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Enzymatic Active Release of Violacein
Present in Nanostructured Lipid
Carrier by Lipase Encapsulated in
3D-Bioprinted
Chitosan-Hydroxypropyl
Methylcellulose Matrix With
Anticancer Activity
Ignacio Rivero Berti
1
, Boris E. Rodenak-Kladniew
2
, Sergio F. Katz
1
, Eva Carolina Arrua
3
,
4
,
Vera A. Alvarez
5
, Nelson Duran
6
,
7
and Guillermo R. Castro
3
,
7
*
1
Laboratorio de Nanobiomateriale, CINDEFI, Departamento de Química, Facultad de Ciencias Exactas, CONICET (CCT La Plata),
Universidad Nacional de La Plata (UNLP), La Plata, Argentina,
2
Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP),
CONICET-UNLP, CCT-La Plata, Facultad de Ciencias Médicas, La Plata, Argentina,
3
Max Planck Laboratory for Structural
Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC), Partner Laboratory of the Max Planck Institute
for Biophysical Chemistry (MPIbpC, MPG), Centro de Estudios Interdisciplinarios (CEI), Universidad Nacional de Rosario, Rosario,
Argentina,
4
Centro de Investigación y Desarrollo en Materiales Avanzados y Almacenamiento de Energía de Jujuy-Univ. Nac., de
Jujuy, Argentina,
5
Grupo de Materiales Compuestos Termoplásticos (CoMP), Instituto de Investigaciones en Ciencia y Tecnología
de Materiales (INTEMA), Facultad de Ingeniería, Universidad Nacional de Mar del Plata (UNMDP), CONICET, Mar del Plata,
Argentina,
6
Laboratory of Urogenital Carcinogenesis and Immunotherapy, Department of Structural and Functional Biology,
Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil,
7
Nanomedicine Research Unit (Nanomed), Center for Natural
and Human Sciences (CCNH), Universidade Federal do ABC (UFABC), Santo André, Brazil
Violacein (Viol) is a bacterial purple water-insoluble pigment synthesized by Chromobacterium
violaceum and other microorganisms that display many beneficial therapeutic properties
including anticancer activity. Viol was produced, purified in our laboratory, and
encapsulated in a nanostructured lipid carrier (NLC). The NLC is composed of the solid
lipid myristyl myristate, an oily lipid mixture composed of capric and caprylic acids, and the
surfactant poloxamer P188. Dormant lipase from Rhizomucor miehei was incorporated into the
NLC-Viol to develop an active release system. The NLC particle size determined by dynamic
light scattering brings around 150 nm particle size and ζ≈−9.0 mV with or without lipase, but the
incorporation of lipase increase the PdI from 0.241 to 0.319 (≈32%). For scaffold development,
a 2.5 hydroxypropyl methylcellulose/chitosan ratio was obtained after optimization of a
composite for extrusion in a 3D-bioprinter developed and constructed in our laboratory.
Final Viol encapsulation efficiency in the printings was over 90%. Kinetic release of the
biodye at pH = 7.4 from the mesh containing NLC-lipase showed roughly 20% Viol fast
release than without the enzyme. However, both Viol kinetic releases displayed similar profiles at
pH=5.0,wherethelipaseisinactive.ThekineticreleaseofViolfromtheNLC-matriceswas
modeled and the best correlation was found with the Korsmeyer-Peppas model (R
2
= 0.95)
with n<0.5 suggesting a Fickian release of Viol from the matrices. Scanning Electron
Microscope (SEM) images of the NLC-meshes showed significant differences before and
Edited by:
Rosaria Ciriminna,
National Research Council (CNR), Italy
Reviewed by:
Deniz Yildirim,
Çukurova University, Turkey
Amiya Kumar Panda,
Vidyasagar University, India
*Correspondence:
Guillermo R. Castro
grcastro@gmail.com
Specialty section:
This article was submitted to
Green and Sustainable Chemistry,
a section of the journal
Frontiers in Chemistry
Received: 06 April 2022
Accepted: 07 June 2022
Published: 07 July 2022
Citation:
Rivero Berti I, Rodenak-Kladniew BE,
Katz SF, Arrua EC, Alvarez VA, Duran N
and Castro GR (2022) Enzymatic
Active Release of Violacein Present in
Nanostructured Lipid Carrier by Lipase
Encapsulated in 3D-Bioprinted
Chitosan-Hydroxypropyl
Methylcellulose Matrix With
Anticancer Activity.
Front. Chem. 10:914126.
doi: 10.3389/fchem.2022.914126
Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 9141261
ORIGINAL RESEARCH
published: 07 July 2022
doi: 10.3389/fchem.2022.914126
after Viol’s release. Also, the presence of lipase dramatically increased the gaps in the interchain
mesh. XRD and Fourier Transform Infrared (FTIR) analyses of the NLC-meshes showed a
decrease in the crystalline structure of the composites with the incorporation of the NLC, and
the decrease of myristyl myristate in the mesh can be attributed to the lipase activity. TGA
profiles of the NLC-meshes showed high thermal stability than the individual components.
Cytotoxic studies in A549 and HCT-116 cancer cell lines revealed high anticancer activity of the
matrix mediated by mucoadhesive chitosan, plus the biological synergistic activities of violacein
and lipase.
Keywords: Violacein, nanostructured lipid carriers, Lipase, chitosan, 3D-bioprinter, controlled release, Violacein
active release
1 INTRODUCTION
Bacterial metabolites make up a great pool of molecules that, in
addition to having a wide range of biological activities, are
relatively simple and inexpensive to produce in a sustainable
way (Venil et al., 2020). In 2020, the GLOBALCAN reported
more than 19 million new cases and 10 million deaths.
Additionally, 80% of new cases occur in individuals over
50 years of age, so as the world population ages, the
prevalence of these diseases grows accordingly (Ferlay et al.,
2020). Violacein (Viol) is a violet-purple pigment synthesized
by numerous Gram-negative bacterial strains that have
demonstrated beneficial antibacterial, antifungal, trypanocide,
antiviral, and antitumoral activities, among others (Durán
et al., 2021). Particularly, Viol inhibits the expression of some
cell markers associated with proliferation (e.g., cyclin-dependent,
mitogen-activated kinases, etc.), inhibit metalloproteinases,
enzymes essential to metastatic processes, and induces tumoral
cell death by different mechanisms mentioned recently (Durán
et al., 2016).
Viol is practically insoluble in aqueous media and its high
hydrophobicity correlated well with the parameter XLogP3-aa =
2.7. XLogP3-aa is a statistical parameter used to estimate the
hydrophobicity/hydrophilicity of a molecule considering the
contribution of each atom, hydrogen bridges, and the terminal
groups (Cheng et al., 2007). The Viol insolubility in aqueous
media is a serious limitation for its prospective application as a
therapeutic agent because of low bioavailability. Besides, it is
estimated that between 50% and 90% of the new drugs in
development and 40% of the drugs currently available in the
market present the same drawback (Liu, 2018). Thus, Viol could
be a promising new drug but also an excellent therapeutic agent if
a suitable drug delivery system is developed. Since Viol is a
hydrophobic molecule, lipid systems can be considered as a
potential best drug carrier (Rivero Berti et al., 2020). Among
them, liposomes, and lipid nanoparticles are probably the most
popular structures employed for the development of drug
delivery systems because of their simple preparation, non-
toxic, easy scale-up, and reproducibility. However, liposomes
are thermodynamically unstable, with high structural
dependence on environmental conditions, unknown cargo
concentration, and unpredictable kinetic release. Meanwhile,
solid lipid nanoparticles display several advantages, such as
improving the solubility of poorly soluble drugs, and also
increasing circulation time, thus avoiding possible resistance
mechanisms displayed by tumor cells, such as efflux pumps
(Creixell and Peppas, 2012). Solid lipid delivery systems
propose platforms that are relatively simple to prepare,
extremely stable, have high loading efficiency for hydrophobic
drugs and have high biocompatibility (Scioli Montoto et al.,
2020). Although SLNs have a high encapsulation efficiency, in
some cases when the nanoparticles mature, the lipids of the
matrix crystallize, and the drug is expelled during the process
with the consequent loss of loaded drug. In nanostructured lipid
carriers (NLC) systems an oily lipid is added to the solid lipid core
to introduce entropy and prevent and/or reduce lipid
crystallization (Gordillo-Galeano and Mora-Huertas, 2018). In
the present work, myristyl myristate (MM) was selected as the
main lipid component for the NLC, since MM has shown
advantages in NLC preparation such as emulsification
enhancer, effective thickener, low acute toxicity in oral or
dermal tests, and its extensively used in personal care and
cosmetic products (Islan et al., 2016;Shilling, 1990;Berti,
et al., 2019). Also, a commercial mixture of caprylic and
capric acids was employed in the NLC preparation because it
is an oily liquid lipid at RT and used as a thickener to improve the
texture and stability of emulsions, it is biodegradable and
commonly used in pharmaceutical and cosmetic formulations
(Islan et al., 2016).
However, one of the main problems in cancer therapies for the
delivery of nanoparticles containing hydrophobic and/or
environmentally sensitive drugs is the low attachment of
nanoparticles to the cancer cell surfaces, insufficient drug
penetration, and inadequate drug release kinetics (Wang et al.,
2018). The consequent drug release failure can result in molecular
degradation under physiological conditions (i.e., lytic enzymes),
fast clearance, and/or formation of insoluble complexes (i.e., π-
stacking) that result in low drug bioavailability and could produce
also undesirable side effects. A potential solution could be the
development of a stimuli-responsive controlled release system
(SRCRS), which will be particularly useful for the treatment of
solid tumors. Among the main advantages of SRCRS is the
circumvention of premature drug clearance, high accumulation
on targeted cells or tissues, and proper stimuli-responsive drug
release with controlled kinetics (Rahim et al., 2021). Importantly,
SRCRS reduces the drug amount to be administered and gets the
Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 9141262
Rivero Berti et al. Violacein Active Release
same therapeutic results, which is a substantial advantage for
improving the individuals’quality of life, and principally for
oncological patients. The use of enzymes for the development of
SRCRS to release the cargo in place and with appropriate kinetics
was described. Enzymes such as proteases, glycosidases, and
lipases were tested for the release of growth factors
(i.e., hepatocyte growth factors and VEGF), insulin,
doxorubicin, nitric oxide, etc. from nanoparticle and hydrogel
devices (Chandrawati, 2016;Wang et al., 2018;Shahriari et al.,
2019).
Specifically, Viol release from the NLC during nanoparticle
disintegration can be affected because the hydrophobic character
of the biodye could be stuck to the lipids, and consequently Viol
bioavailability will be reduced. In this sense, the inclusion of an
inactive but dormant “alive”lipase in the NLC can improve Viol
release from the lipids by hydrolyzing the ester linkages of the
lipids in the presence of a physiological environment. Lipase from
R. miehei showed optimum activity at temperatures between 37°C
and 40°C, in the presence of surfactants, but the optimal pH is
strain-dependent and goes from the alkaline range (i.e., pH
between 8.0 and 8.8) to the acid one (i.e., pH = 5.0–5.4) (Takó
et al., 2017). Both simultaneous conditions are relevant for cancer
treatment since solid tumors show high temperatures compared
to the nearby normal tissue because of high metabolic rates, and
also an acid surrounding environment due to the exacerbated
production of lactic and acetic acids (Rahim et al., 2021). In
addition, lipase released from the NLC can act on the membrane
surface of cancer cells, not only disturbing its structure and
function but also hydrolyzing lipids that could facilitate the
diffusion of Viol into the cells. Moreover, the presence of
lipase released from the NLC could catalyze the hydrolysis of
triglycerides present in the serum, reducing the formation of
precancerous lesions as well as colon, pancreatic, and prostate
cancers (Chandra et al., 2020).
On the other hand, medical implants provide a novel platform
from which the drug can be released over time. Medical implants
show at least two obvious advantages: first, the release is site-
specific (or locoregional), reducing the adverse side effects
resulting from the systemic drug circulation and allowing a
better quality of life for the patient since no other tissues and
organs will be in contact with the drug. Second, they guarantee
the patient’s adherence to the treatment (Ahmed et al., 2019). In
the specific case of solid tumors, whenever possible, resection
surgery is the first treatment option, but combined therapies are
key in cancer treatment, and surgery is frequently associated with
adjuvant chemotherapy treatments (Wolinsky et al., 2012).
Three-dimensional bioprinters can be used for the
development of organoids, tissue engineering, scaffolds for
drug delivery, tissue printing, and novel hybrid devices
(Stanisz et al., 2020). Particularly, a 3D bioprinter was
designed and constructed in our laboratory to develop
different matrix structures for drug delivery used as patches or
implants for the treatment of different pathologies (Katz and
Castro, 2019). The treatment of solid tumors with 3D-bioprinted
matrices has the advantages of personalized medicine because it is
possible to adjust the implant to the characteristics, size, and
location of the tumor. In addition, 3D implants can be placed as
close as possible to the tumor surfaces adjusted to the tumor
microenvironment or also in the cavity left by the resected tumor
after a surgery that reduces the drug circulation in the body and
consequently, the administered drug amount and its adverse
effects.
Two biopolymers were selected for the development of a 3D-
bioprinted matrix: chitosan (Chi) and hydroxypropyl
methylcellulose (HPMC) because of their advantages. Chi is
composed of ß-linked N-acetyl-D-glucosamine and
D-glucosamine randomly distributed. Chi possesses a residual
positive charge making the biopolymer mucoadhesive and cell-
adhesive, very relevant properties to enhance the attachment of
the biopolymer gels to the cell surfaces, which increases tissue
permeability and drug residence time. In addition, Chi displays
antimicrobial and antioxidant activities. Chi is nontoxic,
biocompatible, and biodegradable by mammalian enzymes,
making the biopolymer an excellent candidate for biomedical
applications in the form of gels, nanoparticles, fibers, and films
(Shariatinia and Jalali, 2018).
Cellulose is the most abundant biopolymer found in nature
and is composed of linear ß-D-glucose units of different lengths,
but its limited solubility reduces applications. HPMC is a derivate
of methylcellulose containing propylene glycol ether. Also,
HPMC is a nontoxic polymer, considered generally recognized
as safe (GRAS) by the FDA, and approved as a food additive by
the EU (Siepmann and Peppas, 2012).
In the present work, a platform of stimuli-responsive
controlled release system (SRCRS) based on NLC containing
lipase as a trigger agent and covered with Chi was integrated into
a polymeric matrix composed of Chi and HMPC. The composite
polymeric matrix was optimized for stability and to be extruded
by a 3D bioprinter developed and constructed in our laboratory.
The purpose of the 3D-printed matrix was to be used as an
implant. Violacein was selected as a hydrophobic drug model
entrapped in NLC for the potential treatment of solid tumors.
The matrix system was characterized using biophysical
techniques, TEM, FTIR, TGA, and dispersive light. The release
of Viol from the matrices was studied by structured kinetic
models. Cytotoxicity in vitro was studied in A549 and
HCT116 cancer cell lines.
2 MATERIALS AND METHODS
2.1 Materials
Violacein [3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-
3H-pyrrol-3-ylidene)-1,3-dihydro-2H-indole-2-one] was
synthesized by Chromobacterium violaceum CCT 3496 and
used following earlier techniques reported elsewhere. Briefly,
an isolated colony of C. violaceum from an agar plate was
inoculated in 250 ml Erlenmeyer containing 100 ml of a
medium composed of 5 g l
−1
D-glucose, 5 g l
−1
peptone, and
2gl
−1
yeast extract dissolved in MiliQ water. The vessel was
incubated at 150 rpm on a rotatory at 30°C for about 36 h. Later
the bacterial culture was centrifuged at 10,000 × g for 15 min at
5°C, and the supernatant was discarded. Violacein was purified
from the cells using a Soxhlet extractor. The Viol purity was 98%
Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 9141263
Rivero Berti et al. Violacein Active Release
purity determined spectroscopically (data not shown) (Mendes
et al., 2001;Berti, et_al., 2019).
Lipase, from R. miehei lyophilized powder (Lip), 4-
nitrophenyl palmitate (pNPP), Poloxamer 188 (Kolliphor®
P188), chitosan (Chi, poly (D-glucosamine) deacetylated
chitin, MW ≈161 kDa), and (hydroxypropyl)methylcellulose
(HPMC, MW ≈26 kDa) were bought from Sigma-Aldrich
(Buenos Aires, Argentina). Myristyl myristate a solid lipid at
RT (MM, Crodamol®MM, MP = 36–40°C), and cetyl palmitate
an oily triglyceride with saturated fatty acids (Crodamol®GTCC-
LQ, MP = −4°C) were a generous donation of CRODA®
(Argentina). All other chemicals, solvents, and reagents
employed in the present work were of analytical grade.
2.2 Lipase Activity and Stability
Measurements
Lipase activity was measured by the release of the colored
compound 4-nitrophenol from 4-nitrophenyl palmitate
(pNPP) as previously reported (Romero et al., 2012). Briefly,
pNPP was pre-dissolved in 1:4 acetonitrile-isopropanol
supplemented with 0.1% (w/v) Triton X-100. The pNPP
solution was dispersed in a 10 mM buffer supplemented with
0.2% (w/v) Arabic gum to reach a final concentration of 500 µM.
One ml of pNPP was placed in a 48-well plate to run the
enzymatic reaction. A lipase sample was added, and the
solution’s absorbance was measured at 405 nm at 37°C every
minute for 20 min to determine 4-nitrophenol concentrations.
Lipase activity was displayed as µmol of 4-nitrophenol released
per second.
The stability of lipase was determined at different pH values by
incubating the enzyme in different buffers at room temperature,
and then its activity was evaluated using buffer phosphate (pH =
7.4) and 37°C for 1 h.
2.3 Nanostructured Lipid Carrier and
Nanostructured Lipid Carrier With Lipase
Preparation
NLCs were synthesized by ultrasonication (Rodenak-Kladniew
et al., 2017). Concisely, a lipid phase containing 400 mg of the
solid lipid myristyl myristate (MM), 100 µl of a commercial
mixture of caprylic and capric acids (Crodamol®GTCC), and
20 µmol Viol was melted in a 70°C bath. Later, 20 ml of 4.0% (w/
v) poloxamer 188 adjusted to pH = 5.0 with acetic acid/sodium
acetate buffer was mixed with the lipid phase and sonicated in an
ultrasonic processor (130 W, Cole-Parmer, United States) at 70%
amplitude for 15 min.
The Viol remaining in the NLC was assayed in a 500 µl
sample of the NLC-Viol formulations taken and filtered in
100 kDa cut-off filters (Amicon®Ultra- 0.5 ml, Merck
Millipore, Ireland). The absorbance of the filtered solution
was measured at 580 nm.
For formulations containing lipase (Lip), 5.0 ml of NLC
dispersion was incubated with enough lipase to produce an
activity of 2.0 µmol of 4-nitrophenol/s at 5°C overnight with
an agitation of 150 rpm with a magnetic stirrer.
2.4 Nanoparticle Measurements
The average hydrodynamic diameter (D
H
), polydispersity index
(PdI), and Z-potential (ζ) were determined for NLC-Viol and
NLC-Viol-Lip samples diluted 1/100 [2.0% (w/v) initial
concentration and 0.02% (w/v) final concentration of MM] in
ultrapure water using a Zetasizer Nano ZS series (Malvern
Instruments, United Kingdom). The determinations were
performed at a 633 nm (He-Ne) laser at a 173°measurement
angle at 25°C by triplicate.
Transmission electron microscopy (TEM) images of the
nanoparticles were acquired in a Jeol-1200 EX II-TEM
microscope (Jeol, Columbia, MD, United States). NLC
suspensions were diluted 1/100 [2% (w/v) and 0.02% (w/v)
initial and the final concentration of MM, respectively] in
ultrapure water or 10 mM phosphate buffer (pH = 7.4)
followed by spreading a 10 µl sample on a 400 mesh Cu grid.
The excess sample was removed with filter paper. A
phosphotungstic acid drop was added to the samples for
contrast enhancement.
2.5 Biopolymer Ink Preparation and
Extrusion
A weighted amount of 200 mg chitosan was hydrated in 1.0 ml of
10% acetic acid to form a homogenous paste. Later, 1.5 ml of NLC
formulation with or without Lip was added and mixed, and then,
500 mg of HPMC was added, and the ink was thoroughly mixed
and charged in a sterilized 5 ml syringe with Luer lock
(TERUMO®, Philippines). The loaded syringe was later
centrifuged at 7,000 × g for 20 min to eliminate any air
bubbles from the 3D ink. A conic plastic tip or nozzle (Fish
Dispensing, China) with a 0.41 mm exit orifice was attached to
the syringe before extrusion.
The 3D meshes were produced by extrusion in a 3D printer
designed in our laboratory. Briefly, the 3D printer consists of an
extruder that precisely pushes ink out of the syringe, a positioning
system for the movements on three axes, and a controller. The
extruder is composed of a socket for the syringe and a bipolar
Nema 17 stepper motor with a high precision planetary system;
the movement on the three axes is provided by two bipolar Nema
17 stepper motors coupled with 8 mm THSL screws and anti-
backlash nuts. The open-source electronics used contain a
16 MHz ATmega 2560 processor that is commanded with
Marlin firmware.
The 3D meshes produced by 3D bioprinting were submerged
for 10 min in a 3.0% tripolyphosphate (TPP) solution, washed
with ultrapure water, and saved in the fridge for biophysical
characterization. Samples of TPP solution and ultrapure water
used for washing were taken to determine Viol concentration.
Figure 1 shows a simplified workflow of NLC and 3D-mesh
production.
2.6 Entrapment Efficiency of the Meshes
Entrapment efficiency was measured for both matrices
prepared with NLC-Viol (Mesh-Viol) and meshes prepared
with NLC-Viol-Lip (Mesh-Viol-Lip). This was conducted by
two methods. The indirect method was carried out by
Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 9141264
Rivero Berti et al. Violacein Active Release
measuring Viol in the TPP solution and in the water used to
wash the 3D meshes, and then subtracting the violacein
amount in those solutions from the initial amount of drug,
using the following equation: Eq. 1
EEindirect(%)
mVioli−(cViolTPPsol ×VTPPsol)
−(cViolww ×Vww)
mVioli
× 100% (1)
where mViol
i
is the initial amount of Viol present in the preparation,
cViol
TPPsol
is the concentration of violacein in the TPP solution,
cViol
ww
is the concentration of Viol in the ultrapure water after
washing, and V
TTPsol
and V
ww
are the volumes of those solutions.
On the other hand, the direct method consisted in adding
3.0 ml of ethanol to a weighted amount of Mesh-Viol or Mesh-
Viol-Lip followed by sonication in an ultrasonic cleaning bath for
30 min. The procedure was performed twice with ethanol
replacement. The efficiency was then calculated by the
following equation: Eq. 2
EEdirect(%)cViol1×V1+cViol2×V2
mVioli
× 100% (2)
where mViol
i
is the initial amount of Viol added to the
preparation, cViol
1,
and cViol
2
are the concentration of Viol in
the first and second ethanol extraction, and V
1
and V
2
are the
volumes of those solutions.
2.7 Violacein Release From the 3D-Printed
Meshes
Four meshes were weighed and placed in 30 ml of 10 mM phosphate
buffer at pH = 7.4 and 37°C, in duplicate. Every hour a 1.0 ml sample
of the media was taken and replaced with an equal volume of fresh
buffer. The samples were later filtered in 100 kDa cut-off filters
(Amicon
®
Ultra- 0.5 ml, Merck Millipore, Ireland). Filtered
solution absorbance was determined at 580 nm to evaluate Viol’s
release. The results were expressed as a fraction of a total load of Viol
released versus time. The Viol release profiles were analyzed by
structured kinetic release models (Supplementary Table S1).
2.8 Physicochemical Characterization of the
3D Meshes
2.8.1 Fourier Transform Infrared Spectroscopy
FTIR spectra were performed in freeze-dried in a Nicolet 6700
model (Thermo Scientific CT, United States) spectrometer
coupled with an attenuated total reflectance (ATR) accessory.
Scans were performed 32 times for each sample in the range from
600 to 4,000 cm
−1
with a 4 cm
−1
resolution.
2.8.2 X-Ray Diffraction
Diffraction patterns of the freeze-dried samples were obtained in
an Analytical Expert instrument using Cu-K radiation (θ=
1.54 Å) from 2θ=10
°to 70°in continuous mode with 0.07°
step size.
2.8.3 Thermal Analysis
Thermogravimetric analysis (TGA) was performed in a
Shimadzu TGA-50 instrument. All assays were carried out in
the range of 20°C–900°C at a heating rate of 10°C/min under an
N
2
atmosphere.
Differential scanning calorimetry (DSC) was carried out on a
Perkin Elmer Inc., Model Pyris 1 (Waltham, MA, United States)
instrument under an N
2
atmosphere. The heating rate of 10°C/
min was used in the range of 0–250°C. All samples were
previously frozen-dried.
2.9 Scanning Electron Microscope Image
Acquisition
SEM analysis of 3D-printed meshes was run with freeze-dried
samples that were then covered with a gold layer thickness of
15–20 nm using a Balzers SCD 030 metallizer. Images were
obtained in a Philips SEM 505 scanning electron microscope
and processed in an image digitizer program (Soft Imaging
System ADDA II, SIS). Later, the images were analyzed with
ImageJ
®
software (NIH, United States), and the surface roughness
was estimated by the standard deviation of grey values in the
image histogram (Wang et al., 2005).
FIGURE 1 | Simplified workflow to produce the 3D meshes.
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Rivero Berti et al. Violacein Active Release
2.10 Cytotoxic Studies
Human colorectal carcinoma HCT-116 and lung
adenocarcinoma epithelial A549 cells were cultured in
Minimal Essential Medium (MEM, HyClone, CA,
United States) supplemented with 10% fetal bovine serum
(Internegocios S.A., Argentina) and 1.0% penicillin/
streptomycin (Gibco Invitrogen Corporation,
United States) in 5.0% CO
2
at 37°C. The viability of the
cells was assayed using MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] assay (Mosmann, 1983).
HCT-116 and A549 cells (4.0 × 10
3
cells/ml) were seeded in
96-well plates and incubated under standard conditions for
24 h. Then, the cells were exposed to 0.5, 2, or 4.0 µM of free
Viol (prepared in DMSO lower than 0.05% (v/v) final
concentration), or equivalent amounts (µM of Viol or mg/
ml of MM) of NLC and NLC-Lip with and without Viol for
24 h. The medium was discarded and incubated in the
presence of 100 µl of 500 μg/ml MTT solution prepared in
MEM with 3 h incubation. Formazan was dissolved in 100 µl
DMSO. The microplates were stirred for 5 min and readings
of absorbance at λ= 560 nm were determined in a microplate
reader (Beckman Coulter DTX 880 Multimode Detector,
United States). A reduction in cell viability by more than
30% was considered a cytotoxic effect (Doktorovova et al.,
2014). A conditioned medium was used to assay Mesh-Viol
and Mesh-Viol-Lip. That is, meshes with and without viol,
and with and without Lip were incubated in MEM
supplemented as before for 48 h. Then, the media was used
to incubate A549 and HCT116 cells for 24 h. Later, MTT
colorimetric assays were performed as previously described.
3 RESULTS AND DISCUSSION
3.1 Lipase Activity and Nanostructured Lipid
Carrier Preparation
The lipase activity was evaluated at different pH values at room
temperature. The high activity of lipase from R. miehei was
detected at pH = 7.4, but no enzyme activity was observed at
pH = 5.5 (Figure 2). However, 95% of enzyme activity was
recovered at pH = 7.4 after lipase incubation at pH = 5.5 for
6 h, indicating a reversible enzyme inhibition. Based on this
result, the synthesis of NLC containing lipase was performed
at pH = 5.5.
Encapsulation efficiency of Viol in the NLC-Viol without and
with lipase was near 100% since Viol concentration in the filtered
solution was below the limit of detection (Supplementary
Material Table S3).
3.2 Nanostructured Lipid Carrier Size and
Z-Pot
The average hydrodynamic diameter (D
H
), PdI, and Z-Pot (ζ)
are shown in Supplementary Table S2. The addition of lipase
does not significantly alter the mean particle size (154.3 nm for
NLC-Viol and 151.5 nm for NLC-Viol-Lip; p>0.05), but it
does significantly alter the PdI (0.241 for NLC-Viol and 0.319
for NLC-Viol-Lip; p<0.05). The ζvalue is close to zero, as
might be expected from the fact that the surfactant used to
stabilize NLC, P188, is neutral in charge. The ζchange between
formulations with and without lipase is significant (p<0.05)
but still very small. Results are summarized in Supplementary
Table S2.
TEM images for NLC-Viol-Lip before and after incubation in
a 10 mM phosphate buffer (pH = 7.4) for 6 h are shown in
Figure 3. Before incubation NLC-Viol-Lip showed an average
diameter of 93.6 nm based on 36 measurements from
micrographs (Figure 3A). After incubation aggregates were
observed (Figure 3B, arrows). These aggregates can be
explained since lipase activity probably weakens the structure
of the nanoparticle by catalyzing lipid hydrolysis, and therefore
remnant NLC produces aggregate structures.
3.3 Biopolymer Ink Preparation and
Extrusion
Different proportions of Chi and HPMC were tested to create an
appropriate polymer mixture to be extruded by the 3D bioprinter.
The final polymer concentrations for the bioink were 6.6% (w/w)
Chi and 16.6% (w/w) HPMC, equivalent to HPMC/Chi: 2.5 ratio.
Lower HPMC content resulted in an unstable printed bioink,
while higher HPMC concentrations drastically reduced the cross-
linking with TPP.
Maximum printing speed that did not produced errors in ink
continuity found was 400 mm/min. For the nozzle diameter used
(0.41 mm) a layer height of 0.33 mm was found optimal, since
layer heights less than that, caused the nozzle to damage the lower
layer and larger layer sizes prevented the correct adhesion of the
developing layer on the lower layer. A video illustrating the
printing process can be found in Supplementary Material.
The two methods used to estimate EE (%) gave differences
lower than 10% among them (Supplementary Table S3).
However, the average EE (%) of Mesh-Viol and Mesh-Viol-Lip
was 93.8% and 92.3%, respectively. These values are very close
considering the several experimental steps for the production of
the drug delivery devices (i.e., sonication, mixing, cross-linking,
and washing).
FIGURE 2 | Lipase activity measured at pH = 7.4 and 37°C after
incubation for 6 h at room temperature at the indicated pH (white, □)and
lipase activity assayed at the indicated pH and 37°C (black, ■).
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Rivero Berti et al. Violacein Active Release
3.4 Violacein Release From the Meshes
The difference between the two Viol release profiles of matrices
with or without lipase is obvious (Figure 4). Lipase strongly
stimulates the release of Viol from the polymeric matrix, reaching
60% release at pH 7.4 after 2 h incubation. While only 20% of Viol
was released from the matrix without lipase under the same
experimental conditions. The kinetic release of Viol from Mesh-
Viol and Mesh-Viol-Lip was analyzed with typical structured
kinetic models (Supplementary Table S1). The best correlations
of Viol release were found with Higuchi (R
2
= 0.94), Korsmeyer-
Peppas (R
2
= 0.95) and Baker-Lonsdale (R
2
= 0.95) tested models
for the matrix without lipase. While the best correlation of Viol
release from the matrix containing lipase was observed in the
Korsmeyer-Peppas model (R
2
= 0.95) (Supplementary Table S5).
The kinetic of Viol release from the matrices and Baker-Lonsdale
fit for Mesh-viol and Korsmeyer-Peppas for Mesh-Viol-Lip are
displayed in Figure 4. The “n”parameter in the Korsmeyer-
Peppas model can be used to predict the potential mechanism
involved in the drug release (Supplementary Table S1). In this
case, Mesh-Viol fitted an n= 0.17 and Mesh-Viol-Lip an n= 0.46;
both cases fall below n<0.5 condition. Considering these
experimental values, the Korsmeyer-Peppas model predicts a
Viol release based mostly on Fick’s law, i.e., only based on the
diffusional mechanism (Ritger and Peppas, 1987).
Additionally, Viol releases from the Mesh-Viol and Mesh-
Viol-Lip matrices were performed at pH = 5.0. No significant
differences were found between the Viol release in both matrices
(Supplementary Figure S1). These results are indicative of the
relevant lipase role in Viol release. However, the Viol release was
faster than in pH = 7.4 buffer, with around 65% of the drug
released in the first 2 h, which can be expected since Chi solubility
is greatly enhanced at lower pH (Kou et al., 2017).
3.5 Mesh Physicochemical Characterization
3.5.1 Scanning Electron Microscope Micrograph
Results
The SEM images display the matrix structure after exposing the
3D prints to 10 mM phosphate buffer (pH = 7.4) for 24 h
(Figure 5). The SEM images showed a relaxed matrix
structure with mesh gaps expanded, determined by the ImageJ
software (Supplementary Table S4). Meshes before drug release
did not exhibit any significant difference (p>0.05) and are
presented as one single group. However, changes in the structure
of the meshes with and without Lip after Viol release were
significant (p<0.05). The Mesh-Viol-Lip displayed a big
variation in the matrix structure that can be attributed to the
presence of lipase in the matrix. This loosening in polymeric
structure can also be observed in the microstructure at higher
magnifications (×1,000) (Figure 5). Mesh-Viol-Lip shows a larger
number of cavities between polymer areas. Also, the roughness of
Mesh-Viol-Lip estimated by the grey histogram confirmed this
result (Supplementary Table S4). A comparative analysis of the
standard deviations of the matrices showed that Mesh-Viol and
Mesh-Viol-Lip before drug release possess smother surfaces (SD
= 35.5) than Mesh-Viol or Mesh-Viol-Lip after the release (SD =
40.1 and 48.3 respectively).
FIGURE 3 | (A) TEM micrography of a 1:100 dilution of NLC-Viol-Lip, and (B) TEM micrography of a 1:100 dilution of NLC-Viol-Lip after it was incubated at a pH =
7.4 for 6 h. Arrows in (B) point aggregates.
FIGURE 4 | Violacein kinetic release from mesh (C), and mesh
containing lipase (▲) at pH = 7.4 respectively. Baker and Lonsdale model fitfor
mesh (−−,R
2
= 0.95) and Korsmeyer-Peppas model fit(−− −−,R
2
= 0.95) for
mesh containing lipase. Upper and lower 95% confidence interval for
each fit(·····).
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Rivero Berti et al. Violacein Active Release
3.5.2 X-Ray Diffraction Analysis
Figure 6 shows XRD patterns for both 3D meshes after 24 h
incubation in release buffer (pH = 7.4) as well as their pure
components. P188 showed a high degree of crystallinity when
pure, with two intense and characteristic peaks at 19.1°and 23.3°,
and a dimmer broader peak between 25.5°and 27.0°(He et al.,
2011). Chi showed a small peak at 19.7°and a dim broad peak at
9°, while HPMC exhibited a wide peak between 15°and 25°.
However, none of these peaks appear in the mixtures, indicating a
loss of the crystalline structure of the polymers in the composite.
Mesh-Viol and Mesh-Viol-Lip show a small compound peak at
21.6°and 23.7°(Figure 6 inset); these peaks also appear and are
characteristic of the MM diffraction pattern, although in much
higher intensity, which indicates that a few amounts of MM
remain in crystals after NLC preparation.
3.5.3 Fourier Transform Infrared Analysis
FTIR spectra for both 3D meshes as well as their major
constituents incubated in phosphate buffer (pH = 7.4) for
24 h are displayed in Figure 7. P188 showed an intense peak at
2,877.32 cm
−1
attributed to C-H aliphatic stretching, a smaller
peak at 1,342.23 cm
−1
attributed to in-plane O-H bending, and
a sharp peak at 1,099.24 cm
−1
, and a smaller peak at
960.39 cm
−1
corresponding to ether C-O symmetric and
asymmetric stretching vibrations (Lal and Datta, 2014).
HPMC spectrum shows a broad peak between 3,100 and
3,600 cm
−1
corresponding to O-H stretching vibrations,
2,894.67, and 1,049.10 cm
−1
in agreement with C-H and
C-O stretching respectively (Furqan et al., 2017). Chi also
showed a broad O-H stretching vibration band between 3,100
and 3,600 cm
−1
, but partially overlapped with broadband due
to asymmetric/symmetric N-H bonds. The band at
2,871.53 cm
−1
was assigned to C-H axial stretching, while
the 1,641 cm
−1
band was attributed to -C=O stretching in
the acetamide groups, and the 1,556 cm
−1
to the trans-
secondary amides (Acosta-Ferreira et al., 2020;Peng et al.,
2020); additionally, the 1,019 cm
−1
band was attributed to C-N
primary amine stretch (Peng et al., 2020). MM showed three
sharp peaks at 2,848.39, 2,915.88, and 2,954.45 cm
−1
,
attributed to asymmetric, symmetric stretching of C-H
bonds on −CH
2
and C-H bond asymmetric stretching on
−CH
3
groups respectively. Another relevant sharp peak was
found at 1731.8 cm
−1
, corresponding to ester carbonyl
stretching vibrations (Castro et al., 2021). Finally, both 3D
meshes showed a complex broad peak between 1,150 and
900 cm
−1
, from overlapping P188, HPMC, and Chi peaks in
that band of the spectrum, and broadband between 3,000 and
3,500 cm
−1
from O-H and N-H vibrations in HPMC and Chi.
However, the 3D meshes containing Lip (Mesh-Viol-Lip)
exhibited weaker peaks at 2,848.39 and 2,915.88 cm
−1
,the
bands presented by MM. Furthermore, Mesh-Viol-Lip did
not show the characteristic peak at 1731.8 cm
−1
attributed
to −C = O stretching of the ester group, while it appeared
in the 3D mesh without Lip (Mesh-Viol). All these points
could reflect a loss in MM content in Mesh-Viol-Lip due to
lipase activity in the 3D mesh.
FIGURE 5 | SEM images for the 3D-Mesh previous release (A), Mesh-Viol and Mesh-Viol-Lip released in phosphate buffer (pH = 7.4) after 24 h incubation (B) and
(C) respectively.
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Rivero Berti et al. Violacein Active Release
3.5.4 Thermogravimetric Analysis
The thermal stability of the 3D meshes after release was studied.
Figure 8 shows the percentage mass loss versus temperature.
Both 3D meshes lose 10% of their weight between 0°C and 125°C,
which is due to remnant water in the samples; similarly, Chi and
HPMC lose 9% and 3% of their weight respectively.
The maximal HMPC weight loss was at 346°C, and before
400°C more than 90% of its mass was lost. This process is
normally attributed to the decomposition of cellulose esters,
and volatilization of these degraded residues (Hasanin et al.,
2022). On the other hand, Chi-MMW, weight loss after 250°Cis
due to further dehydration, deacetylation, and
depolymerization (de Britto and Campana-Filho, 2007). MM
maximum weight loss was found at 283°C; this step is normally
attributed to volatilization rather than thermal degradation (Xu
et al., 2022).
Between 125°C and 290°C Mesh-Viol and Mesh-Viol-Lip lose
36.5% and 35.2% of their mass respectively, which coincides with
the acute mass loss of MM. Moreover, after finishing the essay,
FIGURE 6 | Diffractograms for Mesh-Viol-Lip, Mesh-Viol, MM (myristyl
myristate), Chi-MMW (medium molecular weight chitosan), HPMC
(hydroxypropyl methylcellulose), and P188 (Poloxamer P188). Mesh-Viol and
Mesh-Viol-Lip were incubated for Viol release in phosphate buffer (pH =
7.4) for 24 h before performing the analysis. Inset is a detail for Mesh-Viol-Lip
and Mesh-Viol between 10°and 40°.
FIGURE 7 | FTIR spectra for Mesh-Viol-Lip, Mesh-Viol, MM (myristyl
myristate), Chi-MMW (medium molecular weight chitosan), HPMC
(hydroxypropyl methylcellulose), and P188 (Poloxamer P188). Mesh-Viol and
Mesh-Viol-Lip were incubated in release buffer (pH = 7.4) for 24 h before
performing the analysis.
FIGURE 8 | Thermogravimetric analysis for Mesh-Viol-Lip, Mesh-Viol,
MM (myristyl myristate), Chi-MMW (medium molecular weight chitosan),
HPMC (hydroxypropyl methylcellulose), and P188 (Poloxamer P188). Mesh-
Viol and Mesh-Viol-Lip were incubated in phosphate buffer (pH = 7.4) for
24 h before performing the analysis.
FIGURE 9 | DSC thermograms for Mesh-Viol-Lip, Mesh-Viol, MM
(myristyl myristate), Chi-MMW (medium molecular weight chitosan), HPMC
(hydroxypropyl methylcellulose), and P188 (Poloxamer P188). Mesh-Viol and
Mesh-Viol-Lip were incubated in phosphate buffer (pH = 7.4) for 24 h
before performing the analysis. Y-axis represents heat flow (exothermic is up).
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Rivero Berti et al. Violacein Active Release
the Mesh-Viol has a remnant of 32.4% of its weight, 31.7% of
Mesh-Viol-Lip, and 25.0% of Chi. Since none of the other
constituents remain at this temperature, the meshes exhibit
greater thermal stability than pure materials, suggesting a
chemical interaction between the biopolymers. In the
Supplementary Material,afigure showing weight derivative
(%) versus temperature is provided (Supplementary Figure S2).
3.5.5 Differential Scanning Calorimetry Analysis
The heat flow from the 3D mesh samples and their main
constituents versus temperature, “up”curves in the Y-axis
representing the exothermic process, and “down”curves showing
the endothermic process, are displayed in Figure 9.MMshowsa
sharp intense peak at 41.7°C, corresponding to its melting
temperature (T
m
), with an area under peak, that is, a melting
enthalpy (ΔH
m
) of -205 J/g, both results are consistent with the
bibliography (Aydn and Okutan, 2011), a small shoulder can be
attributed to a pleiomorphism transition. Similarly, T
m
for P188 is
53.4°C(He et al., 2011), and a ΔH
m
of −145 J/g. The broad
endothermic peak between 50°Cand100
°CinHPMCandChi-
MMW can be attributed to a loss of water (El Maghraby and
Alomrani, 2009). DSC analysis for mixtures, Mesh-Viol and Mesh-
Viol-Lip showed a sharp and small peak near MM T
m
, at 40.2°Cand
39.9°C respectively. This T
m
depression can be explained by the
Gibbs-Thomson equation (Supplementary Equation S1)and
(Supplementary Material)(Han et al., 2008). Based on the
Gibbs-Thomson equation, as the radius of the nanoparticle
decreases, so does its melting point. Also, a bigger and wider
peak appears before 100°C. Mesh-Viol-Lip small peak was
smaller, while the big broad peak was bigger, which may indicate
a change in Mesh lipid and water content after drug release.
3.6 Cytotoxicity Assays
Figure 10 A and B show cytotoxicity for different NLC
formulations and free Viol. At 0.5 µM (filled in black) NLC-
Viol showed a greater effect than free Viol. On the contrary, the
cytotoxic effect was greater in 2.0 µM free Viol (filled in grey).
This was observed for both cell lines tested. This phenomenon
was already reported in previous works where Viol was
nanovehiculized (Berti, et_al., 2019;Berti et al., 2022).
Additionally, in both cell lines tested NLC-Viol-Lip showed
more cytotoxicity at 2.0 µM (filled in grey) than NLC-Viol (p<
0.05), even though at this concentration lipase and empty NLC
showed no toxic effects. This result revealed that a more efficient
biocide effect between NLC, Lip, and Viol could be attributed to the
increase of free biodye due to nanoparticle degradation by lipase
activity. However, other effects could contribute to this result.
Recently, Viol was described as a membrane disruptor in an
attempt to explain its various biological effects (Gupta et al.,
2021). Considering this hypothesis, the activity of lipase and the
activity of violacein could produce a positive effect at their site of
action. Additionally, the incorporation of Lip into the formulation
can impact positively on the treatment of solid tumors since it was
found that lipase activity is suppressed or absent in some breast,
colorectal, and lung tumors; therefore, lipase activity may have a
tumor-suppressive effect (Sun et al., 2013).
Figure 10C shows the cytotoxicity assay for A549 and
HCT116 cell lines when they were incubated in a medium
that was in contact with 3D-printed meshes. CM-control
represents a medium without treatment, that is, without 3D-
printed mesh, which was incubated for the same period as the
treated media. This experimental procedure was considered the
“aging”of the medium alone, which presents a reduction in
viability in the HCT116 cell line (p<0.005). However, the
FIGURE 10 | Cell viability For NLC formulations and free Viol in A549 (A)
and HCT-116 (B) cell lines, respectively. Cell viability on conditioned
medium (C).
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Rivero Berti et al. Violacein Active Release
biopolymeric matrix alone without violacein or lipase (Mesh) did
not further reduce viability when compared with CM-control (p>
0.5). Besides, both the lipase effect (Mesh-Lip) and violacein effect
(Mesh-Viol) significantly decreased the cell viability (p<0.005).
Additionally, the major cytotoxic effect on the HCT116 cell line
was observed with Mesh-Lip-Viol, being greater than that of
Mesh-Lip or Mesh-Viol (p<0.05 in both cases).
4 CONCLUSION
Recent strategies for the treatment of several diseases could
involve the use of enzymes, with more than 100 potential
candidates. However, marketed enzymes applied in
biomedicine are still scarce (Bosio et al., 2016). The emergence
of nanotechnology opened a new landscape for the development
of efficient devices used in the treatment and diagnosis of diverse
pathologies, particularly cancer. In this regard, diverse
approaches of nanoparticles containing enzymes to target
tumors were recently reviewed (Li et al., 2020;Kapalatiya
et al., 2022). Smart nanoparticles containing enzymes were
developed using different strategies such as decorating
nanoparticle surface with specific enzymes acting over the
tumor surface or a ligand of nanoparticle surface for releasing
covalently linked molecules, nanoparticles loaded with prodrugs
enzyme-activable, and nanoparticles with enzyme-degradable
core for drug controlled release. In all cases, the effect of the
environmental conditions to trigger the drug is crucial,
particularly with enzymes located on the surface of the
nanoparticle which could be exposed to extreme conditions
such as high shear rate and acid pHs produced by the high
metabolism of tumor cells (i.e., lactic, and acetic acids) that can
inactivate and/or denature the biocatalyst. In the present work,
NLC contains a hydrophobic potential drug, Viol, and a latent
lipase that can be activated under physiological conditions by
hydrolyzing the lipid structure of the nanoparticle with
consequent release of the biodye with controlled kinetic. Also,
active lipase could act over cancer cells’membrane facilitating the
contact of Viol within the cells and the entrance into the
cytoplasm. Particularly, the amount of enzyme can be
optimized to control the lipid hydrolysis and consequently
change the amount of Viol release. Additionally, the NLC
coated with Chi could guarantee the attachment of the NLC
to the surface of the tumor cells (Ahmad et al., 2017).
The progress of 3D scaffolds made of biopolymer is a new trend
for personalized medicine not only for the study of the
development of cancer tumors but also to find novel strategies
for cancer therapy, particularly after tumor resection and/or
intensive treatments when surgeries are not advised (Augustine
et al., 2021). In previous work, bacterial cellulose scaffolds
containing SLN loaded with doxorubicin were successfully
applied in-vivo to treat an orthotopic breast cancer mouse
model. The results indicate a substantial decrease in tumor
growth and metastasis, as well as drug amount by the
treatment with a doxorubicin-NLC-loaded scaffold (Cacicedo
et al., 2018).
The novel trends in biomedicine are involving the
development of more complex structures for tissue
engineering and repair, drug delivery, and organ
fabrications. Among them, the 3D bioprinter allowed for
the printing of hydrogels for the construction of 3D
matrices (i.e., scaffolds) with defined and unique properties.
Considering a personalized tumor therapy, the use of a 3D-
bioprinter allows tailoring scaffolds with defined chemical
composition and dimensions (i.e., size and thickness). In
addition, the inclusion of the NLC into the 3D Chi-HPMC
printed matrix will help to reduce the dispersion of the
nanoparticles in the body. Also, the contact of the 3D
scaffold containing the NLC-Viol with the solid tumor can
be associated with a decrease in the administered drug amount
which reduces the undesirable side effects of cancer drugs and
increases patient comfort.
Finally, a novel Stimuli-Responsive Controlled Release
System platform based on a 3D bioprinter and NLC
containing a hydrophobic molecule actively released by an
enzyme was developed. The main advantages of the platform
are the versatility of the system based on: 1) the development of
the composition of an appropriate nanostructured lipid carrier
according to the physicochemical properties of the drug; 2) the
incorporation of lipase in the NLC-drug that can control the
kinetics of the drug release and the amount of the drug; 3) the
inclusion of the NLC-drug-enzyme in a biodegradable 3D
matrix that can be tailored based on the tumor
characteristics; 4) the 3D matrix containing the drug could
be placed on the tumor site avoiding drug dispersion in the body
and/or inactivation and/or undesirable side effects; 5) the 3D
matrix containing chitosan, a mucoadhesive and cell-adhesive
polymer, can increase the contact with the tumor cells; 6) the
presence of lipase and chitosan in the formulation could
produce a positive effect, facilitating the entrance of the drug
into the cells since both components could act over cell
membranes just hydrolyzing lipids and interfering with the
transport cell mechanisms.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
IR, main experimental work, design, and data analyses and
curation, writing, and drawing. BR-K, cellular assay
experiments, and data analysis, writing. SK, 3D bioprinter
experiments. EA, characterization of nanoparticles and
discussion. VA, experimental determination of biophysical
properties of materials. ND, critical review, and text edition
and discussion. GC, supervision, experimental design, writing,
review and editing, funding acquisition.
Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 91412611
Rivero Berti et al. Violacein Active Release
FUNDING
Financial support by UNLP (X815), CONICET (PIP 0034), and
ANPCyT (PICT 2016-4597; PICT 2017-0359) is gratefully
acknowledged.
ACKNOWLEDGMENTS
Financial support by UNLP (X815), CONICET (PIP 0034), and
ANPCyT (PICT 2016-4597; PICT 2017-0359) is gratefully
acknowledged. We would also like to thank Lucia S. Carnaghi
for her help along with the development of the
experimental work.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fchem.2022.914126/
full#supplementary-material
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