MATERIALS FOR LIFE SCIENCES

Synthesis, characterization and antibacterial activity

of thymol-loaded polylactic acid microparticles

entrapped with essential oils of varying viscosity

Agni Kumar Biswal1 , Isha Vashisht1 , Aamir Khan1 , Shivangi Sharma1 , andSampa Saha1,*

1Department of Materials Science and Engineering, Indian Institute of Technology, Delhi, India

Received: 9 December 2018

Accepted: 1 April 2019

� Springer Science+Business

Media, LLC, part of Springer

Nature 2019

ABSTRACT

Double emulsion technique (W1/O/W2) was used to achieve PLA (polylactic

acid)-based microparticles loaded with thymol (hydrophilic antibacterial active)

with high encapsulation efficiency ([ 90%) that was later released in water in

controlled fashion. In order to enhance the release of thymol from semicrys-

talline slow degrading PLA matrix, pores were introduced into the particles by

incorporating minute quantity of essential oils (EOs) such as castor, mustard,

olive and coconut oils of varying viscosity (from 128 to 13 mPa S). Strikingly, it

was found that the pore size of microparticles (pore size 1.2–0.53 lm and

specific surface area 7.5–2.1 m2/g) has been influenced by the viscosity of EOs.

The plausible mechanism of formation of pores using EOs was also explored. As

expected, the release rate of thymol did get accelerate with the increase in pore

density and its release mechanism can well be explained with the help of power

law model. In addition to that, these porous particles were demonstrated to act

as an effective carrier for continuous delivery of thymol with enhanced inhibi-

tory activity probably due to synergistic action of EOs and thymol. Therefore,

EOs employed here served dual purposes. These PLA-based microparticles

composed of completely natural ingredients can be exploited as active food

packaging material to prolong the shelf life of food.

Introduction

Traditionally, active compounds were directly added

to food formulation for protecting food from micro-

bial spoilage, thereby enhancing food shelf life. But

these methods suffer from several limitations such as

more amount of additives are needed to protect food

materials from deterioration, raw/fresh food may

lose their taste/flavor, and also there may be some

complex reaction between food and actives [1–3].

Consequently, there is a demand in food industry for

polymer packaging from which active compounds

are diffused onto the food surface in order to prevent

Address correspondence to E-mail: ssaha@polymers.iitd.ac.in

https://doi.org/10.1007/s10853-019-03593-7

J Mater Sci

Materials for life sciences

bacterial growth. In this respect, controlled release

packaging (CRP) technology can be beneficial where

actives, e.g., antibacterials, are released from pack-

aging matrix in continuous fashion to inhibit bacterial

growth for prolonged period [4]. Controlled release

packaging is an up-growing domain of the food

industry [5]. Several polymeric forms [5, 6] have been

utilized for packaging, such as sachets, polymeric

coating [7], polymeric films [8, 9] and polymeric

microparticles [10]. Among these materials, poly-

meric microparticles have been less studied com-

pared to polymeric films. One of those few reports

includes a system developed by Incarnato and his

group, comprised of antioxidant (a-tocopherol)microencapsulated PLA (polylactide) microparticles

for the active packaging of food materials [10]. In our

previous publication, antibacterial (benzoic acid) was

encapsulated into hollow PLA microparticles using

emulsion solvent evaporation method and it was

shown that the controlled release of benzoic acid

inhibited the bacterial growth over 1 month [11].

Inspired by all these reports, we have decided to

focus our current work on PLA-based porous

microparticles as packaging materials. Furthermore,

among the biodegradable polymers, PLA micropar-

ticles have received extensive attention as drug

delivery vehicle as well as food packaging matrix

because they are suitable for body/food contact as

per EU (Commission Regulation EU No. 10/2011)

and US (FCS Notifications No. 178/2002 and No.

475/2005) legislation [12]. In food industry, the use of

natural additives with antibacterial properties

instead of chemical additives has been immensely

increased due to their no side effects on human

health [13–15]. Among the plethora of naturally

occurring antibacterial, thymol (2-isopropyl-5-

methylphenol), a monoterpene phenol derivative of

cymene, is a well-known natural antibacterial agent

[16]. Thymol displays strong antimicrobial activity

against food-borne pathogenic organisms such as

E. coli, monocytogenes, typhi, S. aureus, and Candida

albicans [17] and hence can be considered for food

packaging applications [18]. In addition to thymol,

among all natural resources, essential oils (EOs) also

show sufficiently high antimicrobial behavior. They

are complex mixture of volatile components such as

terpenoids, monoterpenoids, sesquiterpinoids and

their oxygenated derivatives, which can easily diffuse

across cell membrane to induce biological reactions

[19]. Essential oils have wide application as

therapeutic agents, as complementary medicines and

as food preservatives [20, 21]. Use of essential oil as

additives in edible films and coating in food pack-

aging has been extensively exploited in the last few

decades. For example, Wu and his group incorpo-

rated cinnamon essential oil containing nanolipo-

somes as an additive in gelatin films which not only

improved the antibacterial property of gelatin film

but also controlled its release rate [22]. Moreover,

these essential oils have also been used in combina-

tion with another antibacterial in order to enhance

the antibacterial efficacy of the system [23, 24].Taking

into account these previous works, herein combina-

tion of thymol and one of several essential oils with

varying viscosity was used as model antibacterials

co-encapsulated in PLA-based microparticles by

employing W/O/W double emulsion solvent evap-

oration method. It is well known that suitable com-

bination of emulsion solvent evaporation and phase

separation by using non-solvent may yield porous

particles which might help in controlling as well as in

accelerating the active release from the particles [25].

In this study, it was hypothesized that minute

quantity of these essential oils may act as non-solvent

and hence may result pores in PLA particles and

these pores can be tunable depending upon the vis-

cosity of the oils employed. Originality of the work

mainly lies here in the hypothesis. Therefore, in

addition to show a synergistic antibacterial effect,

these oils may also help in controlling the release of

antibacterial (thymol). In this paper, along with a

fixed concentration of thymol, EOs such as castor oil,

mustard oil, olive oil and coconut oil with viscosity

ranging from 128 to 13 mPa S (to cover a wide range

of viscosity which encompasses most of the edible/

medicinal oil [21]) were employed individually into

PLA microparticles to study the effect of viscosity of

oils on the pore formation, and a probable reason

behind the formation of porous particles was also

discussed here. All formulations were thoroughly

characterized, and their release profiles were studied

at neutral buffer. An attempt was also made to

understand the release mechanism with the help of

power law model. Finally, the antibacterial activity of

all the particles was assessed over time against E. coli

using plate spreading method.

J Mater Sci

Materials and methods

Materials

Polylactide (grade 3052D) was purchased from Nat-

ureWorks, USA. PVA (poly(vinyl alcohol)) cold, with

molecular weight 125000 g/mol, was procured from

Central Drug House (CDH), India. Thymol (99.6%

pure) was purchased from Loba Chemie. Dichlor-

omethane (DCM) (HPLC grade) was obtained from

Fischer, India. Essential oils such as coconut, castor,

olive and mustard oil were purchased from Marico,

Sisla laboratories, Zetun Australian and Babaji

Udyog, respectively, and their viscosity was deter-

mined by parallel-plate rheometer. The materials for

antimicrobial study such as nutrient agar and luria

broth were purchased from Hi-Media. The bacteria

Escherichia coli (E. coli) and BL21 DE3 gold strain were

obtained from Agilent Technologies, USA.

Fabrication of microparticles

Porous PLA microparticles were prepared using a

simple water/oil/water double emulsion technique.

At first, 5 wt% of PLA (0.3 g) solution was prepared

in 6 mL of DCM and 0.15 g of thymol was added to

the solution. A previously prepared 1 mL of 0.1 wt%

PVA aqueous solution was added dropwise to the

above mixture to make w/o primary emulsion. To

this emulsion, 0.01 mL of one of the four essential oils

(castor/mustard/coconut/olive) was added under

constant stirring and the whole emulsion was stirred

for 20 min. The resultant solution was then added to

50 mL of 0.25 wt% (w/w) PVA solution containing

1 mL DCM and emulsified under mechanical stirring

at 250 rpm for 4 min at room temperature. After the

formation of emulsion droplets, 150 mL of 0.25 wt%

(w/w) PVA solution was added and stirred using

overhead stirrer for 4 h. After complete removal of

DCM, the solid microparticles were collected and

washed for several times. The microparticles were

then freeze-dried and stored for further

characterizations.

Characterizations

Morphological analysis

The particle size and surface morphologies (includ-

ing cross section) of microparticles were analyzed

using scanning electron microscope, Zeiss EVO50 at

20 kV. In order to see inside of the particles, before

analysis, samples were dipped into liquid nitrogen

and cross-sectioned by a razor blade. The gold coat-

ing of samples was carried out using a sputter coater.

The particle size distribution was determined using

Image J software.

Viscosity and interfacial tension measurements

Viscosity of PLA solutions (5 wt% in 200 mL DCM)

with and without EOs (333.3 lL) was measured using

Brookfield Viscometer. The interfacial tension

between PLA solutions changing different EOs and

aqueous phase (0.1 and 0.25 wt% PVA solutions) was

measured by Tensiometer (K100, Kruss) using Du

Nouy ring method.

Brunauer–Emmett–Teller (BET) analysis

BET specific surface areas of polymeric microparti-

cles were measured by BET method (N2 adsorption)

using Quantachrome Autosorb IQ instruments at

77.35 K.

Thermal analysis

Thermal properties of microparticles were investi-

gated by using differential scanning calorimeter

(Q200, TA instrument) under nitrogen atmosphere.

All the samples were oven-dried before experiment.

Then, samples were heated from 20 to 200 �C at a

heating rate of 10 �C/min and cooled to 30 �C. Again,

samples were heated up to 200 �C keeping the same

heating rate. The resultant thermal transitions were

recorded in the thermograms.

Distribution of active ingredient in microparticles

Distribution of thymol across the surface and within

the bulk of microspheres was investigated by using

confocal Raman microscope (Gloucestershire, GL 127

DW, UK). The particles (whole and cross sections)

were irradiated at 785 nm near-infrared diode laser

with a scanning range of 3200 to 100 cm-1.

Encapsulation efficiency

The encapsulation efficiency of thymol-loaded

microparticles was determined by using UV–Vis

J Mater Sci

spectroscopy. 20 mg of thymol-loaded microparticles

(20 mg) was dissolved in DCM, and the solvent was

evaporated to dryness. Subsequently, PBS solution

(2 mL) was added and stirred for 30 min to facilitate

partitioning of thymol into PBS (aqueous). Mixture

was allowed to keep undisturbed for 2 h to separate

DCM and aqueous phase. The aqueous phase was

withdrawn and analyzed by UV spectrophotometer

(T90 ? UV/VIS spectrometer, PG instruments Ltd.)

for determining the thymol contents. Prior to this

measurement, a calibration curve of thymol was

made in PBS solution. The encapsulation efficiency

(%) was calculated according to the given equation. A

control experiment was also done to find out the

extraction efficiency of thymol from DCM to water

following the literature procedure [11]:

Encapsulation efficiency %ð Þ

¼ Experimental active agent loading

Theoretical active agent loading� 100:

In vitro release of thymol from microparticles

Release study of thymol was carried out in PBS buffer

of pH 7 at 37 �C and 200 rpm in an orbital shaker.

20 mg of microparticles was dispersed in 5 mL of

PBS buffer in vial and kept under the above condi-

tion. After each time interval, 2.5 mL of solution was

withdrawn from each vial and then 2.5 mL of fresh

buffer was added to the vial. Concentration of

released thymol from each withdrawal was deter-

mined by UV–Visible spectroscopy at 274 nm, and

release percentage was calculated by the following

equation:

Assessment of antibacterial activity of microparticles

Antimicrobial activity of samples A–E was studied

against Escherichia coli by using plate spreading

method [11]. At first, minimum inhibitory concen-

tration (MIC) of free thymol as well as thymol-loaded

microparticles having different EOs required to

completely inhibit the bacterial growth was deter-

mined. The MIC value of free thymol was evaluated

by taking its concentrations ranges from 100 to

500 lg/mL in Luria broth solution. To determine the

particle concentration for each sample (sample A–E)

to completely inhibit the bacterial growth, various

concentrations of particles ranging from 20 to 100 mg

in 1 mL of luria broth solution were kept in incubator

shaker at 200 rpm and at 37 �C for 24 h and then

1 mL of supernatant was taken out from each con-

centration. 10 lL of a freshly prepared E. coli culture

having bacterial concentration of 8 9 108 cells/mL

was inoculated into extracted samples and incubated

for 24 h. 50 lL of incubated solution was spread onto

the previously prepared nutrient agar petri plates

and left it over for drying. Finally, the plates were

kept for incubation and the bacterial growth was

calculated and recorded as CFU/mL. The particle

concentration to fully inhibit bacterial growth was

estimated. After finding out these concentrations, the

same concentration was used for determining their

long-term antibacterial activity over 10 days using

the same plate spreadingmethod asmentioned above.

After extraction of sample at each interval, fresh luria

broth solution was added to the stock solution to

replace the extracted volume. After this, the incubated

bacterial solutions taken at various time intervals were

diluted by PBS solution of pH 7 to have several dilu-

tions ranging from 101 to 108 times. 50 lL of each

samples was plated onto nutrient agar petri plates and

incubated under the same condition as mentioned

above. The number of colonies was counted and

reported as colony-forming unit per mL (CFU/mL) or

as log (CFU/ml). All experiments were run in tripli-

cates. At the same time, a set of samples A to Ewithout

thymol-loaded particles were also tested.

Results and discussion

Fabrication of microparticles

In a typical experiment, polymer solution (O) was

prepared by dissolving the polymer (PLA) and thy-

mol in a volatile organic solvent (dichloromethane).

The aqueous 0.1% (w/v) PVA solution (W1) was

added dropwise to the resultant polymer solution

% Active agent release ¼ Amount of active agent in supernatant

Amount of total active agent used in encapsulatedmicroparticles� 100:

J Mater Sci

(O) followed by addition of one of the essential oils

(castor, mustard, coconut and olive) to make (W1/O)

emulsion. Then, the entire emulsion was poured into

another PVA solution (0.25%, w/v) (W2) and emul-

sified to form a W1/O/W2 double emulsion using an

overhead stirrer. Finally, nascent emulsion droplets

were hardened by subsequent removal of solvent in

order to make microparticles. According to SEM

investigations, thymol along with one of the four

essential oils (EOs)-loaded microparticles has

spherical morphology with porous surfaces, as

shown in Fig. 1. The variation in particle size and

pore size was observed for different essential oils

with varying viscosity, and the data are summarized

in Table 1. It was assumed that stability of the inner

aqueous phase would play a pivotal role in generat-

ing particles with porous morphology. The particle

size distribution histogram (histogram not shown

here) suggested that the size of thymol- and EO-en-

capsulated microparticles was found to be higher

(i) (ii)

(iii) (iv)

(v)

Figure 1 SEM micrographs

for (i) sample A, (ii) sample B,

(iii) sample C, (iv) sample D,

(v) sample E and their

respective zoom in images of

the surfaces (insets).

J Mater Sci

compared to the particles without the addition of EO

presumably due to the inclusion of EO that might

have stabilized the inner aqueous droplet (Fig S1,

discussed later). Furthermore, close examination of

SEM images of all microparticles revealed the gen-

eration of porous surfaces only with the microparti-

cles which contained EO. From the cross-sectional

view of particles (Fig S2), it can be understood that

distribution of pores was throughout the bulk as well

as on surface of microparticles except for the sample

A. Based on SEM images analyzed by ImageJ soft-

ware, the pore size and pore density on the surface of

microparticles were calculated and are displayed in

Table 1. Strikingly, it was observed that porous

morphology of these particles did show a correlation

with viscosity of EOs, because it was ascertained

from SEM images that castor oil with the highest

viscosity (128 mPa S) resulted microparticles with

the highest pore size and pore density, followed by

mustard (23.2 mPa S), olive (18.49 mPa S) and coco-

nut oil (13.07 mPa S), respectively (Fig. 1ii–v,

Table 1). Surface area calculated from BET analysis

further supported this trend (Fig S3, Table 1) by

showing almost linear fit to the curve obtained by

plotting specific surface area versus viscosity of EOs.

In order to understand the effect of EOs, only thymol-

loaded PLA particles were also fabricated without the

addition of essential oil and smooth spheres were

formed with non-porous surfaces (Fig. 1i). Taken

together all the facts discussed above, the plausible

mechanism of formation of pores in the microparti-

cles in the presence of EOs can be explained as fol-

lows (Scheme 1). Double emulsions (W1/O/W2) are

a complex system composed of dispersed oil droplets

(i.e., polymer and essential oil solution in DCM)

which contain even tinier inner water droplets (W1).

There can be two ways by which pores might be

formed onto the surface of the particles. In case of

W/O/W double emulsion, surface pores can be

generated by the inner aqueous droplets (W1) with

progressive evaporation of DCM. But, as per litera-

ture reports, inner aqueous volume of 12% (used in

this study) may not be sufficient to generate pores

onto the viscous polymer surface layer by dispersing

inner aqueous droplet [26]. Moreover, no surface

pores can be found on the particles fabricated with-

out EOs (sample A). However, for samples with EOs,

evaporation of DCM may also lead to generate

additional tiny droplets inside the dispersed oil

droplets due to the phase separation of essential oil (a

non-solvent for PLA [27] due to significant differ-

ences in solubility parameter of PLA and EOs [28])

Table 1 Viscosity of essential oils (EOs), particle size, pore size, surface area and encapsulation efficiency of thymol-loaded

microparticles

Sample name

(composition)

Name of

EOs

employed

Viscosity of

EOsa (mPa S)

Particle

sizeb

(lm)

Pore sizeb

(lm)

Pore densityb (number

of pores/lm2) 910-2

Specific

surface areac

(m2/g)

Encapsulation

efficiencyd (%)

Sample A (PLA/

thymol)

No EOs – 193 ± 42 – – – 90.0

Sample B (PLA/

thymol/castor)

Castor oil 128 207 ± 61 1.20 ± 0.12 5 ± 0.28 7.51 94.5

Sample C (PLA/

thymol/mustard)

Mustard oil 23.2 216 ± 54 0.84 ± 0.13 1 ± 0.05 3.3 99.3

Sample D (PLA/

thymol/olive)

Olive oil 18.49 195 ± 53 0.77 ± 0.12 0.7 ± 0.10 2.0 97.8

Sample E (PLA/

thymol/coconut)

Coconut oil 13.07 221 ± 52 0.53 ± 0.05 0.4 ± 0.04 2.1 95.2

aViscosity of oils was measured by using parallel-plate rheometerbParticle size, pore size and pore density were measured from SEM images using ImageJ software; average pore number was obtained by

counting only the visible side of the microparticles in SEM images; so, the pore number in the whole particle should be approximately

double of the reported valuescSpecific surface area was measured by BET method, and the values are average of three runsdEncapsulation efficiency was determined from UV/Vis spectrophotometer, and the values are average of three runs

J Mater Sci

from polymer phase [23] (see arrows in Fig S1). Ini-

tially, polymer (PLA) and EOs were well soluble in

dichloromethane (DCM). As evaporation of dichlor-

omethane was progressed, both polymer and EO

concentration got increased that resulted the phase

separation of EO and polymer due to their immisci-

bility [29]. Then, initially formed tiny EO droplets

within emulsion droplet may coalesce into small EO

droplets. It was postulated that the porous skin layer

of particles resulted from the inclusion of inner

aqueous droplets stabilized by these small EO dro-

plets along with PVA (surfactant) within the polymer

skin layer which was solidified at the outer interface

[26]. This kinetic arrest traps inner water phase to

yield pores/voids after evaporation of water. To

further understand the water droplet stabilization by

EO, the viscosity of organic phase and interfacial

tension between organic and water phases (which

contain 0.1 wt% and 0.25 wt% PVA solution as inner

water and outer water phase, respectively) were

measured for all the samples (samples A–E, Table SI).

It is clear from Table SI that the value of interfacial

tension between water and organic phase is sub-

stantially higher in the presence of EOs for 0.1 wt%

PVA solution than the interfacial tension between

organic phase with EOs and water phase comprising

0.25 wt% PVA due to higher surfactant concentration

at the interface. This may drive the EO droplets

toward the outer water phase from the inner water

phase [30, 31]. But, there is insignificant change in

interfacial tension among the various EOs or no oil at

that interface. In this scenario, viscosity of the organic

phase plays the pivotal role for arresting the inner

water droplet. As expected, viscosity of organic

phase increases with the increase in viscosity of EOs.

Hence, it can be assumed that the EO droplets will be

kinetically trapped at the skin for stabilizing inner

water droplet because of high viscosity exerted by the

oil phase leading to formation of greater pore size

and pore density in the case of castor oil having the

highest viscosity. This is clearly evident in Fig S1b

(see the red arrows). For the similar reason, coconut

oil with least viscosity would favor the formation of

particle skin with minimum pore size and density.

Similar observation was also reported by Kim et al.

[32]. They have observed the arresting of 2-methyl

pentane droplet onto the surface of oil (PLGA in

DCM) droplet similar to particle-stabilized emulsion

owing to the fast extraction of DCM. In our system

also, amount of DCM was kept low (3.3 vol%) which

was marginally higher than its solubility limit (2

vol%) [33] in water so that quick evaporation of DCM

can take place, because, after diffusing through water

only, DCM will be evaporated at water/air interface.

This accelerated DCM evaporation in turn decreased

the time allowed for coalescence of inner water dro-

plets (W1) with the external water phase (W2) and

hence increased the chance of pore formation by

retention of inner aqueous phase (W1). Furthermore,

in another report, Shi et al. [34] also concluded that

the rate of solvent removal plays a key role in

resulting porous structure of microspheres. Finally,

during freeze-drying process under high-vacuum

inner water droplets stabilized by EO droplets along

with PVA and PLA were evaporated and converted

into holes/pores (Scheme 1). However, when these

inner EO droplets were not included into the skin

layer (interfacial region), particles with non-porous

surfaces were formed (sample A). Hence, thymol-

loaded particles with no addition of EO rendered

non-porous surface. But, double emulsion was also

necessary to make the porous shell layer. In our

Scheme 1 Schematic

representation of the formation

of porous microparticles.

J Mater Sci

previous work, it was found that without inclusion of

inner aqueous droplet, no pores can be found onto

the surface of microspheres [23]. Therefore, it was

essential to add EO in the oil phase of double emul-

sion to make particles with porous surface. After

cross-sectioning the particles, it was found that inside

the microspheres, pores were uniformly distributed

throughout the particles even in the case of particles

having no EO (Fig S2(i)). Because of fast removal of

solvent, polymer precipitation will proceed from

surface to the core of microspheres. Hence, inner

aqueous droplet will be trapped inside the micro-

spheres by high-viscous polymer layer as discussed

above. This will finally develop into small pores

inside the microspheres after evaporation of water

[25] even in the case of sample A. It was also noted

that for high-viscous castor oil, pore density and size

were quite bigger compared to the rest of EOs. This

could be attributed to the high-viscous oil phase

(PLA along with EO in DCM) which might have

stabilized the inner water (primary emulsion) droplet

against coalescence by providing strong interfacial

tension [25] (see green arrow in Fig S1b). Therefore,

coconut oil has least viscosity among the four EOs

and displayed minimal porosity (Fig S1e) due to ease

of migration of low-viscous EO toward outer water

phase leading to reduced number of trapped inner

water droplets at the surface

Encapsulation efficiency of thymoland in vitro release study

The encapsulation efficiency of thymol was quite

high and found to vary from* 94 to 99% for samples

B–E (Table 1). A marginal decrease in encapsulation

efficiency was found for castor oil-loaded particles

probably due to its larger pore size compared to

others. However, the low encapsulation efficiency of

thymol for non-porous sample (sample A) implied

the role of hydrophobic interaction of EO with thy-

mol resulting in higher retention of it even with

porous surfaces. In order to predict particles’

behavior as active packaging materials, the in vitro

release study of thymol was conducted for samples

A–E at 37 �C in neutral PBS buffer (pH 7); the same

conditions were used for the bacterial growth during

antibacterial study [35]. As shown in Fig. 2, all sam-

ples displayed a high initial burst release of[ 35%

within 24 h because of adsorption of thymol onto the

surface of particles. Castor oil-loaded particles

showed highest burst release since the pores were

largest among all, and hence the diffusion rate of

thymol was high, especially in comparison with no-

oil-loaded particles having non-porous surface mor-

phology. A large initial release was definitely a plus

for antibacterial packaging applications to provide

immediate protection from bacterial contamination

[36]. After the initial burst, a slow and continuous

release was obtained from internally trapped thymol

irrespective of the nature of samples. As expected,

castor oil-loaded particles with the highest porosity

showed a relatively faster and maximum % of

release (* 65%) compared to its analogs. However,

complete release was not attainable for any samples,

probably due to strong interaction between thymol

and PLA matrix (discussed later). Similar trend was

observed in releasing thymol in water from other

polymeric films [18, 37]. It was noteworthy that

release of thymol from non-porous surface was not

hugely different from the porous particles, partly due

to achievement of considerable porosity inside the

particles and also being a small molecule, diffusion of

thymol remained largely unaffected by the pore size.

Another important observation was that the encap-

sulation of thymol (volatile) in these particles pre-

vented its evaporation to air in its dried form and

hence increased its storage stability [36]. There was

no change in weight of particles over a long period of

time (15 days) when the dry particles were stored in

open air.

Several approaches were reported in the literature

to understand the release kinetics of entrapped agent

Figure 2 In vitro release study of samples A, B, C, D and E in

PBS (pH 7) at 37 �C. All experiments were run in triplicates, and

the average values are shown with error bars.

J Mater Sci

from polymeric matrix. In this respect, many

researchers utilized a classic semiempirical power

law model (Eq. 1) that can be fitted well with the

experimental results to understand the release phe-

nomena predominantly occurred by diffusion,

degradation or a combination of both [38–40]:

Mt

M1¼ ktn; ð1Þ

where Mt and M? are cumulative release of thymol

at time t and infinite, n was a release exponent which

mainly defines the release mechanism, and k was the

rate constant. Such model was applied in our system

too and found to be well-fitted by the experimental

data (Fig S4, Table 2). It was interesting to note that

the release of thymol was characterized by low

release exponent value (n\ 0.2) that proportionally

increased with the porosity of the particles. Same

trend was observed for the value of rate constant

(k) which was least for non-porous particles. As per

the model, for spherical particles, when

n value\ 0.20, release kinetics would follow combi-

nation of both diffusion and erosion path. These

observations suggest that after initial burst release of

surface-adsorbed thymol, water ingression through

the pores was increased marginally for the porous

particles which favored its release slightly over the

non-porous particles. This might be due to strong

aggregation of thymol in PLA matrix that needed to

be solubilized before being released in pure diffusive

mode. Hence, it did not follow pure Fickian release

mode. This indirectly implied the existence of strong

attractive forces between thymol and polymer matrix.

Moreover, according to our hypothesis of pore for-

mation, pores would be surrounded by hydrophobic

EO layer which may significantly inhibit water

ingression through the pores resulting marginal

increase in release rate. In addition, the low n and

k values implied that the PLA degradation did not

happen significantly within the span of testing per-

iod, otherwise release would have been much accel-

erated resulting in an increased n and k values [41].

Active–polymer interactions: analysisof thermal transitions by DSCand spectroscopic investigations by Ramanmicroscopy

It was evident from the release study that there exists

a strong interaction between thymol and PLA matrix.

In order to investigate the type of interactions

between active and polymer, the following investi-

gations were executed. Figure 3 displays the DSC

heating traces of samples A–E. The second heating

traces were recorded after removing the thermal

history of all the samples at 200 �C. All the experi-

mentally determined values of phase transition tem-

peratures such as Tg (glass transition temperature),

Tcc (cold crystallization temperature) and Tm (melting

point) are displayed in Table SII. Introduction of

thymol (melting point 49 �C) into PLA matrix sig-

nificantly affects the crystallization temperature (Tcc).

However, Tg of all samples remains almost unaf-

fected even after the addition of essential oils,

implying the absence of any plasticization/anti-

plasticization effect induced by thymol and essential

oils [11, 42]. Probably, molten thymol acts as sticky

additive which sticks to polymer melt and decreases

the chain mobility to inhibit crystallization and hence

Table 2 Release kinetic parameters (n, k) and correlation

coefficient (R2) calculated by fitting experimental results (Fig

S2) to Eq. 1

Sample Name n k R2

Sample A 0.174 0.34 0.976

Sample B 0.151 0.437 0.965

Sample C 0.145 0.399 0.965

Sample D 0.149 0.387 0.963

Sample E 0.153 0.373 0.968

Figure 3 DSC thermograms of pure PLA, samples A, B, C, D

and E.

J Mater Sci

significantly lowers the crystallinity due to strong

interaction between additive and polymer chain [43].

Furthermore, the absence of melting peak of crystal-

lites of thymol indicates the inhibitory effect on thy-

mol crystallization induced by PLA matrix due to

appreciable interaction between the two [44, 45]. In

addition to that, thymol- and EO-loaded PLA parti-

cles exhibit one broad melting peaks instead of two

peaks observed for pure PLA samples (because of

polymorphic crystalline transition [46, 47]). This

indirectly implies the inhibition of PLA crystalliza-

tion due to significant attractive forces between active

(primarily thymol) and polymer matrix. To further

investigate about the possible interaction between the

thymol and PLA matrix, Raman microscopy was

carried out for all the samples (Fig. 4). After analyz-

ing all the Raman spectra obtained from samples A–

E, noticeable shift of peaks was observed for both

PLA and thymol portions. For example, carbonyl

stretching frequency of PLA has been shifted from

1772 cm-1 (pure PLA) to * 1767 cm-1 for thymol-

and EO-loaded PLA particles presumably due to

H-bonding between ester group of PLA and hydroxyl

group of thymol. Similarly, shifting of characteristic

peaks from thymol portions such as 957 cm-1 (aro-

matic =C–H stretching from thymol) to 961 cm-1,

1262 cm-1 (–C–O stretching from thymol) to

1273 cm-1 and 1624 cm-1 (aromatic –C=C stretching

of thymol) to 1620 cm-1, respectively, for active loa-

ded particles is clearly evident in Fig. 4. Shifting of

these peak positions definitely indicated the preva-

lence of hydrophobic interactions between the aro-

matic ring of thymol and polymer matrix in addition

to H-bonding between them [48, 49].

Evaluation of antibacterial activity

As per literature information, thymol shows antibac-

terial activity against various Gram-positive and

Gram-negative bacteria [36, 50–52]. Herein, E. coli

(Gram-negative bacteria) was chosen to demonstrate

thymol’s antibacterial activity and MIC (minimum

inhibitory concentration) was determined and found

to be 350 lg/mL. This value was in close agreement

with the literature value reported by Cosentino and

group [36] who found a range from 225 to 450 lg/mL

as MIC for E. coli. In order to demonstrate the

antibacterial activity of thymol-loaded PLA particles,

appropriate concentration of thymol-loaded particles

(samples A–E) was first found out at which complete

inhibition of bacterial growth (E. coli) takes place using

plate spreading method (Fig S5) and summarized in

Table SIII. It was evident from these values that con-

centration of thymol-loaded particles was enormously

high compared to the concentration of free thymol due

to the slow release of thymol in water from PLA par-

ticles within the span of testing period (24 h). Similar

observation was made by Esposti et al. [37], who

demonstrated antibacterial activity of thymol released

from acrylic resin. Interestingly, among all the sam-

ples, thymol along with EO-loaded particles showed

lower particle concentration than that of particles with

no oil (sample A). This definitely implied the syner-

gistic antibacterial action of EOs along with thymol

[53]. Among all the EO employed, olive oil displayed

theminimumparticle concentration and hence sample

E can be considered as the most potent particles in

terms of antibacterial action. As the amount of thymol

released was almost equal in all cases within the span

of testing time (24 h), it can be assumed that the con-

siderable difference of reduction in particle concen-

tration among the samples was primarily originated

from essential oils which are well-known natural

antibacterial even at very low concentration. TheFigure 4 Raman spectra for pure thymol, pure PLA, samples A,

B, C, D and E starting from bottom to top.

J Mater Sci

strong antimicrobial activities in olive oil were mainly

due to the presence of long-chain a,b-unsaturatedaldehydes [54], and the reported MIC value of this oil

ranges from 10 to 60 lg/mL [55, 56] for complete

inhibition of bacterial growth. Similarly, the antimi-

crobial activity of mustard oil was attributed to the

presence of allyl isothiocyanate (AITC) [57], MIC of

which ranges from 50 to 1000 lg/ml for complete

inhibition of bacteria [57, 58]. Furthermore, it was also

reported that castor and coconut oil too have the

antimicrobial property because of the presence of

several fatty acids like Ricinoleic acid [59], lauric acids

[60], etc., and their MIC values were found to be in the

range of 50–250 lg/mL [61] and 60–128 lg/mL [62],

respectively. Therefore, it can be assumed that the

antibacterial effect of these oil-loaded microparticles

was due to synergistic combinations of thymol along

with the small amount of EOs [49]. The prolonged

antibacterial activity was also measured for all the

samples against E. coli over a period of[ 1 week. The

data accumulated from the antibacterial tests were

calculated by the ANOVA and student t tests, keeping

the significant level\ 0.05, 0.01 and 0.001 on various

samples (Fig. 5, Fig S6). Significant reduction in bac-

terial growth was observed for all the samples (except

sample A) till 11 days due to appreciable release of

thymol from porous particles (Fig. 5, Fig S6). More-

over, particles having no EO showed bacterial growth

inhibition only till 5 days due to low release of thymol

(especially after initial burst) in addition to no-oil effect

from non-porous particles (Fig S6). Apart from this,

E. coli was also incubated with the samples having no

thymol (Fig S7) and it was found that the bacterial

growthwas slightly less than the control. These results

implied that the enhanced antibacterial activity was

only shown by those particles which contain both

essential oil and thymol probably due to synergistic

action of the two actives. Overall, it can be inferred that

continuous and most efficient bacterial growth inhi-

bition effect was observed over a week from porous

PLA particles having olive/mustard oil (with low

particle concentration for complete inhibition) along

with thymol loaded into it.

Conclusions

A facile method based on W/O/W double emulsion

technique was developed for the fabrication of thy-

mol-encapsulated PLA-based microparticles. In order

to accelerate the release of thymol inwater, pores were

generated onto the surface of microparticles by

incorporating minute quantity of essential oils (castor,

olive, mustard and coconut) of varying viscosity.

Interestingly, it was realized that the pore density and

pore size can be easily varied by altering the viscosity

of added essential oils, e.g., being themost viscous EO,

castor oil containing particles showed maximum pore

density and pore size. Presumably, stabilization of

inner aqueous phase by essential oil droplet (phase

separated) along with PVA and PLA was considered

to be responsible for the surface pore formation. The

release mechanism of thymol from these variably

porous particles canwell be explainedwith the help of

power lawmodel. Furthermore, the present study also

demonstrated the effectiveness of thymol-loaded

particles entrapped with essential oils as antibacterial

carrier with enhanced inhibitory activity probably due

to synergistic action of thymol and essential oils. Since

the particles made of completely natural ingredients

displayed sustainable antibacterial activity over

10 days, these can be attractive as active food pack-

aging material for prolonging the food shelf life.

Figure 5 Antimicrobial activity of samples A, B, C, D and E

against E. coli expressed in bacterial growth inhibition %, and the

data are processed using ANOVA one-way statistical analysis and

are significant at p value\ 0.001.

J Mater Sci

Acknowledgements

The research leading to these results has received

funding from the Department of Science and Tech-

nology (DST), India, under Extramural Research

Grant: SB/S3/CE/068/2015, and IIT Delhi, New

Delhi, India.

Electronic supplementary material: The online

version of this article (https://doi.org/10.1007/s108

53-019-03593-7) contains supplementary material,

which is available to authorized users.

Compliance with ethical standards

Conflict of interest Authors have no conflict of

interest to declare.

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