pharmaceutics

Article
Injectable Hyaluronan-Based Thermoresponsive Hydrogels for
Dermatological Applications
Si Gou 1,2 , Alexandre Porcello 3 , Eric Allémann 3 , Denis Salomon 4 , Patrick Micheels 5 , Olivier Jordan 3
and Yogeshvar N. Kalia 1,2, *

1 School of Pharmaceutical Sciences, University of Geneva, 1211 Geneva, Switzerland; [email protected]

2 Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1211 Geneva, Switzerland
3 KYLYS Sàrl, 34, Route de la Galaise, c/o FONGIT, Plan-les-Ouates, 1228 Geneva, Switzerland;
[email protected] (A.P.); [email protected] (E.A.); [email protected] (O.J.)
4 Clinique Internationale de Dermatologie Genève SA, 1201 Geneva, Switzerland; [email protected]
5 Private Practice, 8, Chemin de la Fontaine, Chêne-Bougeries, 1224 Geneva, Switzerland;
[email protected]
* Correspondence: [email protected]

Abstract: Most marketed HA-based dermal fillers use chemical cross-linking to improve mechanical
properties and extend their lifetime in vivo; however, stiffer products with higher elasticity require
an increased extrusion force for injection in clinical practice. To balance longevity and injectability,
we propose a thermosensitive dermal filler, injectable as a low viscosity fluid that undergoes gelation
in situ upon injection. To this end, HA was conjugated via a linker to poly(N-isopropylacrylamide)
(pNIPAM), a thermosensitive polymer using “green chemistry”, with water as the solvent. HA-L-
pNIPAM hydrogels showed a comparatively low viscosity (G0 was 105.1 and 233 for Candidate1
and Belotero Volume® , respectively) at room temperature and spontaneously formed a stiffer gel
with submicron structure at body temperature. Hydrogel formulations exhibited superior resistance
against enzymatic and oxidative degradation and could be administered using a comparatively
lower injection force (49 N and >100 N for Candidate 1 and Belotero Volume® , respectively) with a
Citation: Gou, S.; Porcello, A.;
32G needle. Formulations were biocompatible (viability of L929 mouse fibroblasts was >100% and
Allémann, E.; Salomon, D.; Micheels,
~85% for HA-L-pNIPAM hydrogel aqueous extract and their degradation product, respectively), and
P.; Jordan, O.; Kalia, Y.N. Injectable
offered an extended residence time (up to 72 h) at the injection site. This property could potentially
Hyaluronan-Based
be exploited to develop sustained release drug delivery systems for the management of dermatologic
Thermoresponsive Hydrogels for
Dermatological Applications.
and systemic disorders.
Pharmaceutics 2023, 15, 1708.
https://doi.org/10.3390/ Keywords: dermal fillers; hyaluronic acid; thermoresponsive; poly(N-isopropylacrylamide); ex vivo
pharmaceutics15061708 skin model

Academic Editor: Natasa

Skalko-Basnet

Received: 14 April 2023 1. Introduction

Revised: 2 June 2023 Injectable biomaterials have become a hot topic as they have been used to address
Accepted: 6 June 2023 problems associated with aging skin, e.g., decreased skin elasticity, facial wrinkles, and
Published: 11 June 2023 collagen degradation [1]. Dermal fillers can achieve rapid facial rejuvenation through
simple and short procedures and have been widely investigated [2,3]. Among the different
materials, hyaluronic acid (HA)-based fillers account for the largest number of products
with more than 2.6 million minimally invasive procedures conducted in the US alone in
Copyright: © 2023 by the authors.
2020 [4,5]. This can be attributed to the non-toxicity of HA, its biocompatibility and easy
Licensee MDPI, Basel, Switzerland.
reversibility [6,7].
This article is an open access article
distributed under the terms and
Natural HA is composed of repeating alternating units of D-glucuronic acid and N-
conditions of the Creative Commons
acetyl-D-glucosamine, all connected by “β-linkages, GlcA β (1→3) GlcNAc β (1→4) [8]. It
Attribution (CC BY) license (https:// is synthesized as a compound of high molecular weight in both the epidermis and dermis
creativecommons.org/licenses/by/ but rapidly undergoes degradation induced by oxidative stress and hyaluronidases. The
4.0/). half-life (t1/2 ) of unmodified HA in the skin is about 12 h [9–14]. The fragmented HA of lower

Pharmaceutics 2023, 15, 1708. https://doi.org/10.3390/pharmaceutics15061708 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2023, 15, 1708 2 of 18

molecular size is a potent inducer of inflammation and angiogenesis, which can elicit adverse
effects such as erythema, slight oedema, hematoma, itching, and pain [15–20]. Therefore, HA
used in dermal fillers is routinely cross-linked to improve mechanical properties and in vivo
residence time [21–23]. It was reported that the lifetime of the filler material is dependent
on the type and density of the cross-linking agents [24]: 1,4-butanediol diglycidyl ether
(BDDE) is the most widely used crosslinker in commercial HA dermal fillers, and a high
cross-linking degree generally results in a stiffer product with higher elastic modulus (G0 )
that potentially extends the residence time in vivo.
However, the high cross-linking degree was associated with hypersensitivity reactions
since the unreacted cross-linking agents or their by-products could be toxic [25–27], and
the increased number of modifications would possibly lead to decreased biocompatibility
of HA-based materials as the reaction conditions (e.g., heat, alkaline conditions) are prone
to degrade HA gels and release small HA fragments with potential safety issues [28–31]. In
addition, the increased extrusion force required for intradermal injection can be an issue
in clinical practice. It is crucial yet complicated to develop a biocompatible product with
an optimal balance between biocompatibility, prolonged residence time and injectability.
To date, issues reported with HA-based dermal filler products on the market include
difficulties in precise injection, unsatisfying/suboptimal volumizing effect and the need for
repeated injections [32].
Recently, a novel technology that involved a specific conjugation of the thermorespon-
sive polymer, poly(N-Isopropylacrylamide) (pNIPAM), to the linear HA backbone via a
cyclooctyne linker (L) was reported [33,34]. Due to the desolvatation of the hydropho-
bic pNIPAM moieties above a defined lower critical solution temperature (LCST) and to
the presence of the linker, the synthetic HA-L-pNIPAM copolymer spontaneously forms
submicron spherical particles above the LCST [33–36]. These domains act as inter-chain
crosslinkers, resulting in a sol–gel copolymer transition driven only by physical interactions,
avoiding the use of chemical cross-linking agents. Preliminary research has shown the
formation of microgel structures upon subcutaneous injection in mice that precisely confine
the HA-L-pNIPAM hydrogel at the injection site, offering an extended residence time with
a lower risk of migration [37].
In this study, the in situ physical crosslinking technology was employed to design
products for a durable and precisely controlled skin correction. The specific aims were
(i) to develop a new family of HA-L-pNIPAM copolymers with optimized physical char-
acteristics as implantable biomaterials for dermatological application in terms of rheo-
logical properties, cohesivity, injectability, and in vitro resistance to oxidative stress and
hyaluronidase-mediated degradation, (ii) to evaluate cytocompatibility of the candidate
formulations using the L929 cell line in vitro, and (iii) to investigate their subcutaneous dis-
tribution pattern as a function of time after implantation in an ex vivo porcine skin model.

2. Materials and Methods

2.1. Materials
Laboratory-grade hyaluronic acid (HA) sodium salt (1500–1750 kDa) was purchased
from Contipro a.s. (Dolní Dobrouč, Czech Republic). Sulfo-Dibenzocyclooctyne-PEG4 -
amine (Sulfo DBCO-PEG4-NH2) was bought from Click Chemistry Tools (Scottsdale, AZ).
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS),
azide-terminated poly(N-isopropylacrylamide) (pNIPAM-N3 ; 15 kDa), hyaluronidase from
bovine testes (Type VI-S), hydrogen peroxide (30% w/w), toluidine blue, Dulbecco’s Modi-
fied Eagle’s Medium—high glucose (DMEM), embryoMax L-glutamine solution (100X),
foetal bovine serum (FBS), and the antibiotic–antimycotic solution (100X) were purchased
from Sigma-Aldrich (St Louis, MO, USA). Dialysis membranes (Biotech CE Dialysis Tubing
300 kDa) were acquired from Repligen (Waltham, MA, USA). The Alcian blue PAS stain kit
was purchased from Abcam (Cambridge, UK). Belotero Balance® and Belotero Volume®
were purchased from Merz Pharma (Geneva, Switzerland) and used as reference products.
Pharmaceutics 2023, 15, 1708 3 of 18

2.2. Synthesis of HA-L-pNIPAM Copolymers

HA-L-pNIPAM copolymers (HA-L-pNIPAM0.10, HA-L-pNIPAM0.25, HA-L-pNIPAM0.50)
were synthesized using a slightly modified procedure based on a previous publication [33,34].
The scheme for the green chemistry synthetic route of the HA-L-pNIPAM copolymers is
presented in Supplementary Materials (Figure S1). Briefly, HA (1500–1750 kDa) was solubilized
in distilled water at 0.2% (w/v) under magnetic stirring. After dissolution of HA, EDC (5 eq.
COO− ) and NHS (5 eq. COO− ) were added at intervals of 15 min. The pH was adjusted to 5.5
using a 0.1 M NaOH and a 0.1 M HCl and monitored using a Metrohm pH gel electrode (Herisau,
Switzerland). Then, Sulfo-DBCO-PEG4-amine, previously dissolved in distilled water, was
added (0.1 eq. COO− for HA-L-pNIPAM0.10 , 0.25 eq. COO− for HA-L-pNIPAM0.25 , and
0.5 eq. COO− for HA-L-pNIPAM0.50 ) and was stirred overnight (12 h) at room temperature
for amidation. The intermediate product (HA Sulfo DBCO-PEG4) was dialyzed thrice
against a 5% (w/v) NaCl solution (MWCO: 300,000, 3 h, at ambient temperature) and
then three times against distilled water before being transferred to a round bottom flask.
Then, the DBCO group of the linker reacted via copper-free azide-DBCO click chemistry
with pNIPAM-N3 (15 kDa, 1 eq. DBCO). The pH was adjusted to 7 using a 0.1 M NaOH
and a 0.1 M HCl. The reaction was allowed to proceed for 12 h under stirring at room
temperature. The final product was dialyzed thrice against a 5% (w/v) NaCl (MWCO:
300,000, 3 h, ambient temperature) and three times against distilled water before being
frozen at −80 ◦ C, lyophilized (Freeze Dryer Alpha 1–4 LD plus, Christ, Osterode am Harz,
Germany; 48 h, 1.5·10−1 mbar, −80 ◦ C) and stored at 4 ◦ C.
The intermediate product (5 mg) and the final product (10 mg) were digested with
hyaluronidase (300 IU/mL) using D2 O as a solvent; then, their chemical structures and
degrees of substitution (DS) were determined from the 1 H NMR spectra acquired on a
Bruker Avance Neo 600 MHz NMR spectrometer at ambient temperature. The DS was
calculated using the integration ratio of methyl protons of HA (δ 2.00 ppm) with the
aromatic protons of the DBCO group (7.67 ppm) for the intermediate product (DS1 ) and
the integration of the methyl protons of pNIPAM (δ 1.13) with the aromatic protons of the
DBCO group (7.67 ppm) for the final product (DS2 ).

2.3. Physical Characterization of HA-L-pNIPAM Hydrogels

2.3.1. Rheological Properties
Rheological behaviors were determined on a HAAKE Mars Rheometer™ (Thermo
Scientific, Waltham, MA, USA) equipped with a Peltier cone-plate C35 2◦ /Ti rotor. Measure-
ments were performed on 420 µL samples with a sample hood to reduce evaporation. First,
the synthetic HA-L-pNIPAM copolymers were dissolved in PBS under agitation overnight;
then, centrifugation was performed at 10,000 rpm for 20 min at 4 ◦ C. The storage modulus
(G0 ) and loss modulus (G00 ) of the obtained formulations (3%, w/v) were assessed as a
function of temperature using a ramp from 22 ◦ C to 37 ◦ C with a heating rate of 0.04 ◦ C/s
and a constant oscillatory frequency of 0.7 Hz, simulating the forces to which a filler is
exposed in vivo from gravity and muscular movements [38].
Then, three HA-L-pNIPAM hydrogel candidates were selected for further physical and
biological characterization. Briefly, Candidate 1 (2%, w/v) and Candidate 2 (2%, w/v) were
formulated by directly dissolving the synthetic HA-L-pNIPAM0.10 and HA-L-pNIPAM0.25
in PBS, respectively, whereas Candidate 3 (2%, w/v) was prepared as a combination of
HA-L-PNIPAM0.50 and non-derivatized linear HA (1500–1750 kDa) with a ratio of 3:1
(w/w). The G0 and G00 values of the three HA-L-pNIPAM hydrogel candidates and the
two reference products (Belotero Balance® and Belotero Volume® ) were also determined
at 22 ◦ C and 37 ◦ C with a constant oscillatory frequency of 0.7 Hz. Shear stress was set to
1.0 N/m2 in all experiments to remain in the linear viscoelastic region (LVE).

2.3.2. Injectability
The injection force profile of the three HA-L-pNIPAM hydrogel candidates and the
two reference products (Belotero Balance® and Belotero Volume® ) were determined using
Pharmaceutics 2023, 15, 1708 4 of 18

a Texture Analyzer TA.XT. Plus (Stable Microsystems Ltd., Surrey, UK). A total of 300 µL of
each sample was placed in a 1 mL syringe (Schott TOPPAC® , 5 mm internal diameter, with
Luer-Lok™, Schott, Mainz, Germany) and extruded through needles of 30G, 32G, and 34G
(13 mm, Needle Concept, Biarritz, France) at a speed of 2 mm·s−1 at 22 ◦ C, respectively.
The maximum force (N) allowed for extruding the samples was set to 100 N.

2.3.3. Accelerated Degradation Assays

The evolutive rheological behaviors of the HA-L-pNIPAM candidate formulations and
the commercial reference products under oxidative stress and enzymatic degradation were
determined on a HAAKE Mars Rheometer™ equipped with a Peltier cone-plate C35 2◦ /Ti
rotor. To generate oxidative stress, 100 µL of hydrogen peroxide 30% (w/w) was added to
400 µL of each sample. The elastic modulus (G0 ), viscous modulus (G00 ) and tangent delta (δ)
values were measured as a function of time during 12 min at a constant oscillatory frequency
of 0.7 Hz. The delay between the addition of H2 O2 and the first measurement was two
minutes. For accelerated enzymatic degradation, 100 µL of hyaluronidase (100 U/mL) was
added to 400 µL of each sample. Enzyme-mediated degradation was carried out using the
same method as describe above. As a control condition, instead of H2 O2 or hyaluronidase,
100 µL of the PBS buffer was added to 400 µL of the sample and assessed at the beginning
of the experiments. A sample hood was used during all the measurements to minimize
evaporation. Shear stress was set to 1.0 N/m2 in all experiments so as to ensure that the
measurements were performed in the linear viscoelastic region (LVE).

2.4. Biological Evaluations of HA-L-pNIPAM Hydrogels

2.4.1. In Vitro Biocompatibility Evaluation Using a L929 Cell Line
Sample preparation. HA-L-pNIPAM candidate formulations (aqueous extracts) and
their enzymatic degradation products were tested for cytocompatibility. The extraction
dilution method was chosen for the preparation of nondegraded polymer samples as
described by the norm ISO 10993-5 regulation. Briefly, the extraction procedure was carried
out in the complete medium containing MEM, 10% FBS, a 1% antibiotic–antimycotic
solution. A total of 0.5 mL of each formulation was dissolved in a 5 mL medium under
continuous agitation at 37 ◦ C for 24 h to obtain an aqueous extract. Degradation products
were prepared by incubating 1 mL of each candidate formulation with 100 µL hyaluronidase
(100 IU/mL, type I–S, Sigma) at 37 ◦ C for 4 h, followed by heat inactivation. Raw HA
(1500–1750 kDa, 2%, w/v) was also tested as a non-derivatized control.
Cytotoxicity test. L929 cell line (murine fibroblast) was purchased from Sigma-Aldrich
(St Louis, MO, USA) and grown in culture flasks containing minimum essential medium
(MEM), supplemented with a 10% foetal bovine serum (FBS), a 1% antibiotic–antimycotic
solution at 37 ◦ C in a humidified 5% CO2 atmosphere and monitored daily using an
inverted microscope. Subcultures were performed twice a week when a confluence of
80% was observed. For cytotoxicity tests, cells were seeded into 96-well culture plate (flat
bottom, Costar, Corning, Inc.) at a density of 1 × 105 cells per well. After 24 h, the culture
media were replaced with a 100 µL sample; a negative control (only MEM with 10% FBS)
and a positive control (0.1% SDS solution) were included to validate the viability protocols.
The assay was carried out in triplicate. All plates were incubated at 37 ◦ C in a humidified
5% CO2 atmosphere, and a WST-1 assay (Roche Applied Science) was performed after 24 h
of incubation to quantitatively determine cell viability as previously described [35].

2.4.2. Evaluation of Tissue Integration in Ex Vivo Porcine Skin Model

Ex vivo porcine skin model. Porcine ears were obtained from a local slaughterhouse
(Loëx, Switzerland) and used within approximately 4 h of sacrifice. The ears were thor-
oughly cleaned first with running water, then with soap to eliminate microorganisms
present on the surface. The skin surface was then shaved and disinfected three times with
an aseptic solution (3 × 90 s). The front root part of the ear, which connects to the head
and contains abundant fat tissue, was first cut out using a scalpel; then, skin biopsies were
Pharmaceutics 2023, 15, 1708 5 of 18

performed using a 16 mm diameter punch (Berg & Schmid HK 500; Urdorf, Switzerland)
and anchored in a Millicell® cell culture insert (d = 12 mm; Merck KGaA, Darmstadt,
Germany) with the epidermis facing up. The inserts were positioned in a 12-well plate
filled with 3 mL of culture media in each well. The details of culture medium are listed
in Supplementary Materials (Table S2). All manipulations were performed in a 30 cm
radius of a flame, with the operator wearing a face mask, gloves and over-blouse to limit
airborne contamination. The tools were disinfected with an alcohol solution and flame. The
viability of the ex vivo model was previously reported as being up to 72 h by histological
observation (H&E staining). In this study, skin biopsies without injection (serving as the
blank control group) were harvested at T0 and T72 and subjected to H&E staining for the
evaluation of structural integrity.
Subcutaneous injection. The culture plate was incubated at 37 ◦ C and a 5% CO2 for
2 h allowing the skin tissue to reach an equilibrium state. The slightly oversized biopsies
(d = 16 mm) were immobilized in the insert (d = 12 mm), which facilitated the subcutaneous
injection. A total of 50 µL of each candidate formulation and reference product (raw linear
HA and commercial reference) was injected ex vivo through the skin into the subcutaneous
fat compartment using a BD Luer-Lok™ 1 mL syringe with a 30G needle. The penetration
depth was controlled to be approx. 4 mm and injections were performed by the same
researcher under the same aseptic conditions described above; an infrared lamp was used
to maintain the ambient temperature at ~40 ◦ C. The skin biopsies were incubated at 37 ◦ C
and a 5% CO2 and the culture media were changed every 24 h.
Histology analysis. Skin biopsies that received candidate formulations were collected
at pre-determined time points T0 (immediately after injection) and T72 , while those im-
planted with raw linear HA and commercial products were harvested at T0 and served
as control for the validation of histological staining (H&E and AB/PAS). The protocols
are presented in Supplementary Materials (Table S3). Each sample was embedded in OCT
and snap-frozen in isopentane chilled with dry ice (−72 ◦ C). In the frozen state, a series of
cross-sections with a thickness of 20 µm were obtained using a cryotome (Thermo Scientific
CryoStarTM NX70; Reinach, Switzerland). Lamellae containing the implanted materials
under visual observation were then placed on Superfrost® positively charged glass slides.
The blade and sample temperatures were −35 ◦ C and −25 ◦ C, respectively. The slides were
stained following the protocol and were mounted using the Eukitt® mounting medium
with a cover slide on the top. Observation was performed in the Bioimaging Core Facility
(Faculty of Medicine, University of Geneva) with the Axioscan Z1 brightfield microscope
in automatic scan mode.

3. Results and Discussion

3.1. HA-L-pNIPAM Copolymer Synthesis
The HA-L-pNIPAM copolymers were synthesized via green chemistry synthetic routes
given the aqueous solubility of the sulfonated DBCO-PEG4 -NH2 linker [33,34]. The final
yield was ~70–80%. The structural characteristics of the HA-L-pNIPAM copolymers were
investigated by 1 H NMR spectroscopy; the spectra are presented in Supplementary Ma-
terials (Figure S2). The aromatic protons of DBCO enabled the calculation of the DS1 of
the amidation from the NMR spectra, with mean values of 1.1, 2.8 and 6.6% for HA-L-
pNIPAM0.10 , HA-L-pNIPAM0.25 , and HA-L-pNIPAM0.50 , respectively. These results agree
with those of previous reports indicating that amidation of HA with EDC/NHS in water led
to a relatively low DS [9,39], and that the DS1 can be modulated by adjusting the amount
of linker. The DS2 for the azide-DBCO grafting with the CH3 groups of pNIPAM units was
~90% for the three HA-L-pNIPAM copolymers; the high substitution degree benefited from
the hydrophilicity and flexibility of the PEG spacer present in Sulfo DBCO-PEG4 -NH2 . The
content of each component in the final products calculated from the DS is presented in
Supplementary Materials (Table S1).
 The three tested HA-L-pNIPAM formulations were all shown to undergo a sharp
increase in storage modulus (G’) values and a decrease in loss modulus (G”) values start-
ing from ~27 °C. This shift occurred within a limited range of less than 2 °C, regardless of
the DS. The G” value reached a plateau at ~36 °C, while the G’ value increased continu-
Pharmaceutics 2023, 15, 1708 ously at higher temperatures. During all the analyses, the G’ value remained superior 6 of to
18
the G” value (tan δ < 1), suggesting that all the tested formulations presented a gel state in
the temperature range of the study. The results confirmed the thermosensitive behavior
of the tested formulations, and the observed thickening effect (viscosity increase) supports
3.2. Physicochemical Characterization of HA-L-pNIPAM Hydrogels
the hypothesis for physical crosslinking of the gel phase that is hydrophobically driven
3.2.1. Rheological Properties
by pNIPAM. Theoretically, the LCST of the system is determined by the physicochemical
Specific
properties of mechanical
the pNIPAM properties (e.g., molecular
polymer (e.g., stiffness, high G0 values
weight, > 50 Pa)
end group), butare
it isrequired
also in-
for injectable volumizing dermal filling materials. Rheological properties of the
fluenced by the environment (e.g., salt content). In the current study, PBS was introduced synthetic
HA-L-pNIPAM
into the system;copolymers
the transitionwere experimentally
temperature, characterized
therefore, by dynamic
was shifted viscoelasticity
to ~27 °C despite the
tests.
fact that the coil-to-globule transition of pNIPAM-N3 (15 kDa) was reported todissolved
HA-L-pNIPAM 0.10 , HA-L-pNIPAM 0.25 and HA-L-pNIPAM 0.50 were simply occur at
in PBS by agitating overnight, and homogeneous formulations (3%, w/v) were obtained
~30–32 °C. Compared to HA-L-pNIPAM0.25 and HA-L-pNIPAM0.50, HA-L-pNIPAM 0.10
after centrifugation. The temperature dependence of their storage modulus (G0 ) and the
showed less significant changes in both moduli, which could be explained by the compar-
loss modulus (G00 ) are presented in Figure 1.
atively lower pNIPAM grafting density.

Figure 1. (A) G’ storage modulus and (B) G” loss modulus of HA-L-pNIPAM hydrogel candidates
Figure 1. (A) G0 storage modulus and (B) G00 loss modulus of HA-L-pNIPAM hydrogel candidates as
as a function of temperature (n = 3, Mean ± SD).
a function of temperature (n = 3, Mean ± SD).

The three tested HA-L-pNIPAM formulations were all shown to undergo a sharp
increase in storage modulus (G0 ) values and a decrease in loss modulus (G00 ) values starting
from ~27 ◦ C. This shift occurred within a limited range of less than 2 ◦ C, regardless of the
DS. The G00 value reached a plateau at ~36 ◦ C, while the G0 value increased continuously
at higher temperatures. During all the analyses, the G0 value remained superior to the G00
value (tan δ < 1), suggesting that all the tested formulations presented a gel state in the
temperature range of the study. The results confirmed the thermosensitive behavior of
the tested formulations, and the observed thickening effect (viscosity increase) supports
the hypothesis for physical crosslinking of the gel phase that is hydrophobically driven
by pNIPAM. Theoretically, the LCST of the system is determined by the physicochemical
properties of the pNIPAM polymer (e.g., molecular weight, end group), but it is also in-
fluenced by the environment (e.g., salt content). In the current study, PBS was introduced
into the system; the transition temperature, therefore, was shifted to ~27 ◦ C despite the
fact that the coil-to-globule transition of pNIPAM-N3 (15 kDa) was reported to occur at
~30–32 ◦ C. Compared to HA-L-pNIPAM0.25 and HA-L-pNIPAM0.50 , HA-L-pNIPAM0.10
showed less significant changes in both moduli, which could be explained by the compara-
tively lower pNIPAM grafting density.
Based on these results, three candidates formulated from the synthetic HA-L-pNIPAM
copolymers were proposed for further investigations into their dermatological application.
Candidate 1 (2%, w/w) and Candidate 2 (2%, w/w) were prepared by directly dissolving
HA-L-pNIPAM0.10 and HA-L-pNIPAM0.25 in PBS, Candidate 3 (2%, w/w) was a combina-
tion of HA-L-pNIPAM0.50 and non-derivatized linear HA (1500–1750 kDa) with a ratio of
3:1. Formulation preparation for the whole study strictly followed the method described
previously since it was crucial to completely hydrate the polymer and to ensure the homo-
geneity of the hydrogel formulation. The HA content in each candidate formulation was
Pharmaceutics 2023, 15, 1708 7 of 18

calculated according to the DS values obtained from 1 H NMR spectroscopy; it is displayed

in Table 1.

Table 1. Summary of three HA-L-pNIPAM hydrogel candidates.

Formulation HA Content
Formulation Copolymer
Concentration (mg/mL) (mg/mL)
Candidate 1 HA-L-pNIPAM0.10 20 15
Candidate 2 HA-L-pNIPAM0.25 20 8
HA-L-pNIPAM0.50 + HA
Candidate 3 20 9.5
(1.5–1.75 MDa)

The rheological properties of the three HA-L-pNIPAM hydrogel candidates were then
determined at 22 ◦ C and 37 ◦ C with a constant oscillatory frequency of 0.7 Hz and shear
stress set at 1.0 N/m2 ; Belotero Balance® and Belotero Volume® were used as references
and were assessed under the same condition, but only at 37 ◦ C. The values of G0 , G00 and
tan δ measured for the three candidates and two commercial products are presented in
Table 2. Above the LCST (at 37 ◦ C), Candidate 1 showed a G0 value in a range compa-
rable to that of the reference Belotero Volume® , while Candidate 2 obtained an approxi-
mately twofold higher G0 value. Given that both candidates had a moderate G00 value, the
00
tan δ (= GG0 ) value of Candidates 1 and 2 were twofold (0.13 vs. 0.23) and threefold (0.08
vs. 0.23) lower than those of Belotero Volume® , respectively. In accordance with the mea-
surements on the synthetic copolymers, the G0 values of Candidates 1 and 2 assessed at
22 ◦ C (below the LCST) were significantly lower than those obtained at 37 ◦ C (for the
candidates and the commercial products) and resulted in increased tan δ values of 0.48 and
0.50, respectively.

Table 2. Rheological properties of HA-L-PNIPAM hydrogel candidates and of commercial products

(n = 3).

HA Content Temperature 00
Formulation G0 G00 Tan δ = G
(mg/mL) (◦ C) G0

Belotero Balance® 22.5 37 53 33 0.62

Belotero Volume® 26.0 37 233 54 0.23
22 105.1 50.1 0.48
Candidate 1 15.0
37 234.1 28.7 0.13
22 167.8 83.8 0.50
Candidate 2 8.0
37 420.2 34.9 0.08
22 66.9 35.3 0.50
Candidate 3 9.5
37 92.5 43.5 0.49

These results suggested that Candidates 1 and 2 could be considered as realistic candidates
for volumizing applications, and the comparatively high HA MW (1500–1750 kDa) employed in
the synthesis of HA-L-pNIPAM copolymers (cf. 800 kDa in Belotero Balance® [40]) contribute to
their mechanical properties. On the one hand, the high G0 at 37 ◦ C endowed the hydrogel with
an excellent ability to regain its initial shape when the dynamic shearing forces were removed
as reported by Gavard Molliard et al. [41], which corresponds to mechanical deformations such
as muscle movements driving dynamic facial motion for a dermal filler in vivo. On the other,
HA-L-pNIPAM hydrogel candidates with a comparatively low viscosity (G0 and tan δ value) at
room temperature (22 ◦ C) would require a lower extrusion force for injection, which would
contribute to accurate and precise injections in the clinic. The spontaneous phase transition
was also associated with Candidate 3, although it displayed viscoelastic characteristics
that are more suitable for more superficial dermal injections, similar to Belotero Balance® ,
 Table 2. Rheological properties of HA-L-PNIPAM hydrogel candidates and of commercial prod
(n = 3).

Pharmaceutics 2023, 15, 1708 8 of 18

Formulation HA Content (mg/mL) Temperature (°C) G’ G” Tan δ =
Belotero Balance® 22.5 37 53 33 0.62
Belotero Volume® 26.0 37 233 54 0.23
due to the presence of non-derivatized linear HA in the system. These results not only
demonstrated the temperature-dependent 22
characteristics 105.1
of the 50.1
proposed HA-L-pNIPAM 0.48
Candidate 1 15.0
hydrogel candidates, but also indicated that the products incorporating such28.7
37 234.1 HA-based 0.13
polymers could be tailored by adjusting the HA 22 MW, the DS, 167.8 83.8
and the final concentration 0.50
Candidate 2 8.0
37
of the polymer to obtain defined viscoelastic properties 420.2 applications.
for different 34.9 All the 0.08
candidate formulations were stored at 37 ◦ C and
22 at 4 ◦ C for 1 month; no significant change
66.9 35.3 0.50
Candidate 3
in rheological 9.5was observed.
properties 37 92.5 43.5 0.49

3.2.2. Injectability
3.2.2. Injectability
The ease of injection is a critical parameter for all types of HA fillers, especially for
The ease of injection is a critical parameter for all types of HA fillers, especially
volumizers. The force in Newtons (N) required to expel the hydrogel as a function of
volumizers. The force in Newtons (N) required to expel the hydrogel as a function o
the stroke distance of the piston in a standard syringe with various needles is reported in
stroke distance of the piston in a standard syringe with various needles is reporte
Figure 2. All samples
Figure 2.could be extruded
All samples couldthrough a 30G
be extruded needle,a Candidates
through 1–3 required
30G needle, Candidates 1–3 requir
a lower plateaulower
forceplateau
(i.e., <25 N) compared
force (i.e., < 25 to compared
N) the two reference
to the products
two (i.e.,products
reference 34 N for(i.e., 34 N
Belotero Balance ® and 42 N for Belotero Volume® ). When a 32G needle was used for the
Belotero Balance® and 42 N for Belotero Volume®). When a 32G needle was used for
injection, the mean
injection, increased
force to close
the mean force to 50 Nto
increased forclose
the three
to 50 candidates (i.e.,candidates
N for ®the three 49 N, 52 N(i.e., 49 N
and 51 N for Candidates 1, 2 and 3 respectively). Belotero Balance
N and 51 N for Candidates 1, 2 and 3 respectively). Belotero Balance required a plateau
® required a pla
force of 78 N, which represented a 2.3-fold increase in comparison with
force of 78 N, which represented a 2.3-fold increase in comparison the 30G needle
withand
the 30G ne
1.5 × more forceandthan Candidates 1–3 with a 32G needle. Belotero Volume ® required more
1.5 x more force than Candidates 1–3 with a 32G needle. Belotero Volume® requ
than 100 N andmorethus was
than considered
100 N and thus not injectable in thisnot
was considered case.
injectable in this case.

Figure 2. Force inFigure 2. Force in Newtons (N) required to extrude

VolumeBelotero Volume ®, Belotero Balance® and
Newtons (N) required to extrude Belotero ® , Belotero Balance ® and the
three candidates as a function of the stroke distance of the piston in a syringe with a 30G, 32G
three candidates as a function of the stroke distance of the piston in a syringe with a 30G, 32G and
34G needle (distance %) at 22 °C.
34G needle (distance %) at 22 ◦ C.

Given that a narrower gauge needle could potentially improve the precision of the
injection and decrease the incidence of pain in clinical practice, notably where indications
are expected to be painful (e.g., increase in lip volume), a 34G needle (external diameter
0.16 mm) representing a 50% thinner needle in comparison with a needle of 30G (external
diameter 0.31 mm) was also tested. It was found that only Candidates 1–3 were able to be
Pharmaceutics 2023, 15, 1708 9 of 18

extruded through this thinner needle. Different injection forces were measured among the
three, proportional to their viscoelastic values, meaning that Candidate 2 with higher values
at room temperature required more force than Candidate 1, and Candidate 1 required more
force than Candidate 3 (i.e., 72 N, 65 N and 55 N, respectively, as plateau force). Surprisingly,
Candidate 3 did not show a significant difference between the 32G and the 34G (i.e., 51 N
and 55 N respectively). Candidates 1 and 2 had a smoother horizontal plateau in the
corresponding force injection profile. This aspect would contribute to facilitating the
injection of the filler product and could be potentially linked to a more homogenous gel
structure. Thus, a HA-based filler that reached clinically relevant values of G0 of more than
400 Pa was injected for the first time through a 34G needle.

3.2.3. In Vitro Degradation

It is considered that dermal filler products are mainly degraded by oxidative stress,
hyaluronidases, and mechanical stress in vivo [6,9,14,27]. Therefore, we compared HA-
L-pNIPAM hydrogels to Belotero Balance® with respect to/in terms of their enzymatic
digestion and oxidative degradation at 37 ◦ C in vitro. After the addition of hyaluronidases,
much more striking effects were observed (Figure 3A–C). Belotero Balance® and Candidates
1 and 2 followed the same decreasing trends in both moduli. Candidate 3 again displayed
a viscoelastic behaviour with high standard deviations in the G0 and G00 values, as well as
an unexpected sixfold increase in the G0 and an increase in the G00 values in the first 40 s
followed by a decrease to 12.97 Pa by the end, leading to a significantly decreased tan (δ)
value. It could be noticed that Belotero Balance® yielded a tan (δ) of 1.15 in response to
hyaluronidase exposure, which was twofold higher than those of Candidates 1–3, indicating
a more fluid-like behaviour for the commercial reference. The storage modulus of Candidate
3 was lower than those of the other two candidates in the control group, in which 100 µL
PBS was added to the candidate formulation instead of 100 µL H2 O2 /hyaluronidases);
however, it yielded a value that was tenfold higher than those of all the other samples after
contact with the enzyme for 10 min.
These results might be explained by the fact that Candidate 3 is a combination of
raw linear HA and HA-L-pNIPAM0.50 . The thermoresponsive behaviour of the highly
grafted PNIPAM resulted in a change in conformation (hydrophobic interactions become
dominant) above the LCST, which hypothetically created condensation and an entangle-
ment of the HA chains, resulting in less available recognition sites (e.g., carboxylic acid
groups for the enzyme). The conjugation of HA carboxyl groups and the formation of
nanometric structures at body temperature are key elements to improve product resistance
to hyaluronidases [33,34]. The higher DS on the carboxylic acid of HA chains resulted in a
filling material that was more resistant with a superior ability to decrease the degradation
rate. Meanwhile, after exposure to hydrogen peroxide (Figure 3D–F), rheological results
showed a plateau for both moduli of Candidate 1 and Belotero Balance® , a slight increase
in the G0 values and a slight decrease in the G00 values for Candidate 2. Therefore, tan (δ)
values of the three samples were stable over the 10 min measurement period. However,
Candidate 3 exhibited a completely different behaviour with a twofold increase in the G0
values and a 30% decrease in the G00 values, resulting in a significant decrease in the tan (δ)
(from 0.82 to 0.32).
Overall, the accelerated degradation assay performed using hyaluronidases and hy-
drogen peroxide showed that hydrogel candidates presented similar or higher storage
modulus (G0 ) values compared to those of Belotero Balance® at the end of the measure-
ments under both conditions, suggesting that the crosslinker-free technology can perform
similarly to a typical crosslinked HA filler in terms of degradation.
Pharmaceutics 2023, 15, 1708 10 of 18
Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 18

Figure 3. Belotero Balance® and the three candidates storage modulus (G’), loss modulus (G”), and
Figure 3. Belotero Balance® and the three candidates storage modulus (G0 ), loss modulus (G00 ), and
tangent delta (δ) normalized to initial values as function of time at 0.7 Hz and 37◦ °C after addition
tangent delta
of 100 µL (δ) normalized(100
of hyaluronidase to initial
U/mL) values
(A, B,as function
and C) (n =of
3; time
Meanat±0.7 HzAfter
SD). and an
37 addition
C after addition
of 100 µLof
100 µL of hyaluronidase (100 U/mL) (A–C) (n = 3; Mean ± SD). After an addition of 100
H2O2 (30% w/w) (D, E and F) (n = 3; ±sd). As a control condition, 100 µL of PBS buffer was added µL H2to
O2
(30% w/w) (D–F) (n
400 µL of the sample.= 3; ± sd). As a control condition, 100 µL of PBS buffer was added to 400 µL of
the sample.
 form of aqueous extract or their degradation product; cytocompatibility was measured by
conducting WST-1 assays.
In the case of extracts (Figure 4), all the samples were not only proved to be non-toxic,
but were also able to support cell proliferation because they had viability values > 100%.
No significant differences in cell viability were found between 24 h and 48 h of culture (p
Pharmaceutics 2023, 15, 1708 11 of 18
> 0.05). For comparison, a 0.1% SDS (positive control) induced high levels of mortality
(viability < 9%), while the diluted extraction samples (twofold and fourfold dilution) in-
duced no cytotoxicity ( see Supplementary Materials (Figure S3). The degradation prod-
3.3.
uctsBiological Evaluation hydrogels
of HA-L-pNIPAM of HA-L-pNIPAM
yieldedHydrogels
viability values ~ 85% (Figure 4); cell viability
3.3.1. In Vitro Biocompatibility on L929
was significantly higher in the presence of the Cell Linedegradation products of non-derivatized
HA (p < 0.05).fillers are categorized as a medical device that must undergo rigorous testing
Dermal
The hypothesis
to determine is that the non-derivatized
its biocompatibility regardless oflinear HA was efficiently
its mechanical, physicaldigested by hy-
and chemical
aluronidase[42].
properties as theTheenzyme had accessoftothe
biocompatibility itsnon-derivatized
backbone. In contrast, the spontaneous
linear HA (1500–1700 KDa) for-
mation
and the of nanostructurehydrogel
HA-L-pNIPAM in the HA-L-pNIPAM
candidates washydrogels
assessed inabove the LCST (observed
a mitochondrial un-
activity assay
der SEM
using in mouse
L929 a previous study [34])
fibroblasts. The hindered
HA-L-pNIPAM access hydrogel
to the cleavage sites and
candidates weretherefore
tested inlim-
the
form of aqueous
ited the extent ofextract
enzyme or digestion.
their degradation product;
As a result, cells cytocompatibility
in contact with these was “partially
measured di- by
conducting WST-1
gested” samples assays.from poor oxygen/nutrient diffusion, leading to a comparatively
suffered
lowerInmitochondrial
the case of extracts (Figure
activity. These 4),results
all theshowed
samplesthat
werethenot only proved tohydrogel
HA-L-pNIPAM be non-toxic,
can-
but wereinalso
didates theirable
testtoforms
support cell proliferation
(aqueous extract andbecause
enzymethey had viability
degradation valuesare
product) > 100%.
com-
No significant
patible with L929differences in cell viability
mouse fibroblasts. werecondition
The test found between
could be 24associated
h and 48 hwith of culture
the in
(p > 0.05). For comparison, a 0.1% SDS (positive control) induced high
vivo scenario considering the fact that dermal fillers were reported as being subject levels of mortality to
(viability < 9%),
swelling and while the
enzymatic diluted extraction
degradation samples
after injection. (twofold
The and fourfold
WST-1 assay dilution)
was chosen to mon- in-
duced
itor thenomitochondrial
cytotoxicity (see Supplementary
activity Materials
of cells after (Figure
exposure S3)). The degradation
to candidate formulations products
in this
of HA-L-pNIPAM
study hydrogelssimple
since it is a relatively yielded
and viability valuesmethod.
cost-effective ~ 85% (Figure 4); cell viability
The assessment of the was
bio-
significantly higher in the presence of the degradation products of non-derivatized
compatibility could be further completed by evaluating their effect on cell growth in terms HA
(p < 0.05).
of cell count, morphology, apoptosis, and cytokine profile.

Figure 4. Effects of raw HA and HA-L-pNIPAM polymers on L929 cells at 24 h of exposure. Data
Figure 4. Effects of raw HA and HA-L-pNIPAM polymers on L929 cells at 24 h of exposure. Data
express the percentage of cell viability with respect to culture media control (n = 3, Mean ± SD).
express the percentage of cell viability with respect to culture media control (n = 3, Mean ± SD).

The hypothesis is that the non-derivatized linear HA was efficiently digested by

hyaluronidase as the enzyme had access to its backbone. In contrast, the spontaneous
formation of nanostructure in the HA-L-pNIPAM hydrogels above the LCST (observed
under SEM in a previous study [34]) hindered access to the cleavage sites and therefore
limited the extent of enzyme digestion. As a result, cells in contact with these “partially
digested” samples suffered from poor oxygen/nutrient diffusion, leading to a compara-
tively lower mitochondrial activity. These results showed that the HA-L-pNIPAM hydrogel
candidates in their test forms (aqueous extract and enzyme degradation product) are com-
patible with L929 mouse fibroblasts. The test condition could be associated with the in vivo
scenario considering the fact that dermal fillers were reported as being subject to swelling
and enzymatic degradation after injection. The WST-1 assay was chosen to monitor the
mitochondrial activity of cells after exposure to candidate formulations in this study since
it is a relatively simple and cost-effective method. The assessment of the biocompatibility
could be further completed by evaluating their effect on cell growth in terms of cell count,
morphology, apoptosis, and cytokine profile.
Pharmaceutics 2023, 15, 1708 12 of 18

3.3.2. Tissue Integration in the Ex Vivo Porcine Skin Model

Porcine skin has been considered as a reliable surrogate for human skin given the
similarities in skin structure and vascularization network, and this supports its use to
predict histological behaviour in human skin after injection. Porcine ears were collected
from a local slaughterhouse after the animal was sacrificed as food supply, respecting the
3R principles—Replace, Reduce, Refine. Experimental setup of the ex vivo porcine skin
model is presented in Supplementary Materials (Figure S4). H&E-stained sections revealed
an intact overall structure of skin explants harvested from the blank control group at T0 and
T72 ; representative images obtained with the Axioscan Z1 brightfield microscope are pre-
sented in Supplementary Materials (Figure S5A). The main anatomical structures—stratum
corneum, viable epidermis, epidermal–dermal junction—and appendages including hair
follicles and secretory glands are well preserved after 3 days of cultivation; however, minor
morphological changes could be noticed, e.g., hyperplasia—a wavy structure with an
increased thickness of the epidermal layer—which is in accordance with previous reports
on human explant skin culture that a general increase in epidermal thickness is noted
over the first week in culture [43,44]. No further deterioration such as epidermal spongio-
sis (vacuolar fluid between the keratinocytes in the epidermis) or epidermal detachment
(stratum basale separated from dermis) was observed in the epidermis despite it being
considered more fragile than dermis due to its non-vascular structures and limited access
to the nutrient supply in culture. Collagen bundles in the reticular dermis layer were seen
to be slightly looser when comparing the samples at T72 to those at T0 . The histological
analysis demonstrated that tissue integrity is maintained unimpaired for up to 3 days in a
culture under the described conditions; therefore, the ex vivo porcine explant skin model
could be used for a short-term investigation.
Alcian Blue (AB) with PAS counterstaining [45–47] was employed to reveal the distri-
bution pattern of the hydrogels and the way they entangle themselves with the surrounding
adipocytes. Results of the validation performed with blank control group (no injection)
and positive control group (subcutaneous injection of natural HA or Belotero Balance® ) are
presented in Supplementary Materials (Figure S5B). Representative AB/PAS-staining im-
ages of the investigated candidates at 0 and 72 h after subcutaneous injection are presented
in Figure 5.
At T0 , all the injected area appeared as an irregularly shaped depot with the material
anchored in the fat lobules. The average dimensions of filler pools and filler spread patterns
varied despite the fact that the same volume (50 µL) was injected in each case. This
could result from the variation in the density of skin tissue at the injection site; therefore,
the force required for injection would be influenced and the injected gel could receive
forces/pressures from the surrounding tissues in a different pattern, which would be a
factor leading to the variations in the swelling of the biomaterials. It could be noticed
that Candidate 1 (Figure 5A) appears to be a continuous gel with a comparatively small
size, while Candidate 2 (Figure 5B) shows larger granules and a continuous texture—
both were distributed evenly throughout the subcutis as large homogeneous pools of HA
material. However, a fibrous gel network embedded with particulates could be observed
with Candidate 3 (Figure 5C), which exhibited the tendency to generate smaller pools and
generously spread within the hypodermis. After incubation with the skin model for 72 h, all
the injected materials were retained beneath the dermis at the injection site, but differences
in the extent of degradation were observed—Candidate 1 (Figure 5D) maintained the depot
of the HA materials with an excellent homogenous distribution, while a significant amount
of Candidate 2 (Figure 5E) was degraded. The superior resistance of Candidate 3 (Figure 5F)
could be attributed to the presence of linear HA. Our hypothesis is that the unmodified
HA was fragmented while embedding in the tissue, but the network constructed by the
HA-L-PNIPAM copolymer was conserved despite its uneven distribution pattern. These
results could be related to the viscoelastic properties and in vitro degradation profile of the
candidates, suggesting that Candidate 1 could be considered as the most promising dermal
filling material.
 x FOR PEER REVIEW
Pharmaceutics 2023, 15, 1708 1313of
of 18

Figure
Figure 5.5. Comparison
Comparison of
of subcutaneous
subcutaneous injection
injection of
of Candidates
Candidates 1–3
1–3 in
in an
an ex
ex vivo
vivo porcine
porcine skin
skin model
model
at T 0 (A–C) and T72(D–F). Light blue indicated the presence of the injected HA gel. Scale bar = 1 mm
at T0 (A–C) and T72 (D–F). Light blue indicated the presence of the injected HA gel. Scale bar = 1 mm
for (A,E) and 2 mm for (B–D,F) .
for (A,E) and 2 mm for (B–D,F).

Upon subcutaneousinjection,
Upon subcutaneous injection,Candidate
Candidate 1 pushed
1 pushed against
against thethe adjacent
adjacent adipocytes,
adipocytes, and
and the displacement and compression of the soft fat lobules
the displacement and compression of the soft fat lobules provided space for the provided space forHAthefiller
HA
filler integration
integration into
into the the subcutaneous
subcutaneous fat, leading
fat, leading to the to the formation
formation of a depot
of a depot in the sub-
in the subcutis as
cutis
shown asinshown
Figurein Figure
5A. 5A. However,
However, a XY planar a XY planar
section section
of the of the skin
skin explants explants
(Figure (Figure
6A) showed
6A)
that showed
the fibrousthat the fibrous
trabecular trabecular
network in the network
hypodermis in the
washypodermis
well preserved,was andwellthepreserved,
injection
and the injection bulks were constrained by the intercommunicating
bulks were constrained by the intercommunicating collagenous trabecular structures collagenous trabec-
with
ular structures with limited movement . The material had a homogenous
limited movement. The material had a homogenous texture and was uniformly distributed, texture and was
uniformly
conformingdistributed, conforming
to the adipose to the
layer of the hostadipose layer ofwithout
tissue border the hostinducing
tissue border without
any alteration
inducing
in the cellany alterationatinboth
morphology the cell morphology
T0 (Figure 6B,C) andat both
T72 T(Figure
0 (Figure 6B–C)
6D,E). and T72 a(Figure
Although slight
6D–E). Although
degradation of thea materials
slight degradation
was noticed of at
thethe materials
interfacewas withnoticed
the hostat tissue
the interface with
after culture
the host
for 72 h, tissue after culture
the homogenous for 72
filling h, the kept
material homogenous
its uniform filling material kept
distribution its and
pattern uniform
was
retained at the injection site. Interestingly, adipocytes appeared to
distribution pattern and was retained at the injection site. Interestingly, adipocytes ap-be diffusing into the
filling material
peared in certain
to be diffusing intoareas (Figure
the filling 6D), suggesting
material a good
in certain areas biocompatibility
(Figure 6D), suggesting of thea
candidates
good and the skinoftissue.
biocompatibility the candidates and the skin tissue.
The interaction between the biological tissue and the injected filler was considered a
critical criterion in the development of the HA dermal fillers. However, due to the new EU
MDR requirements of prohibiting animal testing, an ex vivo porcine skin model developed
in-house was employed for screening the innovative HA-L-pNIPAM hydrogels. In this
study, the three HA-L-PNIPAM hydrogel candidates were injected into the hypodermis
through a 30G needle that penetrated to a depth of approximately 4 mm as described
previously. On the one hand, intradermal injection was reported as having a high risk of
uneven distribution and host tissue response, as well as the formation of visible lumps
under the skin [48]; on the other hand, a systematic imaging study on the subcutaneous dis-
tribution of the three commercially available HA filler formulations provided the validation
Pharmaceutics 2023, 15, 1708 14 of 18

of the excellent safety profile for subcutaneous injection, which was the approach in most
actual clinical practice despite the product claims and indications [1,49]. AB/PAS-staining
allowed for a direct visualization of the injected materials without any further step for
labelling that might potentially change their behaviour. It not only provided evidence
for the similarity of Candidate 1 to commercial dermal fillers in terms of biointegration
immediately after injection (at T0 ) as previously reported [49], but also offered an insight
into the dynamic behaviour of the candidates as a function of time in the subcutaneous skin
layer under physiological conditions. It is of great interest to perform studies to quantitate
Pharmaceutics 2023, 15, x FOR PEER REVIEW 14 of 18
biological responses in the ex vivo skin model to further understand the safety and efficacy
of the proposed formulation.

Figure 6.
Figure 6. Distribution pattern and tissue integration of Candidate 1 at T00 (A –C) and T72
(A–C) 72(D –E) in an
(D,E)
Scalebar
ex vivo porcine skin model. Scale bar==22mmmmforfor (A),
(A), and
and 100100
µm µm for (B–E).
for (B–E).

Based on the original

The interaction between in vitro and ex vivo
the biological dataand
tissue presented herein,
the injected several
filler future areasa
was considered
of focuscriterion
critical have been identified,
in the such asofthe
development thedevelopment
HA dermal fillers.of a dermal
However,filler due
product
to the fornew
lip
enhancement or a bodyoffiller.
EU MDR requirements Physicians
prohibiting animalusetesting,
different antypes
ex vivoof HA to address
porcine skin modelvarious
de-
anatomic sites. The
veloped in-house washigh physicochemical
employed for screening tunability and versatility
the innovative HA-L-pNIPAM of HA-L-pNIPAM
hydrogels.
technology
In this study, the three HA-L-PNIPAM hydrogel candidates were injected into theAhypo-
may make it easy to develop specifications according to anatomic sites. labial
volumizer should exhibit a high shear modulus (i.e., exceeding 100
dermis through a 30G needle that penetrated to a depth of approximately 4 mm as de- Pa) and strong cohesivity.
Furthermore, it should
scribed previously. Onbe theadministered using extremely
one hand, intradermal smallwas
injection needles which
reported as cause
having minimal
a high
discomfort,
risk of uneven thereby facilitating
distribution and clinical usage response,
host tissue [41]. HA-L-pNIPAM
as well as candidates
the formation were oftailored
visible
to reachunder
lumps a highthe
storage modulus
skin [48]; on the G0other
at body temperature
hand, a systematic (i.e.,imaging
up to 420 Pa) while
study on theremaining
subcuta-
injectable through extremely fine 34G needles. Tissue integration
neous distribution of the three commercially available HA filler formulations providedinto the hypodermis on
an adapted ex vivo porcine skin model and a newly developed human
the validation of the excellent safety profile for subcutaneous injection, which was the ap- abdominal skin
model
proachshowed
in mostthe hydrogel
actual distribution
clinical patternthe
practice despite as product
a depot, claims
with retention at the injection
and indications [1,49].
site.
AB/PAS-staining allowed for a direct visualization of the injected materials without and
This innovation could enhance injection precision, reduce patient discomfort, any
minimize
further step post-injection
for labellinghematoma.
that mightThe potentialchange
potentially formulations were not only
their behaviour. It notestablished
only pro-
to be non-toxic
vided evidence and biocompatible,
for the similarity of but also exhibited
Candidate an extended
1 to commercial residence
dermal fillers duration
in terms of at
the site of injection due to the microgel structures of the system above
biointegration immediately after injection (at T0) as previously reported [49], but also of- the LCST [33,34].
This feature suggests that these formulations could be utilized as efficient drug delivery
fered an insight into the dynamic behaviour of the candidates as a function of time in the
systems. They could enable extended and sustained release of therapeutic agents, thereby
subcutaneous skin layer under physiological conditions. It is of great interest to perform
presenting a novel strategy for managing various dermatological conditions (e.g., psoriasis,
studies to quantitate biological responses in the ex vivo skin model to further understand
herpes, acne, and alopecia). With this capability, they could potentially offer an innovative
the safety and efficacy of the proposed formulation.
approach to treatment by providing the drugs where they are most needed and maintaining
Based on the original in vitro and ex vivo data presented herein, several future areas
the therapeutic effect over a longer period. This enhanced delivery system could lead to
of focus have been identified, such as the development of a dermal filler product for lip
enhancement or a body filler. Physicians use different types of HA to address various an-
atomic sites. The high physicochemical tunability and versatility of HA-L-pNIPAM tech-
nology may make it easy to develop specifications according to anatomic sites. A labial
volumizer should exhibit a high shear modulus (i.e., exceeding 100 Pa) and strong cohe-
Pharmaceutics 2023, 15, 1708 15 of 18

better patient compliance and potentially improved outcomes in managing dermatological

disorders.

4. Conclusions
In the present study, the advantages of HA-based thermosensitive fillers regarding
hydrogel injectability, enzymatic degradation resistance and tissue integration were high-
lighted and compared to those of commercial reference products. Copolymer syntheses
were performed in water; it should be possible to scale up the manufacturing process due
to the water-based, robust scheme of the EDC/NHS amidation and the efficiency of the
click chemistry approach. Three different fillers based on HA-L-pNIPAM (i.e., Candidates
1, 2 and 3) were tailored to reach a high storage modulus G0 at body temperature (i.e., from
92 Pa to 420 Pa) while remaining injectable through 34G needles at room temperature.
The ability of the thermosensitive fillers to form microgel structures above the LCST
offers major benefits in comparison to chemically crosslinked HA fillers in terms of in-
jectability for an easier clinical use including the increase in precision and pain reduction.
The resistance of hydrogels to enzymatic and ROS-mediated degradation was higher or
comparable to those of a commercial reference. A mix of linear HA 1.5–1.75 MDa and
highly derived HA-L-PNIPAM0.50 , Candidate 3, with HA contents of 9.5 mg/mL, showed a
higher resistance to enzymatic degradation than the reference product, which was also ob-
served after subcutaneous injection into an ex vivo porcine skin model developed in-house.
Candidate 1 (HA-L-pNIPAM0.10 , HA content of 15 mg/mL) was proposed as the most
promising dermal filling material considering the balance of the HA content, viscoelastic
properties, degradation profile and tissue integration.
The ex vivo and in vitro results presented herein warrant a next step toward in vivo
studies to confirm the advantages of use including the volumizer properties of HA-L-
pNIPAM formulations as a dermal filler product. The superior biocompatibility and
excellent distribution pattern of these HA-L-pNIPAM hydrogels not only confirmed their
potential as dermal fillers, but also opened the possibility for further investigation into
their application as a drug delivery platform for a prolonged and sustained release of
therapeutics. This is planned for future studies.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pharmaceutics15061708/s1, Figure S1. Synthetic route of HA-L-
PNIPAM. Amidation reaction and copper-free azide-DBCO click chemistry reaction; Figure S2. 1 H
NMR spectrum of HA (starting material); HA-Sulfo DBCO (intermediate product); HA-L-PNIPAM
(final product) in D2O; Figure S3. Effects of aqueous extraction obtained from raw HA and HA-L-
pNIPAM polymers on L929 cells at 24 h and 48 h of exposure; Figure S4. Experimental setup of the ex
vivo porcine skin model, skin explant (d = 14 mm, thickness ~1 cm) was anchored in a TC-Inserts
(SARSTEDT, d = 12 mm, Pore Ø = 1 µm, PET), which allowed for injection of 50 µL HA (3%, w/v)
subcutaneously; Figure S5. H&E and Alcian Blue/PAS staining of blank control group (skin biopsy
without injection) at T0 and T72 ; Figure S6. AB/PAS and H&E staining of raw HA and Belotero
Balance® at T0 ; Table S1. Content of each component in the final products; Table S2. Composition
of culture medium; Table S3. Protocol for Alcian blue/Periodic Acid Solution staining; Table S4.
Protocol for Hematoxylin/Eosin staining.
Author Contributions: Conceptualization, E.A., O.J. and Y.N.K.; methodology, S.G., A.P., E.A., O.J.
and Y.N.K.; experiments, S.G. and A.P.; investigation, S.G., A.P.; resources, E.A. and Y.N.K.; data
curation, S.G. and A.P.; writing—original draft preparation, S.G. and A.P.; writing—review and
editing, E.A., O.J., D.S., P.M. and Y.N.K.; supervision, O.J. and Y.N.K.; project administration, O.J.
and Y.N.K.; funding acquisition, O.J. and Y.N.K. All authors have read and agreed to the published
version of the manuscript.
Funding: The project was funded by a grant from the Swiss Innovation Agency (Inosuisse 44531.1
IP-LS).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Pharmaceutics 2023, 15, 1708 16 of 18

Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: O.J. and Y.N.K. would like to thank the Swiss Innovation Agency for the award
of a grant that provided financial support for S.G.
Conflicts of Interest: University of Geneva holds a patent covering the described hydrogel. A.P., E.A.
and O.J. hold shares in KYLYS Sarl.

Abbreviations
HA: hyaluronic acid; pNIPAM: poly(N-isopropylacrylamide); MW: weight-averaged molec-
ular weight; HMW: weight-averaged high molecular weight; ROS: reactive oxygen species; LCST:
lower critical solution temperature; EDC: N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide; NHS:
N-hydroxysuccinimide; DS: degree of substitution; SEM: scanning electron microscopy; G0 : stor-
age/elastic modulus; G00 : loss/viscous modulus; LVE: linear viscoelastic region.

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