Thymol Jounal 2

Folle et al.
J Nanobiotechnol (2021) 19:359

https://doi.org/10.1186/s12951-021-01092-z Journal of Nanobiotechnology
RESEARCH Open Access
Thymol‑loaded PLGA nanoparticles:

an efficient approach for acne treatment
Camila Folle1, Ana M. Marqués2, Natalia Díaz‑Garrido3,4,5, Marta Espina1,6, Elena Sánchez‑López1,6* ,
Josefa Badia3,4,5, Laura Baldoma3,4,5, Ana Cristina Calpena1,6 and Maria Luisa García1,6*
Abstract
Background: Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually
treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we devel‑
oped polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant
properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic
efficacy of this novel nanostructured system were assessed.
Results: Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around
− 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate
penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity
against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin
microbiota. Additionally, the stability and sterility of developed NPs were maintained along storage.
Conclusion: TH-NP showed a promising and efficient alternative for the treatment of skin acne infection, avoid‑
ing antibiotic administration, reducing side effects, and preventing microbial drug resistance, without altering the
healthy skin microbiota. Additionally, TH-NP enhanced TH antioxidant activity, constituting a natural, preservative-free,
approach for acne treatment.
Keywords: Acne, Thymol, PLGA nanoparticles, Skin delivery system, Cutibacterium acnes, Skin microbiota,
Antimicrobial, Antioxidant
*Correspondence: [email protected]; [email protected]

1
Department of Pharmacy and Pharmaceutical Technology and Physical
Chemistry, Faculty of Pharmacy and Food Sciences, University
of Barcelona, 08028 Barcelona, Spain
Full list of author information is available at the end of the article
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Folle et al. J Nanobiotechnol (2021) 19:359 Page 2 of 21
Graphical Abstract
Background preventing colonization of pathogenic bacteria such as

Acne is a common skin disorder, known as Acne vulgaris, Staphylococcus aureus [4]. Concerning Cutibacterium
that affects a large number of the population. Several fac- acnes, previously known as Propionibacterium acnes, it is
tors, such as hormones, diet, stress and environmental a resident bacteria, mainly located surrounding the hair
pollution, among others, may contribute to acne devel- follicle, which is likely to proliferate under unbalanced
opment. These factors trigger hyperactivity of sebaceous function of the sebaceous glands, leading to acne devel-
glands that produce elevated levels of sebum, hyperkera- opment, swelling and inflammation [5]. Hence, C. acnes
tosis by blockage of the hair follicle and, additionally, has a dual activity on the skin microbiota, being a non-
contribute to the excessive microbiota reproduction [1]. pathogen essential for sebum control, as well as an active
The skin is divided into three functional layers that pathogen on acne infection and inflammation. Addition-
surround the hair follicle. The base is found on the der- ally, it maintains the acidic pH of the pilosebaceous folli-
mis–hypodermis junction, just above the fat tissue. Pro- cle by hydrolyzing sebum triglycerides and via propionic
duction of sebum occurs in the sebaceous gland, which acid secretion. Moreover, it has been previously stated
is located inside the dermis. The upper layers of the epi- that acne might be the result of an unbalanced equilib-
dermis are composed by keratinocyte cells surrounding rium between C. acnes and S. epidermidis [6]. Therefore,
the hair top-to-bottom. The external skin layer, stratum a suitable treatment for acne should provide good anti-
corneum (SC), protects the skin from the external envi- microbial activity but acquiring mild effect to the healthy
ronment, being considered the main challenge for several microbiota.
topical drugs penetration. Nanoparticles (NP) are a good approach to enter the
Several microorganisms reside in the skin and can hair follicle and release the antimicrobial agent directly
be classified as resident or transient microbiota. They in the acne lesion [7, 8]. The fat deposit on the subcu-
are normally gram-positive bacteria, not regarded as taneous tissue may behave as a deep compartment for
pathogens, which survive longer on intact skin than the drug, delaying its entry in the blood circulation
gram-negative transient species. The protective micro- [9]. Polymeric nanospheres of poly(lactic-co-glycolic)
biota functions are believed to confer microbial antago- acid (PLGA), form a matrix structure containing the
nism activity and nutrient competition for the stability entrapped active compound. PLGA, approved by the
of the dermal ecosystem, preventing the adherence of Food and Drug Administration (FDA), is known to be
pathogens and maintaining the skin health balanced [2]. safe for dermal applications due to its bioavailability and
Staphylococcus epidermidis is the most abundant colo- biodegradable profile. One of the advantages of PLGA
nizer of human skin and, despite considered a benign NPs entrapping highly volatile compounds is that their
microorganism, it is highly present in acne lesions [3]. It production can be performed at room temperature.
is ubiquitously found mainly concentrated on the upper
layers of the skin and may have a probiotic function by

Moreover, nanotechnology also has several advantages in membrane will lead to cell disfunction and apoptosis,
dermal drug delivery since small particle diameters tend resulting in loss of intracellular contents. Some authors
to penetrate into the deep skin (DS), withdraw the drug suggested that TH antibacterial action is due to the
in a controlled manner and be mainly retained in the increased permeability of bacterial cell membranes [24].
deeper layers [8, 10]. The size and flexibility of PLGA NPs However, other studies suggest that TH is responsible
enable entry into host cells via endocytosis, thus allowing for the inactivation of enzymes implicated in synthesis of
intracellular release. In addition, they are easily phagocy- structural constituents [14]. The extent of bacteria mem-
tosed by host phagocytes [11]. brane damage induced by a compound could be related
In this area, several clinical assays highlight the suc- to its intrinsic hydrophobicity. In the other hand, the
cessful combination of nutraceuticals/cosmetic com- slight hydrophilicity could enhance diffusion through the
pounds and their encapsulation in nanostructured extracellular polysaccharide matrix and cause destabili-
systems for topical acne treatment. In this area, Abd- zation of bacterial biofilms [16].
Allah and colleagues showed successful reduction of acne With the aim to increase and prolong skin penetration
inflammatory lesions with nicotinamide loaded chitosan into the hair follicle, without affecting the entire micro-
nanoparticles [12]. Furthermore, quercetin has also been biota, PLGA NP containing TH (TH-NP) have been
encapsulated into nanovesicles and proven to be effective developed and optimized using the design of experi-
both as antibacterial and anti-inflammatory [13]. ments (DoE) approach. Physicochemical characterization
Among the wide range of nutraceutical compounds, and biopharmaceutical behavior of optimized TH-NPs
thymol (TH), is a multifunctional monoterpene of aro- have been determined. Cytotoxicity, cellular uptake and
matic phenolic structure, with a volatile compound with antioxidant activity of TH-NP have also been assessed in
a strong and characteristic odor. It can be found natu- human keratinocytes cell-line (HaCaT). In addition, anti-
rally occurring in plant extracts or on its white crystalline microbial therapeutic efficacy of this colloidal system was
synthetic form. It is found in Lamiaceae plant species, evaluated in vitro and ex vivo.
especially oreganos and thymes, which present antimi-
crobial, antioxidant and antiseptic properties [14–17]. It Results
is considered safe in cosmetic formulations up to 0.5%, Formulation characterization and optimization
according to the Cosmetic Ingredients Review (CIR) and The optimization of TH-NP was obtained by developing
it is used as preservative in cosmetics and foods [18]. a full composite factorial design of five levels and three
Moreover, TH antioxidant and antimicrobial properties factors. Studied independent variables were the amount
allow cosmetic products to avoid the use of other chemi- of TW and TH as well as pH formulation. The latter was
cal compounds as preservatives. The effects of TH are chosen due both to Thymol pKa (pKa 10.6) which, as pre-
largely attributed to its antioxidant properties, via free viously reported in other studies, can influence in the EE
radical scavenging thus enhancing endogenous anti- [25, 26].
oxidant activities and chelation of metal ions [19]. The The results of the DoE physicochemical characteriza-
antioxidant activity provides an interesting therapeutic tion and the entrapment efficiency of TH-NPs are shown
approach to restore skin homeostasis, maintaining its in Table 1. The average particle size (Zav) values ranged
internal conditions relatively constant and stable, modu- from 162 to 235 nm, being the polydispersity index (PI)
lates the stratum corneum SC barrier function and pre- comprised between 0.06 and 0.12. Based on the criteria
vents skin irritation [20]. for monodispersed systems (PI < 0.1), all the formula-
Bacterial survival depends on membrane lipid homeo- tions presented homogeneity [27]. The surface charge of
stasis and the ability to adjust the lipid composition of TH-NP, measured as Z-potential (ZP), ranged from − 22
bacterial cells in different environments. There are bio- to − 31 mV. This negative ZP is associated with negative
chemical processes that underlie adjustments and are surface charge associated with PLGA, the main NPs com-
responsible for membrane phospholipid homeostasis in pound [25, 28–30]. Moreover, ZP is related to the stabil-
bacteria, controlling the movement of substances across ity of colloidal dispersions, for this reason, the developed
the cell membrane [21]. These processes depend on pro- formulations with values closest to − 30 mV, were con-
teins embedded in a lipid matrix that closely approxi- sidered the most stable. The encapsulation efficiency (EE)
mates a phospholipid bilayer. of designed formulations ranged from 71 to 83%.
graphics® software, are shown in Fig. 1. The statistical

The hydrophobic nature of TH facilitates its interac- Surface response charts of the DoE, performed by Stat-
tion with the lipidic bacterial membrane via direct bind-
ing with biomolecules, such as proteins, providing strong analysis (ANOVA) only presented significant differences
antimicrobial effect by altering its morphology and lead- for the particle size (p < 0.01), influenced by both, pH of
ing to bacterial death [22, 23]. Disruption of bacterial the aqueous phase and ratio of surfactant/pH (Fig. 1A).

Table 1 Values of the 2 3 + star central composite factorial design, parameters, and responses
Independent variables Dependent variables/responses
TH TW pH Zav (nm) PI ZP (mV) EE (%)
Factorial points
F1 −1 −1 −1 217.9 ± 1.2 0.112 ± 0.001 − 30.7 ± 0.9 76.35 ± 1.53
F2 1 −1 −1 234.9 ± 2.1 0.093 ± 0.004 − 29.9 ± 0.4 79.16 ± 0.18
F3 −1 1 −1 176.2 ± 1.0 0.075 ± 0.023 − 27.7 ± 1.3 76.67 ± 2.17
F4 1 1 −1 174.0 ± 0.6 0.081 ± 0.012 − 28.3 ± 0.8 80.07 ± 3.65
F5 −1 −1 1 162.0 ± 0.4 0.072 ± 0.011 − 26.2 ± 0.3 79.64 ± 3.79
F6 1 −1 1 163.5 ± 1.4 0.071 ± 0.009 − 23.6 ± 0.5 73.52 ± 1.83
F7 −1 1 1 183.9 ± 0.4 0.087 ± 0.020 − 29.5 ± 0.1 78.41 ± 2.51
F8 1 1 1 172.4 ± 1.1 0.094 ± 0.009 − 26.6 ± 0.3 76.60 ± 5.60
Axial points
F9 1.68 0 0 174.2 ± 0.6 0.061 ± 0.018 − 25.2 ± 0.4 77.01 ± 3.07
F10 − 1.68 0 0 176.7 ± 1.1 0.083 ± 0.033 − 23.6 ± 0.3 73.55 ± 2.93
F11 0 1.68 0 187.1 ± 0.8 0.024 ± 0.006 − 23.1 ± 0.6 71.38 ± 0.62
F12 0 − 1.68 0 167.4 ± 2.1 0.046 ± 0.021 − 26.1 ± 0.4 76.92 ± 2.47
F13 0 0 1.68 164.6 ± 1.1 0.057 ± 0.010 − 23.1 ± 0.8 72.44 ± 1.66
F14 0 0 − 1.68 202.6 ± 3.5 0.063 ± 0.045 − 22.5 ± 1.1 82.89 ± 6.01
Central points
F15 0 0 0 175.4 ± 2.1 0.053 ± 0.013 − 24.5 ± 0.6 74.94 ± 1.77
F16 0 0 0 176.3 ± 1.9 0.072 ± 0.016 − 25.3 ± 0.7 78.14 ± 0.49
The responses at a fixed TH concentration (2.5 mg/mL) where TH-NP sedimentation was observed by the first
are illustrated for Zav, ZP and EE (Fig. 1B–D), respec- left peak at the bottom of the vial, being reversible by agi-
tively. Results show that the highest EE was achieved at tation. Moreover, it can be observed that at 37 °C the sig-
the lowest pH, while for ZP, the absolute high values were nal greatly decreases, presenting TH-NP destabilization
reached when the pH and surfactant were simultane- at higher temperatures. Additionally, EE was maintained
ously low or high. Considering all the evaluated param- at 4 and 25 °C for 6 months, whereas at 37 °C, decreased
eters, formulation F4, containing 0.25% of TH and 0.4% by 2.5-fold from the initial value. The parameters of all
tween 20 (TW) has been optimized to carry out further storage conditions were within stable criteria, presenting
experiments. better short-term stability when stored at 4 °C. Moreover,
samples presented no microbial growth within storage,
Morphology and stability of TH‑NP confirming the preservative effect of TH.
The morphology of TH-NP was evaluated by trans-
mission electron microscopy (TEM) and it is shown in
Fig. 2A. Moreover, TH-NP maintained their structure for Interaction studies
1 month at 4 and 25 °C and additionally, for 12 months The interactions between TH and PLGA, carried out by
at 4 °C (Fig. 1 of SM). A small particle aggregation takes differential scanning calorimetry (DSC) and X-ray dif-
place after 12 months, which was confirmed by a slightly fraction (XRD), are shown in Fig. 2. DSC thermograms
increased particle size, as indicated in Table 2. of TH showed an endothermic peak at 52 °C, which cor-
This slight aggregation is also related with the decrease responds to its melting transition (Fig. 2B). A minimal
of ZP, since electrostatic forces between surface-charged displacement of endotherm was presented by polymer
NPs decrease when stored in aqueous media. Tem- alone and blank NPs (B-NP). However, TH-NP presented
perature showed to accelerate particle destabilization an onset peak displaced at 40 °C, due to TH-PLGA inter-
by decreased ZP. A slight decrease of the pH value was action. The XRD diffractograms (Fig. 2C) show TH on
also observed, probably due to a partial hydrolysis of the its crystalline form, expressed by the sharp diffraction
polymer. peaks. TH-NP could be observed as non-sharp peaks,
All these phenomena are in accordance with the pre- confirming that TH was dispersed in the polymer matrix
dicted backscattering profile shown in Fig. 2 of SM,

Fig. 1 Factorial design with TH-NP fixed at 2.5 mg/mL TH: A Pareto’s chart for particle size (ANOVA) and surface response for B Zav particle size
(nm), C ZP (mV) and D EE (%)
in its amorphous form (molecular dispersion) and also skin barrier disorders. The corresponding kinetics of
showing a similar profile to B-NP. both skin types are shown in Table 3. The permeation
flux (J) of TH and TH-NP were increased by 2.1- and 2.6-
In vitro drug release fold, respectively, on damaged compared to healthy skin,
The in vitro release profile of TH from TH-NP against where all samples presented significant statistical differ-
free TH, carried out in Franz diffusion cells, is shown ences (p < 0.001) between them. In both cases, damaged
in Fig. 3A. As can be observed, the release of free TH and healthy skins, TH presented significantly (p < 0.001)
through the dialysis membrane was faster, while TH-NP faster penetration rate compared to TH-NP, increased
provided a slow-rate prolonged release. Kinetic data was by 1.6 and 1.3-fold, respectively. The total amount pen-
adjusted to Boltzmann Sigmoidal equation showing that etrated (Ap) was significantly higher (p < 0.001) in dam-
TH reached 50% (V50) of total amount released within a aged skin. In the other hand, the total amount retained
short period of time (1.5 h), while TH-NP only achieved inside the skin (As) was similar for both samples compar-
the same within 23 h. The total amount of TH released in ing healthy to damaged skins and there were significant
24 h was 55% and 35% for TH and TH-NP, respectively, differences (p < 0.001) comparing TH-NP with TH.
presenting statistically significant differences (p < 0.01). The total amount of thymol penetrated within 24 h
(Fig. 3B), was split into stratum corneum (SC) and deep
skin (DS). The amount found in the SC was the same for
Ex vivo skin permeation TH on both skin types and similarly to TH-NP on dam-
The ex vivo skin permeation of TH and TH-NP were aged skin. However, for TH-NP, the retained amount
performed in healthy skin and, additionally, in damaged was significantly higher (p < 0.01) on normal skin, in
skin, where the SC was previously scratched to mimic agreement with the slow-rate penetration, presenting

Fig. 2 A Transmission electron microscopy image of TH-NP. Scale bar: 200 nm, B DSC thermograms of TH-NP, B-NP, and compounds separately, C
X-ray diffraction patterns of TH-NP, B-NP, and compounds separately

Table 2 Physicochemical stability of TH-NP stored at different cell membrane and the nucleus are represented as green
temperatures (4, 25 and 37 °C) measured at 0, 1, 3, 6 and and blue, respectively. In the merged images, it can be
12 months observed that the internalized nanoparticles were mainly
t (months) Zav (nm) PI ZP (mV) pH localized in the cytoplasm.
0 158.8 ± 1.7 0.068 ± 0.027 − 24.7 ± 1.0 4.20

In vitro antioxidant efficacy in HaCaT cells
4 °C 1 158.6 ± 1.8 0.108 ± 0.046 − 20.1 ± 0.6 4.07
The antioxidant activity of TH and TH-NP, performed
3 159.0 ± 2.1 0.101 ± 0.034 − 17.1 ± 0.1 3.90
in HaCaT cells, was successfully achieved by reducing
6 168.6 ± 1.7 0.152 ± 0.033 − 15.3 ± 0.1 3.68
the amount of reactive oxygen species (ROS) generated.
12 204.5 ± 1.3 0.231 ± 0.015 − 11.1 ± 0.7 3.61
While B-NP did not present activity, TH and TH-NP
25 °C 1 162.3 ± 0.4 0.119 ± 0.015 − 19.1 ± 0.1 3.91
showed a 20% and 32% of ROS reduction, respectively,
3 182.3 ± 1.4 0.145 ± 0.020 − 10.3 ± 0.2 3.64
within 2 h treated with H 2O2 (Fig. 4C). Moreover, TH-NP
37 °C 1 177.4 ± 1.2 0.128 ± 0.012 − 15.3 ± 0.6 3.32
was statistically significant compared to TH.
3 216.4 ± 2.8 0.205 ± 0.001 − 8.56 ± 0.3 2.95
In vitro antimicrobial efficacy

delayed entry of the particles. In the other hand, it can be The minimal inhibitory concentration (MIC) was deter-
observed that TH-NP presented significantly (p < 0.001) mined for TH and TH-NP on S. epidermidis being both
higher amounts retained in DS on both skin types, com- 512 µg/mL. For C. acnes, TH and TH-NP displayed
pared to TH. Meanwhile, TH-NP on damaged skin was the same MIC and minimal bactericidal concentration
significantly higher than normal skin (p < 0.01). (MBC) values, being 250 µg/mL and 400 µg/mL, respec-
Ex vivo skin permeation route was studied by con- tively. Therefore, no differences were observed in the
focal microscopy using rhodamine-labelled TH-NP concentrations between samples. However, there were
(R-TH-NP) after 24 h of permeation (Fig. 3C). The results relevant differences between different bacterial strains.
obtained showed that R-TH-NP successfully penetrated In the other hand, clindamycin, a strong antibiotic com-
inside the skin hair follicle, where acne pathogen infec- monly used to treat severe acne, presented MIC < 2 µg/
tion and inflammation occur. The image (Fig. 3C_II) illus- mL for both microorganisms. For this reason, clinda-
trates that R-TH-NP were found concentrated in the hair mycin is able to treat acne. However, it also affects the
follicle and presented delayed entry accumulation in the healthy resident bacteria of the skin.
SC. The bacterial viability reduction, evaluated by determi-
nation of decimal reduction time (D), treated with TH or
TH-NP in a timely manner, is illustrated in Fig. 5A. In the
Cytotoxicity in HaCaT cells case of C. acnes, the decrease of viable bacteria correlated
The cytotoxicity of TH-NP was evaluated on HaCaT with the applied dose. Although the effect of TH and
cells, incubated for 24 h with concentrations up to 1 mg/ TH-NP were similar, the activity of TH-NP was slightly
mL (Fig. 4A). Results showed that TH-NP was not cyto- sustained at lower dosages. At the MIC concentrations,
toxic at concentrations up to 50 µg/mL, as cell viabil- they present minimal reduction activity, whereas, at con-
ity was kept close to the untreated control cells. A 20% centrations higher than MBC, the reduction was boosted
reduction in cell viability was observed at 100 µg/mL and for all tested samples. Meanwhile, S. epidermidis pre-
close to 90% reduction at concentrations ≥ 250 µg/mL. sented very slow viability reduction when incubated
Different results were obtained with washed nanopar- with TH and TH-NP at twice the MIC (Fig. 5B), the
ticles (TH-NP-w), which were not cytotoxic at 100 µg/ same highest concentration tested for C. acnes. It can
mL and caused only a 25% reduction in cell viability at be observed that TH completely reduced S. epidermidis
250 µg/mL. Differences between TH-NP and TH-NP- viability within 8 h, whereas TH-NP treated cultures still
w indicate that the presence of free TW in the samples presented living colonies within 24 h.
could cause toxicity to HaCaT cells. Data of decimal reduction time (D), the time taken to
reduce a decimal part of the bacterial viability, is shown
Cellular uptake of TH‑NP in Table 4. In the case of C. acnes, differences could be
The cellular uptake of R-TH-NP (20 µg/mL) was analyzed observed comparing the variable dosages applied. How-
in HaCaT cells. At this nanoparticle concentration, cell ever, variations between TH and TH-NP were not
viability was over 90%. After 2 h incubation, fluorescence detected. In the case of S. epidermidis, TH-NP presented
was detected by confocal microscopy in cells treated with statistically significant differences compared to TH.
R-TH-NP but not in untreated control cells (Fig. 4B). The Moreover, comparing the activity between both micro-
organisms, at the same dosage, only TH-NP presented

Fig. 3 A Release profile of TH and TH-NP adjusted to Boltzmann Sigmoidal equation. Total amount released in 24 h expressed as % Mean ± SD
(n = 3). Statistical analysis one-way ANOVA t-student test *p < 0.01, B Total amount of TH and TH-NP penetrated in 24 h in healthy and damage skin.
SC: stratum corneum (tape stripping), DS: deep skin (extraction). Values represent the Mean ± SD (n = 3). Statistical analysis for each localization via
one-way ANOVA Tukey’s Multiple Comparison Test. Different letters inside the bars indicates significant differences between groups (*p < 0.01 and
**p < 0.001), C Confocal microscopy images of pig skin R-TH-NP penetration after 24 h: I untreated skin control, II SC and hair follicle, III hair follicle
cross-section
statistically significant differences for S. epidermidis TH-NP resulted on elongated cells, thickened cell enve-
against C. acnes. lope, and blebs formed on the surface.
Moreover, the structure of C. acnes (Fig. 5B), evalu-
ated by scanning electron microscopy (SEM), presents a
rod-shape and smooth membrane. Treatment with TH or

Table 3 Ex vivo skin permeation parameters

Healthy skin Damaged skin
TH TH-NP TH TH-NP
J (μg/cm2/h) 12.68 ± 1.73 8.03 ± 0.55 a 26.43 ± 2.13 a b d 21.03 ± 0.92 a b

Kp (cm/h) 5.07E−03 ± 6.91E−03 3.21E−03 ± 2.19E−04 a 1.06E−02 ± 3.51E−04 a b d 8.41E−03 ± 3.69E−04 a b
Ap (μg/cm2) 106.43 ± 9.96 96.39 ± 12.68 272.36 ± 14.02 a b d 202.58 ± 11.65 a b
As (μg/cm2) 6.19 ± 1.45 10.85 ± 1.12 a c 5.92 ± 0.92 10.83 ± 2.13 a c
At (μg/cm2) 112.61 ± 11.41 107.24 ± 13.80 278.28 ± 14.93 a b d 213.78 ± 13.78 a b
SSD (p < 0.01) a b c d
J: flux, Kp: permeability constant, Ap: total amount penetrated, As: total amount retained inside the skin, At: total amount penetrated and retained inside the skin
Ex vivo antimicrobial efficacy less amount of cellular leakage could be observed. The
The ex vivo antimicrobial efficacy of TH and TH-NP minor effects of TH-NP within 8 h of treatment might be
on C. acnes skin inoculated, as prevention or treatment related to the slow-rate release and penetration.
for 24 h, were successfully determined. In both studies,
all samples presented significant differences against the Discussion
control (**p < 0.001). The activity was found greater as In the present study, TH was successfully loaded into
prevention than treatment (Fig. 6A), where TH-NP pre- PLGA NP. Developed TH-NP presented suitable phys-
sented higher activity than TH, but no statistically signifi- icochemical parameters with excellent stability and
cant differences were observed. In the other hand, for the high EE. DoE analysis showed that Pareto’s diagram
dose-dependent treatment (Fig. 6B), administration of a and surface responses indicated that only the pH of
single dose showed significant differences between TH the aqueous phase and the ratio of pH/TW induced
and TH-NP (#p < 0.05). statistically significant differences on Zav, being the
Although TH-NP had lower effectiveness within one effect of these variables not significant on the EE. The
dose, they performed greater activity than TH when 2 surfactant usually shows statistically significant influ-
or 3 doses were applied ($$p < 0.001). Meanwhile, TH did ence on the EE and morphometry of polymeric NP,
not show statistical differences comparing the 3 dosage depending on the amount applied [27]. TW has good
protocols. Moreover, the highest efficiency of the experi- permeability profile and its amphiphilic properties may
ment was achieved by 3 doses of TH-NP. control the interactions between the active compound
The simulation of skin infection and treatment was car- and the biopolymer carrier [31]. Interaction studies
ried out on ex vivo fresh human skin explants, by inocu- showed that encapsulated TH was present on its amor-
lating C. acnes for 16 h, followed by 8 h treatment with phous form and the thermal transition was affected by
TH or TH-NP. Morphology of C. acnes can be observed TH-polymer interactions [25]. Analyzing the stability
in Additional file 1: Figure S3. Moreover, Figure 6C illus- of TH-NP for the first 3 months, an increase of parti-
trates the untreated skin and the C. acnes inoculated and cle size and a decrease in ZP, depending on the stor-
penetrated within the skin layers. The treatment with TH age temperature, were observed. The decrease of ZP
demonstrated a fast and strong activity towards the bac- induced TH-NP instability in aqueous medium, due
teria membrane, presenting surrounding it, a great loss to a decrease of electrostatic forces between surface-
of intercellular material which may indicate damaged charged NP, generating in some cases, a slight particle
membrane. In the case of TH-NP, a slower effect with aggregation. Moreover, TH-NP at high temperature,
(See figure on next page.)

Fig. 4 A Cell viability of HaCaT keratinocytes after incubation for 24 h with TH-NP and the washed NPs (TH-NP-w) at different concentrations.
Cell viability was assayed by the MTT reduction method; 100% viability was set with the values obtained with the untreated control cells. Values
represent the Mean ± SD (n = 3). Statistical analysis one-way ANOVA Tukey ‘s Multiple Comparison Test, *p < 0.01 versus control and $p < 0.01 versus
TH-NP-w, B Confocal microscopy analysis of HaCaT cells incubated with R-TH-NP: (I) nuclei; (II) membranes; (III) R-TH-NP; (IV) merged (I), (II), and
(III), respectively; (V–VIII-h) 3D-plot of (I)-(IV), respectively, C Time course analysis of ROS production induced by H 2O2 (2 mM) in HaCaT cells treated
with TH or TH-NP. The ROS production (100%) was set with the values obtained with cells challenged with H 2O2 for 2 h. Data were expressed (%) as
the Mean ± SD (n = 8). Statistical analysis was performed by one-way ANOVA Tukey’s Multiple Comparison Test, **p < 0.001 versus controls at 2 h;
$
p < 0.05 and $$p < 0.001 between TH and TH-NP treated cells

Fig. 4 (See legend on previous page.)

Fig. 5 A Bacterial viability reduction of (I) C. acnes 6 h contact with TH or TH-NP (dose dependent) at 0.25, 0.5 and 1 mg/mL, (II) S. epidermidis 48 h
contact with THF or TH-NP at 1 mg/mL. Data is expressed as log10CFU of mean values, B SEM micrographs of C. acnes (I) control and treated for 1 h
with (II) TH or (III) TH-NP. Black arrows indicate bacteria division and white arrows membrane disruption
presented sedimentation that was reversed by agitation, microbial contamination along storage, confirming TH
as also observed by other authors [32]. TH-NP stored antimicrobial preservative activity.
at 4 °C have shown outstanding short-term stability in Biopharmaceutical behavior of TH-NP presented a
aqueous solution. However, in order to extend TH-NP sustained in vitro release profile, while TH reached the
stability, either incorporation in a semi-solid formula- steady state very fast. The ex vivo skin rate of TH was
tion or freeze-drying are recommended [27, 33]. In higher than TH-NP by 1.6 and 1.3-fold, respectively,
addition, it has been shown that TH-NP did not present being faster on damaged than healthy skin. As previously
described [34], these results confirm that the external

Folle et al. J Nanobiotechnol
(2021) 19:359
Table 4 Decimal reduction time (D) for C. acnes and S. epidermidis viability
TH (mg/mL) TH-NP (mg/mL)
0.25 0.50 1.00 0.25 0.50 1.00
C. acnes
r2 0.9654 0.9743 0.9812 0.9964 0.9984 0.9949
k (logCFU/h) 1.00 ± 0.11 2.03 ± 0.19 3.48 ± 0.08 1.02 ± 0.12 1.96 ± 0.43 3.44 ± 0.03
D (min) 60.5 ± 6.5 29.6 ± 2.7 17.3 ± 0.4 59.0 ± 7.0 31.4 ± 6.9 17.4 ± 0.1
S. epidermidis
r2 0.9944 0.9886
k (logCFU/h) 1.75 ± 0.02 0.54 ± 0.01
D (min) 34.2 ± 0.4 111.1 ± 2.9*$
CFU: colony forming units. One-way ANOVA (t test): statistically significant differences *p > 0.001 versus TH, $p > 0.0001 against C. acnes, same dosages

Page 12 of 21
Fig. 6 A Bacterial viability on ex vivo treated skin with TH or TH-NP prevention, B Bacterial viability on ex vivo treated skin with TH or TH-NP
dose-dependent treatment with 3 applied doses at times 0, 12 and 18 h of incubation. Values represent viable count of C. acnes as the Mean ± SD
(n = 3), C TEM images of normal human ex vivo skin: (I) untreated, (II) inoculated with C. acnes (black), treated with (III) TH and (IV) TH-NP. White
arrows indicate the loss of bacterial intracellular material. Scale bars: 2 µm (I), 1 µm (II) and 500 nm (III and IV). Statistical analysis one-way ANOVA
Tukey’s Multiple Comparison Test represent **p < 0.001 compared to control and $p < 0.01/$$p < 0.001 comparing TH and TH-NP.
hydrophobic skin surface was altered, thus enabling sub- kind of compounds are known to be skin penetration
stances to penetrate faster and easier through this barrier. enhancers, by impairing the lipids of the SC [35]. On
The enhanced penetration of TH and TH-NP through the other hand, TW, an anionic surfactant, also provide
the skin may be due to TH properties as well as the TW enhanced permeability through phospholipid mem-
properties. On the one hand, TH is a terpene and these branes, inducing some damage to epidermal membranes

which decrease skin resistance towards the diffusion of In the HaCaT cell line, TH-NP at low concentrations
the active. Some authors also explained that the mecha- did not alter cell viability, presenting no cytotoxicity.
nisms of TW could be attributed to an improvement of The cellular uptake images showed most of the NPs in
the thermodynamic activity adsorption and fusion due to the cytosol but also some particles reached the nucleus
micellar complexation, or decreasing the SC hindrance within only 2 h. This would enable TH-NP to perform its
or modification of its intracellular lipid barriers [36]. The antioxidant activity inside the cells to improve the skin
amount of TH retained inside the skin was higher for healing process on acne lesions [20]. This activity was
TH-NP in both (healthy and damaged) skin types. The confirmed by reducing the ROS generated, since TH-NP
only difference between healthy and damaged skins was improved significantly compared to TH. Moreover, pro-
the highest amount in the SC and DS, respectively. The longed release of TH as well as increased stability of
lesser amount of free TH against TH-NP retained inside TH-NP may also favor enhanced antioxidant activity.
the skin was probably due to its fast-rate penetration. In The antimicrobial activity of TH and TH-NP against C.
agreement with TH-NP slow-rate penetration, confo- acnes was similar and successfully demonstrated in vitro
cal microscopy study confirmed that TH-NP presented and ex vivo. The in vitro activity increased with high con-
delayed entry, accumulating into the layers of the SC. This centrations of TH-NP, and they presented the decimal
means that when the SC was disrupted, the flux of NPs reduction of bacterial viability within 60, 30 and 17 min,
penetration improved, and therefore, the amount inside for MIC, 2 × MIC and 4 × MIC, respectively, confirm-
the deeper layers of the epidermis and dermis increased. ing dose-dependent activity. Additionally, the concen-
Interestingly, TH-NP skin penetration route was through trations used above MBC completely reduced all CFU
the hair follicle, exactly where acne occurs. According to very fast. For the ex vivo experiments, simulating an acne
previous authors [37], the physicochemical properties of infection inside the skin explant, samples resulted more
the active compound as well as the barrier properties of efficient for prevention than treatment, despite, no sig-
the hair follicles define the penetration route (intrafol- nificant differences between samples could be observed.
licular or transfollicular). Polymeric NPs preferentially This can be explained by the fact that part of the amount
accumulate in the follicular entry, in a time dependent of the formulations applied were retained on the SC, and
manner, where the skin penetration through the hair fol- therefore, products had a direct contact with C. acnes
licle is size dependent [8]. In this way, particles with aver- when this was inoculated onto the skin. Meanwhile TH
age diameter of 200 nm are likely to penetrate faster than penetrates faster throughout the skin, providing only
micro-sized particles or free molecules. Therefore, the an immediate effect. For TH-NP, the effectiveness was
smaller the particle size, the higher would be the accu- higher when multiple dosages were applied onto the skin
mulation into the hair follicle, and thus achieving lower providing a slightly sustained effect. These results are in
permeability rates [8, 37]. Studies carried out by Yukuy- accordance with those observed in the biopharmaceuti-
ama et al. [38], indicated that NP stored in the hair fol- cal studies. The same behavior was observed by electron
licle will be cleared by hair growth or sebum production. microscopy in the ex vivo images, where the effect of
Zhu et al.[39], developed PLGA TH microparticles as TH on the bacterial membrane was found stronger than
antimicrobial agents for food preservative application, TH-NP, for 8 h contact inside the skin. For this reason, it
containing particle size ranging for 20 to 70 µm. Due to could predictably act in the same way on skin microbiota
the fact of the particle size selectivity for follicle entry, and therefore, TH-NP would constitute a less aggressive
our developed TH-NP would be more efficient for pen- and more efficient system for such treatment. The in vitro
etrating the hair follicle to treat acne. Furthermore, other SEM images showed modified cells, with greater wall
authors stated that the reservoir of the hair follicle could thickness and the development of blebs [40]. This obser-
store actives 10 times longer than the reservoir of the vation agrees with the previously mentioned mechanism
SC, and also, that hair follicle under movement (in vivo) of antimicrobial activity, by triggering loss of intercellular
would improve NP penetration [37, 38]. Recent studies nutrients.
confirmed that diffusion of polymeric NPs only crossed Concerning S. epidermidis, a more resistant type of
the SC reaching the viable epidermis only after needle microorganism, it presented very slow viability reduc-
puncture [37]. This could be also observed, by confo- tion in contact with TH and TH-NP, compared to C.
cal microscopy in healthy skin, where TH-NP were not acnes, even tested at the highest concentration. Growth
found beyond the SC, unless inside the hair follicle. For was abolished within 8 h for TH, while for TH-NP this
all these reasons, TH-NP are more efficient for acne effect was observed over 24 h. Therefore, if these NP
treatment due to the prolonged penetration and release were administered in vivo for acne skin treatment, at the
inside the hair follicle. same concentrations, it would be less effective towards
the entire skin microbiota, being at the same time a

strong bactericidal against C. acnes. This favors the desir- showed outstanding antimicrobial activity in vitro and
able microbiota-friendly activity, where the antimicro- ex vivo against C. acnes, with minor effects towards S.
bial ingredient will not alter the good functionality of the epidermidis, which promises to be a great microbiota-
healthy skin, acting efficiently against C. acnes and only friendly candidate for acne treatment. Additionally,
mediating S. epidermidis proliferation. Another proof TH-NP adhered into the SC layers would provide good
of concept was previously stated by other authors [41] protection on the acne lesions against external micro-
providing evidence that microbiota shifts notably during bial aggressors. Moreover, the cellular uptake of TH-NP
puberty, increasing predominance of Cutibacterium spe- has also improved the antioxidant activity in keratino-
cies and decreasing abundance of Staphylococcus species. cyte cells, which would be promising on cell regenera-
Meanwhile, in adulthood it remains unstable due to con- tion on the healing process of the acne lesion. Therefore,
stant external environmental changes, suggesting mutual TH loaded nanostructured system has been successfully
beneficial microbiome-host interactions. As a comple- developed and physicochemically characterized demon-
ment, the MICs showed that the concentration needed as strating excellent properties for acne topical treatment.
bactericidal against C. acnes was lower than the minimal
concentration needed to inhibit S. epidermidis growth. Methods
This means that for in vivo conditions, the desirable
PLGA Resomer® RG 504H (consisting of a carboxylic
Materials
effect for acne treatment would maintain the skin bal-
anced. In the case of clindamycin, the MIC for C. acnes terminal group, molecular weight 38,000–54,000 Da and
and S. epidermidis was confirmed to be strongly efficient molar ratio lactide:glycolide 50:50) was purchased from
at a very low dose. This powerful activity is the main chal- Boehringer Ingelheim (Ingelheim, Germany). Thymol
lenge with this type of drug, since it completely abolishes (TH), Tween 20 (TW) and acetone were purchased from
all the microbial content of the skin, therefore, treating it Sigma-Aldrich (Spain). Milli-Q water was obtained from
by unbalancing the healthy conditions for the microbiota, a double distilled Millipore system. All chemicals and
thus, leading to microbial resistance. For this reason, in reagents used were analytical grade (HPLC).
this study we managed to confirm an effective activity
of a natural active, at higher concentration, but that can Preparation of TH‑NP
maintain the microbiota function to rebalance the skin TH-NP containing a matrix structure (nanospheres) were
conditions, maintaining it healthy upon the treatment. obtained by solvent displacement evaporation, described
From another point of view, since the route of pen- by Fessi et al. [42]. Briefly, an aqueous phase containing
etration of polymeric TH-NP into the skin was through TW and an organic phase were prepared. The organic
the hair follicle, the observed activity will be performed phase was made by dissolving PLGA and TH in acetone,
directly on the acne lesion. Therefore, TH-NP perform and it was added dropwise into the aqueous phase under
a protective effect on the healthy skin microbiota, along continuous stirring. Finally, in order to evaporate the
with extending the retention and release of TH, directly organic solvent, a rotatory evaporator (Buchi, Switzer-
to the site of the infection with prolonged activity. land) was used at 30 °C under constant pressure, obtain-
Moreover, the sebum content on the hair follicles could ing TH-NP dispersed in water [43–45].
facilitate absorption and release of TH from TH-NP by
hydrophobic interactions. Nevertheless, since PLGA skin
Optimization of TH‑NP
metabolism occurs by biodegradation into its monomers
TH-NP were optimized using the design of experiments
(lactic acid and glycolic acid), these compounds may help
approach (DoE). A full factorial central design of five levels
to modulate the skin pH, which would contribute to the
and three factors was applied in order to reduce the num-
sebum control.
ber of experiments [46]. This experimental design consisted
of 16 formulations with variable factorial points (− 1/ + 1),
Conclusions
axial points (− 1.68/ + 1.68) and central points (0), each
TH was successfully encapsulated into PLGA NPs with
involving 8, 6 and 2 formulations, respectively. The con-
particle size below 200 nm and high EE with suitable sta-
centration of active (TH), surfactant (TW) and the pH of
bility. Moreover, TH-NP solution did not present micro-
the aqueous phase were selected as the independent vari-
bial growth under a storage period of 12 months, due
ables (Table 5). PLGA was fixed to 9 mg/mL for the entire
to antimicrobial properties of TH. Therefore, they can
experiment. The effect of the independent on the depend-
act as natural preservative system. TH-NP presented
ent variables (morphology, z-potential and encapsulation
a sustained release and slow-rate penetration on skin,
efficiency) has been analyzed [47].
through the hair follicle, with higher amounts retained
inside the skin, compared to TH free. Moreover, TH-NP

Table 5 Factors and levels of DoE independent variables Barcelona, Spain). A pan with indium (purity ≥ 99.95%;
Factors Levels Fluka, Switzerland) was used to check the calibration
of the calorimetric system and an empty pan was used
− 1.68 −1 0 1 1.68
as a reference [25]. Samples were heated from 10 °C to
TH (mg/mL) 1.16 1.5 2.0 2.5 2.84 100 °C at 5 °C/min under a nitrogen atmosphere. Data
TW (mg/mL) 1.32 2.0 3.0 4.0 4.68 were evaluated from the peak areas with Mettler STARe
pH 4.32 5.5 6.0 6.5 7.68 V 9.01 DB software (Mettler-Toledo). The physical state
(amorphous or crystalline) of TH and TH-NP was ana-
lyzed by X-ray diffraction (XRD). Samples were sand-
Physicochemical characterization of TH‑NP wiched between 3.6 µm films of polyester and exposed to
Zav and PI were determined by photon correlation spec- Cu K α radiation (λ = 1.5418 Å) with work power (45 kV,
troscopy, using a ZetaSizer Nano ZS (Malvern Instru- 40 mA). Diffractograms were recorded on a PANalytical
ments; Malvern, UK). The surface charge, measured as X’Pert PRO MPD θ/θ, powder diffractometer of 240 mm
ZP, was determined by electrophoretic mobility using the of radius, using PIXcel detector (active length = 3.347°).
same instrument. The morphology of the particles was The measure time was defined 200 s per step, 2θ/θ scans
accessed by TEM (transmission electron microscopy, JEOL from 2 to 60°2θ with a step size of 0.026°2θ [49].
JEM1010, Tokyo, Japan), using Megaview III (Soft Imaging
Systems, GmbH, Münster, Germany). The negative staining Stability of TH‑NP
was carried out with 2% uranyl acetate. The physicochemical stability of the optimized formula-
Quantitative analysis was performed by reverse-phase tion was followed during storage at different conditions:
high-performance liquid chromatography (HPLC) 25 and 37 °C for 3 months and 4 °C for 12 months. The
by a modification of the method described previ- stability was studied by measuring backscattering of
near-infrared pulsed light (λ = 880 nm), bottom-to-top
System with UV detector, using a Kromasil® column
ously [48]. Studies were carried out in Acquity Waters
analyzer Turbiscan®Lab expert (Formulaction, L’Union,

of the turbiscan cell containing TH-NP, using optical
(C18, 5 μm, 150 × 4.6 mm). The mobile phase con-
sisted of acetonitrile:water under gradient conditions of France), to predict the behavior of the NPs in solution.
30:70/58:42/30:70 during 20 min. TH was determined at Additionally, measurements of Z av, PI, ZP and TEM
wavelength of 275 nm. images were also monitored at selected times. The EE
The encapsulation of TH was measured indirectly by stability was also measured at 6 months of storage.
quantification of unloaded amount. Samples were diluted To analyze the microbial preservative activity of TH
1:10 in Milli-Q water:ethanol (90:10) and centrifuged (Cen- during storage, samples stored for 6 months at room tem-
trifuge 5415C, Geratebau Eppendorf GmbH, Engelsdorf, perature and 12 months at 4 °C where used. For direct
measurement, 0.1 mL of each sample was transferred
device (Amicon® Ultra, 0.5 mL 100 K, Merck Millipore
Germany) for 10 min at 14,000 rpm, using Millipore filter
into the plates or, additionally, samples where diluted
Ltd., Carrigtwohill Co. Cork IRL). The filtered fractions 1:10 in neutralizing solution (Berens Cosmetic Dilu-
were quantified by HPLC, and the EE was determined by ent, Scharlab, UK), then 1 mL was transferred into the
Eq. (1): plates. The total viable count was carried out by inclu-
sion on TSA (Tryptone Soy Agar, Oxoid, UK) for bacteria
Ci − Cs or Sabouraud Dextrose Agar (Oxoid, UK) for fungi and
EE = · 100 (1)
Ci yeasts. Plates were incubated at 35 ± 2 °C for 3 days or at
where Ci is the initial concentration of the active and Cs 28 ± 2 °C for 7 days, respectively. This methodology was
is the concentration of the unloaded amount found in the designed based on specifications of the European Phar-
filtered fraction. macopeia monographs (2.6.12. Microbiological examina-
tion of non-sterile products: total viable aerobic count).
Interaction studies
Interaction studied were carried out by previous ultracen- In vitro release
trifugation of the samples at 15,000 rpm during 30 min of The in vitro release of TH from TH-NP against free TH
TH-NPs (Beckmann-Coulter ultracentrifuge). The possi- was carried out using vertical diffusion Franz cells (FDC-
ble interactions between TH and PLGA were assessed by 400, Vidra-Foc, Barcelona, Spain) with a thermal bath set
differential scanning calorimetry (DSC). Thermograms to 32 °C, to mimic skin in vivo conditions, under constant
were obtained on a DSC823e System (Mettler-Toledo, stirring. For this study, methylcellulose membranes (Dial-
ysis Tubing – ViskingCode DTV12000.03.000, Size 3, Inf

Day 20/32″–15.9 mm, MWCO–12–14.000 Da, Liverpool Ex vivo pig skin penetration was obtained from the ani-
Road, London, UK) were placed between donor/recep- mal house (Bellvitge, University of Barcelona), used in
tor compartments (2.54 cm2). Samples of TH or TH-NP accordance with the protocol approved by the Ethics
Committee of the University of Barcelona. For this study,
phase was filled with Transcutol P®:water (50:50), main-
were added to the donor phase (0.5 mL) and the receptor
rhodamine-labelled PLGA (R-PLGA) was synthesized
taining sink conditions. Aliquots of 300 μL were collected as previously described [43]. R-PLGA was used at 0.01%
at selected times, replaced with the same amount of into TH-NP, added in the organic phase with PLGA.
receptor medium [33]. Data were analyzed by HPLC and R-TH-NP were applied onto the ex vivo pig skin 0.64 cm2
(donor compartment) and allowed penetration for 24 h.
model, Eq. (2), using GraphPad®.
processed with the Boltzmann Sigmoidal mathematical
Skin samples were washed, fixed in PBS containing 4%
paraformaldehyde (PFA) for 4 h, followed by cryoprotec-
Bottom + (Top − Bottom) tion into PBS with 30% sucrose for 24 h, snap-frozen in
Y = (2)
Samples were mounted in O.C.T.® Compound (Tis-
(1 + exp( (V50−X)
Slope )
isopentane at − 50 °C, then kept overnight at − 80 °C.
sue-Tek®, Sakura) and sliced with cryostat microtome
frost® Plus (Menzel-Glaser, Thermo Scientific, USA),

Ex vivo skin permeation (LEICA CM3050 S) at − 20 °C onto glass-slides Super-
covered with Fluoromount G® (Invitrogen, Thermo

Ex vivo human skin permeation was carried out by verti-
cal diffusion Franz Cells, using the same methodology as
described above. Human skin was obtained from abdom- Fisher Scientific, USA). Samples were visualized by con-
inal plastic surgery (Hospital de Barcelona, SCIAS, Bar- focal laser scanning microscopy (Zeiss LSM 880), using
celona, Spain), following a protocol approved by the objective lens 10× 0.45. Images were acquired using Zen
Bioethics Committee of the Barcelona-SCIAS Hospital. Black 2.3 software performing z-stack sections and thus
Skin samples (2.54 c m2, 0.4 mm thick) were clamped into processed with ImageJ software.
the Franz cells with the SC facing up [50]. Previously,
some of the skin samples were scratched with sandpa- Cytotoxicity studies in HaCaT cells
per to mimic damaged skin SC. The donor compartment Human keratinocytes (HaCaT) cells were cultured in high
analyzed by HPLC and processed using GraphPad®. The

was filled (0.5 mL) with TH or TH-NP (0.25%). Data were glucose DMEM (Dulbecco’s Modified Eagle’s Medium
(Thermofisher), supplemented with 10% fetal bovine
skin permeation parameters were calculated by Eq. (3): serum (FBS), 2 mM l-glutamine, 100 units/mL penicil-
lin G and 100 µg/mL streptomycin. Cells were incubated
J = Kp · C0 (3) at 37 °C and 5% C O2 and experiments were performed
where J is the flux, Kp is the permeability coefficient and when cells reached 80–90% of confluence.
C0 is the initial concentration of the active [51]. Cytotoxicity of TH-NP was determined by MTT
The amount retained inside the skin was assessed by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
tape striping and skin extraction techniques. Firstly, bromide) assay, by reduction of tetrazolium salt by intra-
skin samples were washed with sodium lauryl sulphate cellular dehydrogenases of viable living cells. TH-NP
(0.02%) and rinsed with distilled water, dried, cut, and were tested at concentrations up to 1 mg/mL. The assay
weighted. For determination of the amount retained in was also performed with TH-NP previously washed
the SC, tape stripping assay was developed based on pre- thrice (TH-NP-w) to remove excess of free TW (cen-
vious authors with minimal modifications [52]. The first trifugation at 14,000 rpm for 15 min). Briefly, HaCaT
layers of the skin were removed by 7 strips of the same cells were seeded in 96-well plates with 100 μL of cul-
ture medium (DMEM) at a density of 2 × 105 cells/well,
Tegaderm®, 6 × 7 cm, 10u, Spain, S.A.). The strips were
region of the SC using a transparent label dressing (3 M
adjusted in automated cell counter (Countess, Invitro-
added into 4 mL of ethanol and placed into an ultrasonic gen, Thermofisher). Cells were incubated with samples
bath (JP, Selecta) for 20 min for compound extraction. for 24 h. Then, the medium was removed and MTT
For determination of the total amount retained inside the (Sigma-Aldrich Chemical Co, St. Louis, MO, USA) was
deeper layers, the rest of the skin was perforated, added added at 0.25% in PBS. After 2 h incubation, the medium
into 2 mL of ethanol:water (50:50) and then kept in the was replaced by 100 µL DMSO (99% dimethyl sulfoxide,
wavelength of 570 nm in a Modulus® Microplate Pho-

ultrasonic bath for 20 min [51]. The amount of thymol Sigma-Aldrich) [53]. Cell viability was then measured at
extracted was determined by HPLC and calculated using
the recovery factor previously obtained. tometer (Turner BioSystems Inc., Sunnyvale, CA, USA).
To determine the permeation route of TH-NP, verti- Results were expressed as percentage of cell survival rela-
cal diffusion Franz cells were used as described before. tive to untreated cells.

Cellular uptake of TH‑NP and indicator (Oxoid, Basingstoke, UK). Prior to each
seeded in an 8-well µ-slide (Ibidi®) following the same

Cellular uptake of TH-NPs was assayed in HaCaT cells experiment, the inoculum was prepared in PBS adjusted
to 0.5 McF. Microbial count of C. acnes was performed in
methodology as described before. Cells were incubated clostridium reinforced medium (CRM) plates, as recom-
with or without R-TH-NP for 2 h at the indicated con- mended for anaerobia growth, incubated at 37 °C.
centration, using FBS/phenol red free medium. Cell The MIC of TH and TH-NP were determined using the
membranes were stained with wheat germ agglutinin broth microdilution assay [55]. Briefly, double concentrated
(WGA) Alexa-488 (Molecular Probes) at 1 µg/mL for sample dilutions were prepared and added (100 μL) to
15 min followed by fixation with paraformaldehyde 3% double concentrated culture medium (100 μL) in a 96-well
for 25 min. Cell nuclei were stained with 4′,6-diamid- polypropylene microtiter plate (Costar, Corning Incorpo-
ino-2-phenylindole (DAPI, Sigma Aldrich, Spain) at rated, Corning, USA). Inocula were prepared to yield a final
0.5 µg/mL for 15 min. Internalization of NPs in HaCaT concentration of 5 × 105 CFU/mL. For S. epidermidis, 10
cells was assessed by confocal microscopy (Leica TCS µL were transferred to inoculate wells with final TH con-
SPII), 63× oil immersion objective lens [43]. Images centrations of 2 to 1024 µg/mL, followed by incubation at
were processed using Fiji image software. 37 °C for 18 to 20 h. For C. acnes, 20 µL was used to inocu-
late wells with concentrations ranging from 2 to 1000 µg/
mL and the plate was incubated at 37 °C for 48 h under
In vitro antioxidant efficacy in HaCaT cells anaerobiosis. Thus, the MBC was performed by transfer-
The antioxidant activity of TH, TH-NP, and B-NP ring 10 µL of each sample presenting no visible growth of
(blank NPs) was assayed in HaCaT cells by quantifica- C. acnes into BHI plates. These were further incubated as
tion of ROS using the fluorogenic probe H2DCFDA. described before. Growth controls were used for the above
Cells were seeded in 96-well plates at 2 × 105 cells/well experiments: presenting antimicrobial sterility (negative)
(100 µL) for 72 h. Cells were loaded with the fluoro- and absent of antimicrobial (positive). Clindamycin was
genic dye H2DCFDA (2′,7′-dichlorodihydrofluorescein also used as an active control for both microorganisms.
diacetate) at 25 µM diluted in DMEM medium absent The determination of the decimal reduction time assay,
of phenol red and FBS, for 45 min in the dark. This explores the antimicrobial activity of TH and its deriva-
fluorogenic dye passively diffuses into the cells, being tive TH-NP on reducing bacteria viability at determined
deacetylated by intracellular esterase and emits fluores- contact times [56]. For C. acnes, formulations were
cence upon oxidation by reactive oxygen species (ROS) diluted with water up to 250, 500 and 1000 µg/mL rep-
[54]. Then, cells were washed with PBS and incubated resenting the MIC, 2 × MIC and 4 × MIC, respectively.
for 2 h with TH, TH-NP or B-NP. After this period, 10 For S. epidermidis, formulations were used at 1000 µg/
µL of H2O2 20 mM was added to each well. Untreated mL, representing twice as MIC. Inocula were prepared
cells with or without H2O2 were used as positive and in PBS at 1 08 CFU/mL and used to inoculate (100 µL)
negative controls, respectively. Fluorescence was meas- each experimental sample of 10 mL, incubated at 32 °C.
ured at excitation and emission wavelengths of 485 and The determined times were 0, 1, 2, 3 and 6 h or 0, 3, 8,
530 nm, respectively. Data were acquired at times t0 up 24 and 48 h for C. acnes and S. epidermidis, respectively.
to 120 min. Data of the positive control (H2O2) at 2 h, After incubations of each time set, an aliquot of 1 mL of
were used to normalize values (%). Background fluores- each sample was neutralized in 9 mL of Berens diluent
cence of the negative control was subtracted from all (Scharlab, Barcelona, Spain) for 15 min, then, diluted
measurements. in PBS on subsequent 10-folds. Drop count method (10
µL) was performed in CRM and TSA agar plates, for C.
In vitro antimicrobial efficacy acnes and S. epidermidis, respectively, incubated at 37 °C
S. epidermidis was grown overnight 37 °C in Muel- as described previously. Bacterial viability was expressed
ler Hinton Broth (MHB) culture medium (Oxoid, Bas- as CFU/mL against time (h). The decimal reduction time,
ingstoke, UK). Prior to each experiment, the inoculum the time taken to reduce by 10% the initial log10CFU, was
was prepared in PBS adjusted to 0.5 MacFarland (McF) determined calculating the inverse of the slope (1/b).
standard, to obtain a suspension with a cell density at The antimicrobial activity was also evaluated by SEM.
the range of 1.5 × 108 colony forming units/mL (CFU/
media in an incubator shaker (Innova® 4080, New Brun-
For this, C. acnes was cultured for 48 h in BHI culture
mL). Microbial count of S. epidermis was performed in
TSA plates, incubated at 37 °C. The C. acnes was cultured swick Scientific) at 37 °C under anaerobic conditions. The
in Brain Heart Infusion (BHI) medium (Oxoid, Basing- concentrated inoculum was transferred (900 µL) to each
tube containing 100 µL of TH or TH-NP at 0.1% or sterile
using parches (AnaeroGen®, Oxoid, Basingstoke, UK)
stoke, UK) for 48 h at 37 °C under anaerobic conditions

distilled water (control) and incubated in the shaker for was prepared in PBS and skin samples were inoculated
1 h. After samples were centrifuged (10,000g for 5 min), (10 µL). After 30 min, 30 µL of TH or TH-NP were
supernatants were discarded and the concentrated pellets administered as a single or repeated dose (1, 2 or 3), at
were placed into poly-l-lysine coated coverslips and kept preselected times (0, 12 and 18 h), completing a total
at room temperature for 24 h [57]. Samples were fixed for incubation of 24 h at 32 °C, in the presence of humid-
4 h with phosphate buffer 0.1 M pH 7.4, containing 4% ity. Then, skin samples were neutralized and extracted
paraformaldehyde and 2.5% glutaraldehyde, then post- as described above. These were tenfold diluted and
fixed with 1% osmium tetroxide (with potassium ferro- transferred to CRM plates by drop-count method (10
cyanide) for 1 h, at 4 °C. After dehydration with alcohol µL). Plates were incubated under anaerobic conditions
gradients, samples were dried at critical point (Emitech at 37 °C for 48 h. Viable bacteria were expressed as CFU
K850), mounted on a conductor adhesive disc (Carbon per treated skin (Additional file 1).
tabs, Agar Scientific), followed by carbon coating under A simulation of skin infection was performed in
evaporation (Emitech 950). Images were analyzed by fresh human skin explant (Hospital de Barcelona,
SEM (scanning electron microscopy, Jeol JSM-7001F). SCIAS, Barcelona, Spain) and analyzed by transmis-
sion electron microscopy. The fat tissue of skin sam-
Ex vivo antimicrobial efficacy
ples, obtained from human abdominal plastic surgery,
The bacterial viability was evaluated on treated human was removed manually with sterile surgical razors. Skin
skin explants obtained from abdominal plastic sur- samples were cut and placed on a 0.64 cm2 Franz dif-
gery (Hospital de Barcelona, SCIAS, Barcelona, Spain), fusion cell. The receptor compartment was filled with
based on other researcher protocols with modifica- PBS, and the skin was inoculated with 20 µL of C.
tions [58]. Skin samples were cut with a cryostat (Leica acnes (108 CFU/mL) and incubated for 16 h at 32 °C,
Microsystems, Wetziar, Germany) in 0.6 cm2, washed followed by treatment with TH or TH-NP (100 µL) for
with ethanol followed by sterile PBS, for 2 and 10 s, 8 h incubation. For electron microscopy, skin samples
respectively, to remove possible existing bacteria. Once were fixed for 2 h with 4% paraformaldehyde and 2.5%
dried with sterile filter paper, skins were placed into glutaraldehyde in 0.1 M sodium cacodylate buffer (pH
petri dishes (purchased from Fischer Scientific) with 7.4), postfixed with 1% osmium tetroxide for 2 h at
the SC facing up, onto PBS-wet sterile filter paper to 4 °C (all from Sigma Aldrich), stained in 0.5% uranyl
keep dermis moisture. Two experiments (prevention acetate (from Fischer Scientific) for 45 min at 4 °C and
and treatment) were set up for 24 h incubation at 32 °C, finally, dehydrated gradually in 30 to 100% ethanol [40].
in the presence of humidity. A fresh overnight culture Samples were infiltered in EPON resin [Eponate 12
of C. acnes was suspended in PBS (1.5 × 108 CFU/mL) (23.5 g), dodecenyl succinic anhydride DDSA (12.5 g)
and skin samples were inoculated with 10 µL. For the and Methyl nadic anhydride MNA (14 g)] (from Sigma
pre-treatment study, TH-NP or TH were applied on Aldrich). Inclusions were performed gradually diluted
skin samples (30 µL) and incubated at 32 °C for 8 h, fol- in ethanol and finally for 3 h using a catalyst [DMP-30
lowed by inoculation with C. acnes (30 µL) for 16 h. For (2,4,6-tris(dimethylaminomethyl)phenol), 0.37 g] (pur-
the post-treatment study, skin was first inoculated for chased from Sigma Aldrich). Polymerization was car-
30 min and then treated with products for 24 h. At the ried out for 48 h at 60 °C. Blocks were sliced in thin
end of the experiment, skin samples were neutralized sections with Ultracut microtome (LEICA), further
in 1 mL Berens diluent (Scharlab, Barcelona, Spain) fixed on copper grids and stained with uranyl acetate
for neutralization (15 min) followed by extraction for 2% for 10 min. Analysis was performed by TEM and
10 min using a sonication bath (JP, Selecta, Spain). The images were obtained with Megaview III.
extraction method was previously optimized by testing
the control at two extraction times (3 to 15 min), con- Supplementary Information
The online version contains supplementary material available at https://doi.
trolling bacteria viability by sonication process. Positive org/10.1186/s12951-021-01092-z.
controls were also performed using PBS. Tenfold dilu-
tions were performed and 100 µL of each sample was
Additional file 1: Figure S1. Morphology of TH-NP by TEM. (A) 1
spread individually onto CRM agar plates and incu- month at 4 °C (B) 1 month at 25 °C and (C) 12 months at 4 °C. Arrows
bated under anaerobiosis at 37 °C for 48 h. Viable bac- indicate aggregation. Scale bar: 200 nm. Figure S2. Stability behavior
teria count was expressed as log/CFU per treated skin. of TH-NP plotted as light backscattering (%) vs sample height, at several
storage conditions: (A) 4 °C up to 12 m, (B) 25 °C and (C) 37 °C up to 3
The analysis of bacterial viability on dose-dependent months. The scans are shown from the bottom to the top of the vial from
study on treated skin was also performed using the the left to the right, as mean values of hourly measurements for 24 h.
same technique as described above, with further modi- Figure S3. Morphology of C. acnes observed by TEM after negative stain‑
ing. Scale bar 500 nm.
fications [58]. A fresh overnight culture of C. acnes

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1
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RESEARCH Open Access

Thymol‑loaded PLGA nanoparticles:

*Correspondence: [email protected]; [email protected]

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Background preventing colonization of pathogenic bacteria such as

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graphics® software, are shown in Fig. 1. The statistical

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0 158.8 ± 1.7 0.068 ± 0.027 − 24.7 ± 1.0 4.20

In vitro antimicrobial efficacy

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Table 3 Ex vivo skin permeation parameters

J (μg/cm2/h) 12.68 ± 1.73 8.03 ± 0.55 a 26.43 ± 2.13 a b d 21.03 ± 0.92 a b

(See figure on next page.)

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Fig. 4 (See legend on previous page.)

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analyzer Turbiscan®Lab expert (Formulaction, L’Union,

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sue-Tek®, Sakura) and sliced with cryostat microtome

frost® Plus (Menzel-Glaser, Thermo Scientific, USA),

covered with Fluoromount G® (Invitrogen, Thermo

analyzed by HPLC and processed using GraphPad®. The

wavelength of 570 nm in a Modulus® Microplate Pho-

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seeded in an 8-well µ-slide (Ibidi®) following the same

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Acknowledgements Pharmaceutical Sciences. Kerala: Transworld Research Network; 2011. p.

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