegradable aliphatic

polyesters (e.g., PLA, PCL), as well as the non-cyto-

toxicity of their metabolic products [27–29]. The

application of PHB-based nanofibrous scaffolds as

antibacterial electrospun mats could be illustrated by

the incorporation of ZnO nanoparticles. To the best of

our knowledge, the design of fibrous composites

based on PHB nanofibers and ZnO nanoparticles with

antimicrobial properties has not been investigated so

far. In this context, this work addresses the elaboration

of ZnO-embedded PHB fibers by the electrospinning

technique and the production of ZnO-covered PHB

fibers via a simultaneous electrospinning/electro-

spraying procedure. Moreover, a relationship between

physical properties of precursory solutions and final

morphology of corresponding nanofibers is proposed.

The influence of ZnO nanoparticles on crystallization

behavior and thermal stability associated with PHB-

based nanofibrous mats is also discussed. Finally, their

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antibacterial activity against E. coli and S. aureus is

examined as a function of zinc oxide concentration

and their fabrication technique.

Experimental

Materials

A commercially available poly(3-hydroxybutyrate)

wasused (Biomer, �Mn = 144 kg mol-1, �Mw= �Mn = 2.3)

for the preparation of fibrous materials. 2,2,2-trifluo-

roethanol (TFE) was used as a solvent for PHB elec-

trospinning and methanol as a dispersive medium for

ZnO nanoparticles. Both solvents were purchased

from Sigma-Aldrich, and in the latter case, it was pre-

viously distilled over CaCl2 before use. Nano-ZnO

particles with quasi-spherical morphology were syn-

thesized in one of our laboratories by the precipitation

method, and the corresponding characterization

showed a high pure product with an average particle

diameter of 12 nmand adiameter distribution ranging

from 8 to 20 nm (See Support Information for details

related to synthesis and characterization of ZnO

nanoparticles) [20]. To determine the antibacterial

properties, the microorganisms used were Escherichia

coliATCC-25922 andS. aureusATCC-29213; tryptic soy

broth (TSB) was employed as the growth medium.

Determination of conductivity and viscosityof precursory solutions usedfor electrospinning

The conductivity of PHB solutions in the absence and

presence of ZnO nanoparticles was determined with a

Mettler Toledo conductometer (S47K model) at

25 ± 2 �C. It is noteworthy that the solutions were pre-

viously homogenized under stirring for about 15 h. In

turn, the viscosity of the solutions as a function of shear

rate was analyzed by the use of an Anton Paar model

Physica MCR 301 rheometer with a cone-plate configu-

ration (50 mm in diameter, angle of 2� and 0.205 of gap)

at 25 �C.

Generation of fibrous materials

The elaboration of fibrous materials was carried out

with a Linari Engineering electrospinning equip-

ment, whose configuration is depicted in Fig. 1. For

mere electrospinning, a syringe with 10 mL of the

PHB/ZnO mixed solution (in TFE 10 wt/vol%) was

placed in a pump, where the distance between the

needle to the grounded cylindrical drum was equal to

25 cm. Then, the polymer solution was subjected to a

voltage of 25 kV at a fixed PHB solution flow

(2.5 mL h-1); consequently, a PHB jet was generated

and collected on the cylindrical drum (spinning

rate = 700 rpm). In the tandem technique, a PHB

solution in the absence of ZnO nanoparticles was

electrospun under the same conditions as those used

for electrospinning. Simultaneously, another syringe

containing a given amount of ZnO (1–5 wt% respect

to PHB) dispersed in 10 mL of methanol was placed

in a second pump (120� tilted in relation to the

position of the first pump) and subjected to electro-

spraying under the same conditions (flow rate, col-

lector rate, and voltage) used in the case of

electrospinning, where the needle-to-collector dis-

tance was equal to 5 cm. The obtained mats were

characterized by means of several techniques, which

will be described in subsequent sections.

Morphology of PHB and PHB/nano-ZnOmats

Scanning electron microscopy (SEM) was used to

examine themorphology of electrospunmats, i.e., those

obtained by electrospinning and electrospinning/elec-

trospraying processes. Moreover, the average fiber

diameter ( �Df) was calculated by resorting to the ImageJ

software. For this purpose, several micrographs were

used and the diameter was measured in four sections

along 60–70 fibers. In the case of electrospinning-

derived mats, some transmission electron microscopy

(TEM) images were obtained. In turn, the electrospin-

ning/electrospraying-derived fibrous composites were

analyzed by X-ray energy-dispersive spectroscopy

(EDX) in order to obtain the corresponding elemental

mapping.Moreover, the porosity andpore sizes ofmats

were determined by mercury intrusion porosimetry

(MIP) using a Micrometrics Autopore IV equipment.

Thedeterminationofporosity featureswasbasedon the

Washburn equation between the applied pressure

(from 1.03 and 206.8 MPa) and the pore size into which

mercury intruded.

Thermal characterization

The crystallization and melting behavior of the elec-

trospun nanocomposites were investigated by

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differential scanning calorimetry (DSC) using a TA

Instruments (Discover series) calorimeter under the

following conditions: one heating–cooling–heating

cycle between -70 and 200 �C at a heating or cooling

rate of 10 �C min-1 applying nitrogen as the inert

gas. On the other hand, thermal stability of PHB-

based mats was assessed by thermogravimetric

analysis (TGA) employing a TA Instruments (Dis-

cover series) thermobalance. The tests were carried

out at a heating rate of 10 �C min-1 from room tem-

perature to 600 �C under nitrogen atmosphere using

sample masses of about 20 mg.

Antibacterial assays

The antibacterial activity was evaluated by means of

the Japanese Industrial Standard Z 2801, which con-

sists of the immersion of mats in sterile vials with

4 mL of a bacteria suspension containing 4.7 9 104

colony-forming units per milliliter (CFU/mL). It is

important to mention that the bacteria concentration

was preadjusted by a dilution process in tryptic soy

broth. Then, the vials (containing the PHB-based

mat ? bacteria suspension) were sealed and placed

in an incubator during 24 h at 37 �C. After the incu-

bation period, the vials were withdrawn, and an ali-

quot was extracted in order to carry out the cell

counting by means of agar plate technique supported

by optical microscopy. In order to determine the

antibacterial efficiency (AE), Eq. (1) was used:

AE ¼ logBt¼ 24 h

Bt¼ 0 h� log

St¼ 24 h

Bt¼ 0 h

� �ð1Þ

where Bt = 0 h and Bt = 24 h is the quantity (CFU/mL)

of survival bacteria after incubation in the presence of

the blank (PHB without ZnO) for 0 and 24 h,

respectively, and St = 24 h is the quantity of survival

bacteria after 24 h of incubation in the presence of

PHB/ZnO fibrous nanocomposites.

It is important to mention that each test was real-

ized three times, thus the values presented herein

resulted from the average measurement and stan-

dard deviation (SD) was specified when needed.

Results and discussion

Physical properties of PHB precursorysolutions used for electrospinning

The physical properties of the precursory solutions,

more particularly the electrical conductivity and

viscosity, exert a strong effect on the final morphol-

ogy of the materials engineered by electrospinning.

Figure 1 Scheme of devices utilized for designing PHB-based

mats by electrospinning: (1) pump with a syringe containing PHB/

TFE solution with or without ZnO nanoparticles, (2) grounded

collector, (3) high-voltage source, and (4) second pump with a

syringe containing ZnO nanoparticles dispersed in methanol, for

electrospinning/electrospraying tandem technique.

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The incorporation of nanoparticles can alter these

properties; therefore, it may be worth carrying out a

careful study of these parameters, in the case of the

PHB solutions containing ZnO nanoparticles.

In this regard, Fig. 2 shows the dynamic viscosity

of PHB dissolved in TFE, with and without ZnO

nanoparticles, as a function of shear rate. It could be

noticed that the presence of ZnO nanoparticles had

no substantial effect on the viscosity values up to the

nanoparticles content reached 5 wt%. For this par-

ticular ZnO concentration, two regimes could be

perceived in the viscosity versus shear rate plot, the

first one taking place at low values of shear rate

(1–20 s-1) in which the presence of nanoparticles

increased the viscosity of PHB solution, while the

second regime (shear rates higher than 20 s-1) was

characterized by the occurrence of a viscosity plateau.

This rheological behavior could be explained in terms

of aggregation of ZnO nanoparticles at high concen-

tration, which might restrict the diffusion of PHB

chains at low shear rates (first regime), but the effect

of nanoparticles (aggregated and/or separated enti-

ties) on viscosity was insignificant when shear stress

(shear rate) was higher.

In spite of the wide shear rate range analyzed, it is

possible to attain a more realistic comparison calcu-

lating the theoretical shear rate used in the manu-

facture of PHB mats through Eq. (2):

_c ¼ 4Q

pR3; ð2Þ

where _c is the shear rate, Q is the flow rate, and R is

the needle radius.

Considering that Q was equal to 2.5 mL h-1 and

R was equal to 0.445 mm, the theoretical shear rate

was equal to 10.8 s-1. Therefore, taking into account

the results of Fig. 2, the viscosity of the PHB solution

was about 0.89 Pa s with insignificant variations due

to the dispersed ZnO nanoparticles.

The polymer solution conductivity is another cru-

cial parameter that has to be considered, prior to

electrospinning process, especially when it refers to

solutions containing nanoparticles. The PHB precur-

sory solutions with 0, 1, 3, and 5 wt% of nano-ZnO

displayed conductivity values of 20, 21, 20, and 22

lS/cm, respectively. Accordingly, the values of con-

ductivity were nearly identical within a marginal

deviation, regardless of the nano-ZnO content. The

influence of PHB solution viscosity and conductivity

on the final morphology of PHB-based mats will be

disclosed in the next section.

Morphology of PHB and PHB/nano-ZnOmats derived from electrospinning

In order to get a better understanding of the rela-

tionship between physical properties (viscosity, con-

ductivity) of precursory solutions and the final

morphology of PHB-based fibrous materials, several

characterization techniques were used, namely elec-

tronic microscopy (SEM, TEM) and mercury intru-

sion porosimetry. In this regard, Fig. 3 shows the

SEM images of the as-obtained materials, which

exhibited the characteristic fibrous morphology

derived from electrospinning. The fibers were not

completely uniform and possessed a few protuber-

ances, which could be due to an unsuitable viscosity

of PHB in TFE as the blank also displayed surface

defects. Regarding the average fiber diameter ( �Df),

their values were equal to 425 ± 170, 380 ± 140,

480 ± 190, and 440 ± 210 nm for the mats with 0, 1,

3, and 5 wt%, respectively. Moreover, the distribution

of fiber diameters varied from 0.1 to about 1 lm for

all cases, as disclosed in Fig. 3. However, the elec-

trospun composite material with 5 wt% ZnO exhib-

ited a bimodal distribution. Considering that the jet

originated during the electrospinning process could

be perturbed by the presence of a relatively high

concentration of nanoparticles, the fibers might not

be uniform in terms of diameters.Figure 2 Viscosity of PHB solutions with different contents of

ZnO as a function of shear rate.

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Figure 3 SEM micrographs of

electrospun PHB-based mats with

a 0, b 1, c 3, and d 5 wt% ZnO

nanoparticles. The corresponding

fiber diameter distributions are

displayed in the right column.

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The porosity of fibrous materials is also an

important morphological parameter; therefore, dif-

ferent porous features of the as-obtained electrospun

composites based on PHB/ZnO were determined by

MIP (Table 1). In this regard, PHB fibers with dif-

ferent concentrations of embedded nano-ZnO exhib-

ited porosity ratio values around 85 %. A negligible

variation of porosity values between pristine PHB

and PHB-based composites was noticed, which could

be expected due to similar fiber diameter distribu-

tions. On the other hand, the corresponding pore

sizes arising from MIP analyses are gathered in

Table 1, in which pores with sizes ranging from 0.4 to

3 lm were observed for neat PHB fibers and PHB-

based composite fibers with 1 and 3 wt% nano-ZnO,

while for the composite with 5 wt% the range was

shifted toward higher values, i.e., 2–8 lm. As a mat-

ter of fact, pore sizes correspond to interfiber spaces

which generally depend on fiber diameters; the

higher the fiber diameter, the larger the interfiber

space. It should be noticed that the reported porosity

values were suitable for the permeation of certain

amounts of oxygen and moisture needed for wound

healing applications, as they had to be higher than

60 % [30–32].

As the dispersion of ZnO nanoparticles embedded

within PHB fibers is another critical concern, TEM

analysis was carried out. In this regard, Fig. 4 pre-

sents several micrographs derived from PHB mats

with 3 and 5 wt% ZnO as well as the corresponding

EDX spectra in which the typical signals for ZnO are

indicated. In the case of PHB with 3 wt% ZnO, a good

dispersion was observed, since a few nanoparticles

were totally isolated and some aggregates of

100–150 nm in size were present (ZnO particles

appear in black color). Conversely, ZnO aggregates

up to 300 nm were detected when the nanoparticles

content was equal to 5 wt%. The presence of these

relatively large particles probably provoked the per-

turbation of the polymer jet during the electrospin-

ning process, resulting in the aforementioned

bimodality of fiber diameter distribution. More

interestingly, some isolated nanoparticles and/or

aggregates were both inside and close to the fiber

wall (semi-exposed). Therefore, such nanofibrous

materials could be envisioned as wound dressings

with controlled release of nanoparticles, as the semi-

exposed nanoparticles would immediately act as

biocides, while the embedded nanoparticles would

be gradually released/exposed, as the polymer is

degraded.

Thermal properties of PHB and PHB/ZnOmats derived from electrospinning

Electrospun polymers used as wound dressings or

scaffolds have to be sterilized by different techniques.

Dry heat sterilization is the most common procedure,

where high temperature is required (ca. 160 �C).Consequently, electrospun materials with excellent

thermal stability are highly desirable [33, 34]. In this

regard, Fig. 5a shows the degradation patterns

resulting from TGA of electrospun PHB-based mats

with and without nano-ZnO. It could be seen that all

PHB mats had a single degradation step; however,

the degradation process started at higher tempera-

ture for electrospun nanocomposites. This was more

clearly observed in the derivative thermogravimetric

curves depicted in Fig. 5b. Neat PHB fibers exhibited

the degradation onset temperature (To) at 210 �C,while fibers with ZnO nanoparticles presented a To

value of 220 �C, regardless of the metal oxide content.

Additionally, the peak temperature (Tp, which was

related to the maximum rate of mass loss) was

affected by the presence of ZnO nanoparticles, as Tp

of neat PHB was equal to 267 �C, while PHB/ZnO

fibers displayed Tp values equal to 275, 271 and

269 �C with 1, 3, and 5 wt% of incorporated

nanoparticles, respectively, and the variation in Tp

values of composites could be due to the nanoparti-

cles dispersion degree. It was reported in the litera-

ture that the thermal degradation of PHB led to short

chains with carboxylic end groups [15, 28]. Therefore,

such these groups could eventually react with the

Table 1 Porous features of

electrospun PHB-based mats

as determined by MIP

ZnO (wt%) Porosity ratio (%) Average pore size (lm) Pore size range (lm)

0 85 1.98 0.4–3

1 83 1.69 0.4–3

3 86 1.11 0.4–3

5 87 2.24 2–8

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Figure 4 TEM images derived from the electrospun PHB mats with a 3 and b 5 wt% ZnO nanoparticles. The corresponding EDX spectra

are located at the right side of the micrographs.

Figure 5 a TGA curves and b corresponding derivative curves for mats derived from the electrospinning of PHB solutions with (open

circle) 0, (filled circle) 1, (filled triangle) 3, and (open triangle) 5 wt% ZnO nanoparticles.

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hydroxyl groups from ZnO nanoparticles surface

forming carboxylate-zinc oxide adducts by transes-

terification, thus conferring a higher thermal stability.

Besides, TGA could give accurate percentages of

residual chars which were associated with the final

nanoparticles contents, whose values were equal to

1.35, 3.12, and 4.55 wt%. These amounts were in good

agreement with the initial ZnO content.

In order to further study the influence of ZnO

nanoparticles on the PHB crystallization behavior in

electrospun fibers, the latter were subjected to DSC

analysis, whose resulting thermograms are disclosed

in Fig. 6. First heating traces of electrospun mats are

depicted in Fig. 6a, in which a negligible effect of

ZnO nanoparticles on thermal transition was

observed, since glass transition temperature (Tg) and

melting temperature (Tm) values were equal to 64

and 170 �C, respectively, regardless of the ZnO con-

tent. Then, during the sample cooling (Fig. 6b), an

exothermic transition was detected which was

attributed to crystallization of PHB. The crystalliza-

tion temperature (Tc) was considered as the maxi-

mum value of the exothermic peak. Therefore, the

values were equal to 90.5, 88.7, 88.5, and 89.4 �C.

Figure 6 DSC thermograms of mats derived from the electrospinning of PHB solutions with different concentrations of ZnO

nanoparticles: a first heating, b cooling, and c second heating.

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Taking into account these Tc values, it could be

established that the ZnO nanoparticles delayed the

crystallization process to a low extent for the lower

ZnO contents (1 and 3 wt%) while for 5 wt% ZnO,

this phenomenon was negligible. The delay of crys-

tallization may result from the potential interaction

between hydroxyl groups of ZnO and carbonyl

groups of PHB chains in the molten phase, thus

hampering the macromolecular reptation, and con-

sequently the stable crystalline structure formation

[35]. The second heating run (Fig. 6c) consisted of the

occurrence of the glass transition at 0 �C for all

samples, and the melting of the crystalline phase

which was a stepwise process with two endothermic

peaks (Tm) at 155 and 166 �C. When comparing the

two heating cycles, the much higher value of Tg in the

first heating run was associated with the orientation

of PHB chains caused during the electrospinning

process, which provoked a reduction of the free

volume by constraining the polymer chains within

the nanofibers. On the other hand, the presence of

two melting peaks was probably due to the formation

of two types of crystalline structures, one of them less

stable, hence a melting–recrystallization–remelting

process took place. Interestingly, the endothermic

peak at higher temperature was more prominent in

the presence of nano-ZnO, thus suggesting that the

nanoparticles did not promote the formation of

stable crystalline structures; therefore, melting–re-

crystallization–remelting might occur to a larger

extent.

Morphology of mats derivedfrom electrospinning of PHB combinedwith electrospraying of ZnO nanoparticles

Based on the premise that the preparation of

antibacterial composite materials by combination of

electrospinning and electrospraying techniques has

been scarcely explored, a series of mats were engi-

neered by electrospinning of PHB solutions and

electrospraying of ZnO dispersions.

First, the morphology of the as-obtained mats was

analyzed by SEM, as shown in Fig. 7. It could be

observed that PHB fibers had a surface with some

defects, most probably due to the viscosity of PHB

solution, and some particles could be detected over

the fiber surface, i.e., ZnO nanoparticles, but addi-

tional analysis will be given in order to corroborate

this assumption. Regarding the fiber diameter

distribution, in all the cases, fiber diameters ranged

from 200 to 800 nm with �Df of 425, 475, 493, and

498 nm for the mats with 0, 1, 3, and 5 wt% ZnO,

respectively.

Concerning the porosity of mats derived from the

electrospinning/electrospraying technique, neat PHB

fibers exhibited a value equal to 85 %, while PHB

nanocomposites displayed 90, 88, and 88 % porosity

ratios with a ZnO content of 1, 3, and 5 wt%,

respectively. Likewise, pore size distributions were

determined by MIP, and all the obtained plots were

quite similar with pore sizes ranging from 0.4 to

4 lm. Regardless of the ZnO content, it could be

stated that the morphological parameters, namely

fiber diameter and porosity ratio, were quite similar,

as expected, since the PHB solution properties were

theoretically identical to those of neat PHB solution.

The evaluation of ZnO distribution on PHB fibers

was not precise by simple observation of SEM ima-

ges. Therefore, EDX detection was utilized to accu-

rately determine the location of nanoparticles by

means of Zn elemental mapping. Figure 8 displays

such mapping characterization for PHB/ZnO-based

fibrous nanocomposites, in which it could be detected

some particles well dispersed on polymeric fibers, in

addition to, aggregates bigger than 500 nm in the

case of PHB sprayed with 1 and 3 wt% ZnO, and

aggregates up to 2–4 lm size for PHB with 5 wt%

ZnO. At this point, it could be inferred that electro-

spraying was a suitable technique for ZnO nanopar-

ticles deposition onto PHB fibers. In the following

section, TGA of the obtained mats will be exposed in

order to get some insight into the strength of ZnO-

PHB interactions.

Thermal properties of PHB and PHB/ZnOmats derivedfrom the electrospinning/electrosprayingtandem technique

Thermograms derived from TGA and their corre-

sponding derivative curves for PHB-based mats are

plotted in Fig. 9. The same degradation pattern could

be observed for all samples. Interestingly, TGA

thermograms revealed that real ZnO contents notably

differed from the theoretical ones, as residual values

were equal to 0.42, 0.47, and 2.31 wt% for PHB mats

with a theoretical value of 1, 3, and 5 wt% ZnO,

respectively. Such significant differences could be

due to a weak adhesion of ZnO on PHB fibers,

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especially in the case of the aggregates perceived by

elemental mapping. Although hydrogen bonding

might be established between hydroxyl groups on

surface of ZnO nanoparticles and carbonyl groups of

PHB, the ZnO aggregates might be prone to detach-

ment as they possessed a lower contact area with the

PHB fibers compared to isolated ZnO nanoparticles,

thus facilitating the loss of the metal oxide

Figure 7 SEM micrographs

of PHB-based fibers sprayed

with a 0, b 1, c 3, and d 5 wt%

ZnO nanoparticles. The

corresponding fiber diameter

distributions are displayed in

the right column.

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nanoparticles by simple handling of the obtained

mats [36]. Regarding the onset degradation temper-

ature values of mats, neat PHB fibers exhibited a To at

210 �C, and in the case of composites constituted of

PHB/ZnO, the To were equal to 220–221 �C for any

ZnO concentration. On the other hand, the maximum

rate of mass loss was shifted toward higher temper-

atures by the presence of nanoparticles since neat

PHB fiber exhibited a Tp value equal to 267 �C and

PHB/ZnO composite fibers displayed Tp values

around 274 �C regardless of the ZnO content. It is

noteworthy that nanoparticles concentration as low

as 0.4 wt% could increase To and Tp values around 10

and 7 �C, respectively. As mentioned before, it was

hypothesized that the enhancement of thermal sta-

bility of PHB was associated to interactions/reactions

between ZnO surface and carboxylic groups of

degraded PHB chains.

Figure 10 shows the DSC heating–cooling–heating

cycles for the fibrous materials derived from elec-

trospinning/electrospraying. The thermal behavior

of such PHB mats was practically identical for all

samples, i.e., in the first heating cycle, a negligible

effect of ZnO nanoparticles on thermal transition

temperatures was observed: Tg was equal to 63 �Cand Tm of PHB fibers occurred at 170–171 �C. During

Figure 8 Elemental mapping of different regions of PHB mats derived from the electrospinning/electrospraying tandem process with a 1,

b 3, and c 5 wt% ZnO nanoparticles.

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cooling, the crystallization occurred at the same

temperature (*90.5 �C) in the case of PHB sprayed

with 0, 1, and 3 wt% ZnO and at 88.5 �C for PHB with

5 wt% ZnO. Obviously enough, a delay of crystal-

lization was induced by the presence of high ZnO

concentrations, as mentioned previously. The second

heating run exhibited the glass transition at 0 �C for

all samples (lower value than as-obtained fiber due to

loss of orientation), and the melting–recrystalliza-

tion–remelting peaks, whose intensity suggested a

higher portion of unstable crystalline structure for the

PHB mat with 5 wt% ZnO.

Antibacterial activity of fibrous PHB/ZnOnanocomposites

Miscellaneous medical devices undergo bacterial

colonization, thus becoming a source of infections [5].

Escherichia coli and Staphylococcus aureus are typical

pathogenic microorganisms, which can generate a

great number of infections, namely neonatal

omphalitis, necrotizing fasciitis, surgical site infec-

tions, infections after burn injuries, and others [37].

Considering this fact, the mats elaborated in this

work were subjected to antibacterial tests in the

presence of an inoculum constituted of the above-

mentioned bacteria.

Figure 11a, b shows the viable cell counting of both

bacteria as a function of theoretical nano-ZnO con-

centration of both electrospun and

electrospun/electrosprayed PHB-based mats. It

could be noticed that there was a substantial prolif-

eration of both bacteria in the presence of neat PHB

fibers, since the viable cell counting increased from

4.7 9 104 CFU/mL (SD = 0.35 9 104) up to 4.2 9 107

CFU/mL (SD = 0.46 9 107) for E. coli, while S. aureus

population increased from 4.9 9 104 CFU/mL

(SD = 0.52 9 104) up to 5.6 9 105 CFU/mL

(SD = 2.1 9 105). Conversely, the presence of ZnO

nanoparticles provoked a remarkable inhibition of

E. coli and S. aureus populations, and the antibacterial

effect of electrospun PHB/ZnO mats was higher than

that of mats derived from the electrospinning/elec-

trospraying technique. Table 2 gathers the antibacte-

rial efficiency, the theoretical concentration of ZnO

nanoparticles, the experimental one determined by

TGA (real) and the calculated concentration (parts

per million) in the samples subjected to antibacterial

tests. In the case of electrospun PHB-based mats, a

ZnO concentration as low as 97 ppm induced an

antibacterial efficiency (AE) value equal to

3.20 ± 0.15 and 2.10 ± 0.02 for E. coli and S. aureus,

respectively, which was higher than that established

for a bactericidal product (AE value should be higher

than 2, i.e., able to kill 99.99 % of bacteria). Moreover,

an increase in ZnO concentration in the samples did

not trigger further the antibacterial activity against

E. coli, since ZnO concentration equal to 239 and

270 ppm showed AE values identical to PHB fibers

with 97 ppm ZnO. In previous sections, it was

Figure 9 a TGA curves and b corresponding derivative curves for PHB-based mats derived from the electrospinning/electrospraying

tandem technique with (open circle) 0, (filled circle) 1, (filled triangle) 3, and (open triangle) 5 wt% ZnO nanoparticles.

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demonstrated by TEM that ZnO aggregation into PHB

fibers was significant by increasing nanoparticles

concentration; consequently, the surface area prone to

act as biocide was probably diminished. In the case of

S. aureus, there was a slight increase in AE with

increasing ZnO concentrations, thus indicating a low

resistance of this type of bacterium to ZnO nanopar-

ticles, which could be due to structural and chemical

compositional differences of the cell wall [12, 38, 39].

On the other hand, taking into account the norm

employed for antibacterial tests, electrospun/

electrosprayed PHB-based fibers exhibited AE values

(from 1.20 to 1.40), unsuitable for bactericidal mats

against E. coli. However, these mats could be used as

bacteriostatic products (bacteria growth inhibitors),

since they showed excellent growth inhibition (ca.

95–97 %). It should be mentioned that a major extent

of ZnO aggregation was revealed by EDX, and a

probable detachment of nanoparticles could also be

claimed, thus explaining the limited antibacterial

behavior of such electrospun/electrosprayed PHB

fibers.

Figure 10 DSC thermograms of PHB-based mats derived from the electrospinning/electrospraying tandem technique with different

concentrations of ZnO nanoparticles.

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Conclusions

Composite mats based on poly(3-hydroxybutyrate)

nanofibers and ZnO nanoparticles were success-

fully elaborated by either electrospinning of

PHB/nano-ZnO mixed solutions or simultaneous

electrospinning of PHB solutions with electro-

spraying of nano-ZnO dispersions. The incorpora-

tion of nanoparticles provoked slight variations in

viscosity and conductivity, but a significant effect

on the morphological parameters of PHB-based

mats was observed up to higher ZnO concentration

(5 wt%). PHB mats showed better thermal stability

and the ZnO nanoparticles had effect on thermal

transitions only after the highly oriented morphol-

ogy was eliminated by heating. When comparing

both electro-hydrodynamic techniques, mere elec-

trospinning was suitable for the design of ZnO-

embedded PHB fibers with bactericidal effect

against E. coli and S. aureus at nanoparticles con-

centration lower than 1 wt%, while the ZnO-cov-

ered fibers derived from the combination of

electrospinning and electrospraying exhibited bac-

tericidal or bacteriostatic effects against S. aureus or

E. coli, respectively, due to the heterogeneous/

weak deposition of nanoparticles.

Figure 11 Colonies forming units per milliliter (CFU�mL-1) after 24 h incubation of a E. coli or b S. aureus in the presence of

electrospun (gray filled columns) and electrospun/electrosprayed (white filled columns) PHB-based mats. CFU�mL-1 values are located on

the corresponding bar.

Table 2 Antibacterial efficiency values for PHB-based mats derived from electrospinning and electrospinning/electrospraying techniques

ZnOt (wt%)a ZnOr (wt%)b ZnOs (ppm)c AEd

E. coli S. aureus

Se SSf Se SSf Se SSf Se SSf

0 0 0 0 0 0 0 0 0

1 1.35 0.42 97 17 3.20 ± 0.15 1.30 ± 0.40 2.10 ± 0.02 1.50 ± 0.10

3 3.12 0.47 239 29 3.20 ± 0.37 1.20 ± 0.15 3.40 ± 0.60 2.80 ± 0.30

5 4.55 2.31 270 130 3.20 ± 0.19 1.40 ± 0.24 3.30 ± 0.19 2.50 ± 0.28

a Theoretical ZnO content in PHB-based matsb Real ZnO content in PHB-based fibers as determined by TGAc Parts per million of ZnO in the sample subjected to antibacterial tests (according to TGA determination)d Antibacterial efficiency values as calculated by Eq. (1) (see ‘‘Experimental’’ section)e Mats elaborated by mere electrospinningf Mats designed by electrospinning/electrospraying

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Acknowledgements

Financial support through French-Mexican PCP

Program is gratefully acknowledged. The authors

thank CONACyT (Mexico) for providing H. Rodrı-

guez-Tobıas with a Ph.D. Grant. They also thank J.

Cepeda and M. Lozano (CIQA, Mexico) and R. Pires

(CNRS, France) for their technical assistance in issues

related to electron microscopy characterization, as

well as J. Quiroz, C. N. Alvarado, and D. Rodrıguez

(CIQA, Mexico) for their technical support.

Electronic supplementary material: The online

version of this article (doi:10.1007/s10853-016-0119-

x) contains supplementary material, which is avail-

able to authorized users.

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