Pharmaceutics

pharmaceutics
Article
Design of Innovative Biocompatible Cellulose Nanostructures
for the Delivery and Sustained Release of Curcumin
Francisca Casanova , Carla F. Pereira *, Alessandra B. Ribeiro , Eduardo M. Costa , Ricardo Freixo, Pedro
M. Castro, João C. Fernandes, Manuela Pintado and Óscar L. Ramos *
CBQF—Centro de Biotecnologia e Química Fina–Laboratório Associado, Escola Superior de Biotecnologia,

Universidade Católica Portuguesa, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal
* Correspondence: cpfpereira@ucp.pt (C.F.P.); oramos@ucp.pt (Ó.L.R.)
Abstract: Poor aqueous solubility, stability and bioavailability of interesting bioactive compounds is
a challenge in the development of bioactive formulations. Cellulose nanostructures are promising
and sustainable carriers with unique features that may be used in enabling delivery strategies. In
this work, cellulose nanocrystals (CNC) and cellulose nanofibers were investigated as carriers for the
delivery of curcumin, a model liposoluble compound. Nanocellulose modification with the surfactant
cetyltrimethylammonium bromide (CTAB), tannic acid and decylamine (TADA), and by TEMPO-
mediated oxidation were also tested and compared. The carrier materials were characterized in
terms of structural properties and surface charge, while the delivery systems were evaluated for their
encapsulation and release properties. The release profile was assessed in conditions that mimic the
gastric and intestinal fluids, and cytotoxicity studies were performed in intestinal cells to confirm safe
application. Modification with CTAB and TADA resulted in high curcumin encapsulation efficiencies
of 90 and 99%, respectively. While no curcumin was released from TADA-modified nanocellulose
in simulated gastrointestinal conditions, CNC-CTAB allowed for a curcumin-sustained release of
ca. 50% over 8 h. Furthermore, the CNC-CTAB delivery system showed no cytotoxic effects on
Caco-2 intestinal cells up to 0.125 g/L, meaning that up to this concentration the system is safe to use.
Citation: Casanova, F.; Pereira, C.F.; Overall, the use of the delivery systems allowed for the reduction in the cytotoxicity associated with
Ribeiro, A.B.; Costa, E.M.; Freixo, R.; higher curcumin concentrations, highlighting the potential of nanocellulose encapsulation systems.
Castro, P.M.; Fernandes, J.C.; Pintado,
M.; Ramos, Ó.L. Design of Innovative Keywords: nanocellulose; curcumin; delivery systems; sustained release; cytotoxicity
Biocompatible Cellulose
Nanostructures for the Delivery and
Sustained Release of Curcumin.
Pharmaceutics 2023, 15, 981.
1. Introduction
https://doi.org/10.3390/
pharmaceutics15030981 Poor bioavailability of bioactive compounds is an increasingly pronounced challenge
in the development of bioactive formulations. Around 40% of marketed active compounds
Academic Editor: Yunlu Dai
and up to 70% of candidates showing high potential in the pipeline of pharmaceutical,
Received: 30 January 2023 nutraceutical and food industries show hydrophobicity, liposolubility or poor aqueous
Revised: 28 February 2023 solubility, resulting in limited potential and unsatisfactory efficacy when administrated
Accepted: 15 March 2023 orally [1–3]. Bioactive compounds such as lipophilic phenols, carotenoids, lipophilic
Published: 18 March 2023 vitamins or phytosterols have very interesting biological functions, but their low water
solubility and stability result in poor bioavailability, restricting their applications [4,5].
These limitations can potentially be overcome by using enabling delivery systems, which
require carrier materials with desirable properties [6,7]. Certain liposoluble compounds
Copyright: © 2023 by the authors.
have been used as model bioactive molecules to study encapsulation strategies, amongst
Licensee MDPI, Basel, Switzerland.
which special interest has been given to curcumin, a natural phenolic compound with
This article is an open access article
important biological properties, namely antimicrobial, anti-inflammatory, anti-mutagenic
distributed under the terms and
and cholesterol-lowering activities [8–11].
conditions of the Creative Commons
Attribution (CC BY) license (https://
Nowadays, there is a growing need for the utilisation of biocompatible raw mate-
creativecommons.org/licenses/by/
rials for the delivery of liposoluble compounds [12]. Proteins, e.g., whey protein [13],
4.0/). caseins [14], gelatin [15] and soy proteins [16] are biocompatible carrier materials, but
Pharmaceutics 2023, 15, 981. https://doi.org/10.3390/pharmaceutics15030981 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2023, 15, 981 2 of 19
they tend to aggregate and are susceptible to disruption under physiological conditions
in the gastrointestinal (GI) tract [17]. Lipid-based carriers, such as lipossomes [18], emul-
sions [19], nano-structured lipid carriers [20] and solid lipid nano-particles [21] are safe
and attractive carriers; however, these materials might experience undesirable phenomena
(e.g., Ostwald ripening, aggregation, oxidation, gelation) resulting from their physical
and chemical instability. Considerable attention has been drawn to natural polysaccha-
rides, namely chitosan [22], cyclodextrins [23], amylose [24], alginate [25], starches [26],
pectin [27] and cellulose [4,28], due to their abundance, low cost, low toxicity, biocompati-
bility and biodegradability. Cellulose is the world’s most abundant natural polymer and it
can be obtained from several renewable and sustainable plant sources, such as lignocel-
lulosic biomass from industrial and agricultural wastes. Non-plant sources of cellulose
also exist, e.g., cellulose produced by bacteria, algae and tunicates [29–31]. The native
cellulose fiber structure is composed of nanostructured cellulose (NC), which can be further
divided into cellulose nanofibers (CNF) and cellulose nanocrystals (CNC). These have
essentially different extraction procedures, as well as different dimensions, morphologies
and crystalline structures, as reviewed by Casanova et al. (2021) [11]. Still, both CNF and
CNC are composed of repeating β-D-glucopyranose units with three hydroxyl groups per
anhydroglucose unit (AGU). Interest in cellulose nanostructures as carriers in delivery
systems has increased in the past few years due to their unique physicochemical properties,
such as renewability, biocompatibility, biodegradability, high surface area and amphiphilic
nature [11].
Nevertheless, surface modification of cellulose structures may be necessary in order
to optimize the loading and release profile of encapsulated lipophilic compounds such
as curcumin, as ascertained by Zainuddin et al. (2017) and Foo et al. (2019) [4,11,28]. In
fact, one interesting cellulose feature is the surface chemical reactivity and accessibility of
hydroxyl groups for chemical modification, which may provide additional functionalities
to the cellulose molecule [32]. Hydrophobic surface modification [4,28] and functionaliza-
tion of cellulose structures by TEMPO-mediated oxidation (2,2,6,6-tetramethylpiperidine
1-oxyl–TEMPO) [33] have been employed to modulate the encapsulation and delivery of
lipophilic compounds, namely curcumin and carvacrol. Hydrophobic modification with
cetyltrimethylammonium bromide (CTAB, a hydrophobic cationic type surfactant) [28,34,35],
and with tannic acid (TA) and decylamine (DA) [4] have been previously studied for the
encapsulation of lipophilic molecules showing high encapsulation efficiencies superior to
90%. However, the biocompatibility of such delivery systems has rarely been analyzed, and
this information is of crucial importance for the applicability of the systems (considering
that surfactants and amino compounds are being used and are possibly toxic). Further-
more, studies showing the release profile of encapsulated bioactive compounds from such
delivery systems are also scarce.
In the present work, CNC and CNF are investigated as delivery systems for curcumin,
a model liposoluble compound. Hydrophobic modification with CTAB and TADA, and
functionalization by TEMPO-mediated oxidation, were also tested and compared, for
both CNC and CNF, in order to evaluate the influence of the different approaches in the
encapsulation and release of curcumin. This work exploited the physicochemical and en-
capsulation properties of such cellulose-based delivery systems, but also the release profile
of the model lipophilic compound (curcumin) in conditions that mimic the gastrointestinal
fluids (pH, temperature and time of exposure). Moreover, the biocompatibility of the
developed delivery systems through in vitro cytotoxicity studies was also assessed. To the
best of our knowledge, this study is unique as there are no reported studies comparing the
performance of different approaches (including chemical modified materials) simultane-
ously and for both CNC and CNF intended for the encapsulation of lipophilic compounds.
Additionally, the release profile under gastrointestinal conditions of such systems has been
investigated to a far lesser extent, and biocompatibility and toxicity studies, which are vital
to assess the applicability of said systems, are rarely performed.
2. Materials and Methods

2.1. Reagents
The reagents used in the experiments were of analytical grade or higher. Commercial
CNC, CNF and CNF-TEMPO (1.4 mmol COONa/g) were kindly supplied by Cellulose Lab
(Fredericton, Canada). According to the supplier, CNC had a needle-like morphology with
10–20 nm width and 50–400 nm length; CNF had a fibrillar morphology with a fiber width
of 50 nm and lengths of up to several hundred microns; CNF-TEMPO had a fibrillar mor-
phology with a fiber width of 40 nm and lengths of up to several hundred microns. CTAB
(cetyltrimethylammonium bromide) (>99.0%), tannic acid (>99.0%), potassium phosphate
(>99.0%), hydrochloric acid (32%) and dimethyl sulfoxide (>99.9%) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Decylamine (99.0%) was purchased from Thermo
Fisher Scientific (Waltham, MA, USA), sodium hydroxide (>95.0%) and sodium chloride
(>99.5%) were purchased from Labchem (Zelienople, PA, USA) and ethanol (>99.8%) was
purchased from Honeywell (Charlotte, NC, USA).
2.2. Production of CNC-TEMPO

CNC-TEMPO was produced from commercial CNF-TEMPO by a method adapted
from Zhou et al. (2018) [36]. Briefly, a CNF-TEMPO suspension (0.5% w/v) was subjected
to ultrasonic irradiation for 10 min with a JP Selecta CY-500 Ultrasonic Homogenizer
(Barcelona, Spain) in an ice bath to avoid overheating. The processor was equipped with a
cylindrical titanium alloy probe tip of 10 mm diameter. Sonication was performed at 80%
amplitude for 5 min at a time to replace the ice bath (500 W, 20 kHz, pulse durations of
15 s on and 5 s off). Aqueous suspension of CNC-TEMPO was freeze-dried for further use.
CNC-TEMPO was produced with a 96.8% ± 1.73 yield from commercial CNF-TEMPO.
2.3. CNC and CNF Modification with CTAB Surfactant

Commercial CNF and CNC (0.4%, w/v aqueous suspension) were modified with an
aqueous solution of CTAB (2.0 mM) by a method adapted from Zainuddin et al. (2017) [28].
The nanocellulose suspensions were slowly added into the CTAB solution, and the mixture
was heated at 60 ◦ C for 3 h. The reaction was left stirring at room temperature overnight.
The unbound CTAB was removed by centrifugation at 10,000 rpm for 10 min. The pellet
was washed with distilled water, followed by another round of centrifugation. This step
was repeated twice. Aqueous suspensions of CNF-CTAB and CNC-CTAB were freeze-dried
for further use and demonstrated production yields of 87.95% ± 2.01 and 82.45% ± 1.26
from CNF and CNC, respectively. The degree of substitutions (DS) was measured by the
percentage of nitrogen (% N) according to Zainuddin et al. (2017) using the Dumas method
in a Foss Dumatec™ 8000 (Hillerød, Denmark) [28,37]. CNF-CTAB had a substitution
degree (DS) of 0.081 ± 0.011 and CNC-CTAB a DS of 0.124 ± 0.005.
2.4. CNC and CNF Modification with TA and DA

The method of modifying nanocellulose with TA and DA was adapted from Foo
et al. (2019) and Hu et al. (2017) [4,38]. CNF and CNC aqueous suspensions (1%, w/v)
were adjusted to pH 8 by addition of 1 M NaOH solution and then TA was added to the
suspensions at the concentration of 1 mg/mL in the final solution. The mixture was stirred
at 200 rpm for 6 h at room temperature to form NC-TA suspensions. Then, DA was added
at the concentration of 40 mg/mL in the final solution. The resulted NC-TADA suspensions
were stirred at 200× g rpm for 3 h, before being centrifuged for 10 min at 1000× g rpm. The
supernatant was discarded, and the pellet was re-suspended in water, followed by another
round of centrifugation. This step was repeated twice. Finally, the modified nanocellulose
suspensions were collected and freeze-dried. CNF-TADA and CNC-TADA were produced
with yields of 113.15% ± 4.45 and 118.33% ± 2.35 from CNF and CNC, respectively.
2.5. Characterization
2.5.1. Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy
The Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy (ATR-FT-IR)
spectra were recorded using the Frontier™ MIR/FIR spectrometer from PerkinElmer in
a scanning range of 550–4000 cm−1 for 16 scans at a spectral resolution of 4 cm−1 . All
analyses were performed in duplicate.
2.5.2. Powder X-ray Diffraction

Powder X-ray Diffraction (PXRD) analyses were performed on a Rigaku MiniFlex
600 diffractometer with Cu kα radiation, with a voltage of 40 kV and a current of 15 mA
(3◦ ≤ 2θ ≥ 60◦ ; step of 0.01 and speed rate of 3.0◦ /min). The Segal Crystallinity Index (CI,
%) was calculated using the following equation:
It − Ia
CI(%) = × 100 (1)
Ia
where It is the total intensity of the (200) peak for cellulose I at 2θ = 22.5◦ , and Ia is the
amorphous intensity at 2θ = 18◦ for cellulose I [39,40]. All analyses were performed in
duplicate.
2.5.3. Zeta Potential Determination

Zeta potential (0.1%, w/v aqueous suspensions) was evaluated by dynamic light
scattering (DLS) using a Malvern Instrument Zetasizer Nano ZSP (Malvern, UK). The
measurements were made at room temperature (25 ◦ C) using a folded capillary cell at
a constant detection angle of 173◦ . The equipment was equipped with a 10 mW He-Ne
laser with an emission wavelength of 633 nm. Data were acquired and analyzed using the
Malvern Zetasizer v. 7.11 software. All measures were performed in triplicate.
2.5.4. Microscopy Analysis

Morphology was evaluated by Scanning Electron Microscopy (SEM) on a Thermo
Scientific™ Pro Scanning Electron Microscope. Prior to analysis, the samples were placed
in observation stubs covered with double-sided adhesive carbon tape (NEM tape, Nisshin,
Japan) and coated with Au/Pd (target SC510-314B from ANAME, S.L., Madrid, Spain) using
a Sputter Coater (Polaron, Bad Schwalbach, Germany). All observations were performed in
high vacuum with an acceleration voltage of 5 kV. The images presented are representative
of the morphology of the sample.
Particles’ morphology was also evaluated by Transmission Electron Microscopy (TEM),
where 10 µL of the samples were mounted on Formvar/carbon film-coated mesh nickel
grids (Electron Microscopy Sciences, Hatfield, PA, USA) and left standing for 2 min. The
liquid in excess was removed with filter paper and the grid was allowed to contact with a
drop of uranyl acetate 2% (w/v) for 2 min. The liquid in excess was removed with filter
paper. Visualization was carried out on a JEOL JEM 1400 TEM (Tokyo, Japan) at 120 kV.
Images were digitally recorded using a CCD digital camera (Orious 1100W, Tokyo, Japan)
at the HEMS/i3S of the University of Porto.
2.6. Curcumin Loading

CNC as carrier was used for the preliminary curcumin:carrier ratio screening. The
ratios tested were 1:2, 1:3, 1:5 and 1:10 (w/w). Calculated amounts of curcumin were
dissolved in ethanol, and then CNC was added to the curcumin solution to achieve a final
CNC concentration of 2% (w/v) in ethanol (70%, v/v), followed by continuous stirring at
250 rpm for 30 min. The curcumin-loaded particles were collected by centrifugation at
10,000 rpm for 10 min at room temperature. The unbound curcumin that remained in the
ethanolic supernatant was analyzed by Ultraviolet-Visible (UV–Vis) Spectrophotometry
using a BioTek Epoch 2 Microplate Reader (Winooski, VT, USA) and the pellet was freeze-
dried after being washed with distilled water by centrifugation twice. Curcumin loading
into CNF and functionalized materials (CNC-TEMPO, CNF-TEMPO, CNC-CTAB, CNF-

CTAB, CNC-TADA and CNF-TADA) was performed as described above for CNC using
the best performing curcumin:carrier ratio.
2.7. Curcumin Quantification

Curcumin was quantified by UV–Vis Spectrophotometry with detection at 425 nm.
Five external standards of curcumin were analyzed (from 0.8 to 12.5 µg/mL) in duplicate
and a curcumin calibration curve was constructed in ethanol (70%, v/v). The method
presented good linearity with a correlation coefficient of 0.9999, and good precision results
with coefficients of variation values lower than 5%.
2.8. Encapsulation Efficiency, Loading Capacity and Yield

The yield (%) was expressed as the ratio of the final freeze-dried mass obtained and
the mass of the starting materials. The unbound curcumin (Curunbound ) that remained
in the supernatant after curcumin loading was analyzed by UV–Vis Spectrophotometry.
The encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the
following equations:
Cur added − Curunbound
EE(%) = × 100 (2)
Cur added
Cur added − Curunbound
LC (%) = × 100 (3)
f inal mass
2.9. Curcumin Release Profile

A simplified protocol that simulates the gastric and intestinal conditions was adapted
from Gorbunova et al. (2018) and Valo et al. (2011) [41,42]. Fifty milligrams of each
encapsulated particle were added to 20 mL of simulated gastric fluid (SGF) comprising
a NaCl solution at 2 g/L adjusted to pH 2 by the addition of 1 M HCl. The experiment
was performed at 37 ◦ C for 2 h under agitation to simulate the digestion conditions at
the stomach. Aliquots of 150 µL were withdrawn every hour and added to 350 µL of
ethanol, and the extracted medium was replaced with fresh medium. The samples were
centrifuged, and curcumin concentration was determined in the supernatant by UV–Vis
Spectrophotometry. After 2 h in SGF, the samples were centrifuged and 20 mL of simulated
intestinal fluid (SIF) comprising a KH2 PO4 solution at 6.8 g/L adjusted to pH 7 by the
addition of 1 M NaOH was added to the pellet. The experiment was performed at 37 ◦ C
for 6 h under agitation to simulate the digestion conditions of the intestine. Samples were
withdrawn every hour as described above. The samples were centrifuged, and curcumin
concentration was determined in the supernatant by UV–Vis Spectrophotometry. The
percentage of curcumin released was determined and plotted over time. All experiments
were performed in duplicate.
2.10. Cytotoxicity Evaluation

Cell Line Growth Conditions. Human colon carcinoma (Caco-2) cells were obtained
from the European Collection of Authenticated Cell Cultures. Cells were cultured at
37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 as monolayers using Dul-
becco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine and no
pyruvate (ThermoScientific, Massachusetts, USA), supplemented with 10% Fetal Bovine
Serum (ThermoScientific, Massachusetts, USA), Penicillin-Streptomycin-Fungizone (1%,
v/v) (ThermoScientific, Massachusetts, USA) and Non-Essential Amino Acids Solution (1%,
v/v) (MEM NEAA, ThermoScientific, Massachusetts, USA). The cells were used between
passages 28 and 32.
Cytotoxicity Assay. Cytotoxicity evaluation was performed according to the ISO
10993-5:2009 standard in Caco-2 cells [43]. The cells were grown to ca. 80% confluence,
detached using TrypLE Express (ThermoScientific, Waltham, MA, USA) and seeded at
1 × 104 cells/well into a 96-well microplate. After 24 h, the culture media was carefully
removed and replaced with culture media supplemented with curcumin (concentrations
between 0.01 and 0.2 mg/mL) or curcumin encapsulating particles (concentrations between
0.03 and 0.5 mg/mL). DMSO at 10% (v/v) in culture media was used as death control, and
plain culture media was used as growth control. After 24 h of incubation, 10 µL of Presto
Blue reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and
the plate was incubated for 2 h. After this period, fluorescence intensity measurements
(Ex: 560 nm; Em: 590 nm) were performed in a BioTek Synergy H1 Microplate Reader
(Winooski, VT, USA). All assays were performed in quadruplicate. Results were given in
terms of percentage of the cell metabolism inhibition.
2.11. Statistical Analysis

All analyses were performed at least in duplicate and the results are reported as the
mean values and standard deviations. As the data followed a normal distribution by the
Shapiro–Wilk Test, one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s
test (p < 0.05) was conducted to determine the significant differences between mean values
Pharmaceutics 2023, 15, x FOR PEER REVIEW 7 of 20
using the SPSS 28.0 Statistical Software Program (SPSS Inc. Chicago, IL, USA).
3. Results
noticed
3.1. at 2850 cm−1ofand
Characterization 2925 cm
the Carrier −1, also reported by Zainuddin et al. (2017) [28], which
Materials
correspond
3.1.1. to theAnalysis
ATR-FT-IR symmetric and asymmetric CH2 stretching vibrations of the alkyl chain
in CTAB.
Commercial CNC, with
Modification CNFCTAB involves the physical
and CNF-TEMPO, adsorption
and prepared of the surfactant
CNC-TEMPO, CNC-CTAB,mol-
ecules onto the surface of nanocellulose via electrostatic attraction
CNF-CTAB, CNC-TADA and CNF-TADA were structurally characterized in terms of of opposite charges
(Figure 2b).
functional Strongusing
groups electrostatic
ATR-FT-IR interactions were due
analysis (Figure 1). to the binding
Vibrations of the
related cationic
to cellulose
charged headare
modifications of CTAB with
identified inthe negatively
Figure chargedillustration
1. A schematic NC surfaces [28].
of the As the surfactant
molecular products
was non-covalently bound with nanocellulose, no major differences in
obtained by the TEMPO-mediated oxidation of nanocellulose and modification with CTAB the ATR-FT-IR
spectra
and TADA of modified and unmodified
are represented in Figure 2.CNC and CNF were observed.
Figure 1. ATR-FT-IR
Figure 1. ATR-FT-IRspectra
spectraof
ofthe
themodified
modifiedand
andunmodified
unmodifiedcellulose
cellulosenanocrystals
nanocrystals(CNC)
(CNC)and cellu-
and cel-
lulose
lose nanofiber
nanofiber (CNF)
(CNF) materials.
materials. Nanocellulose
Nanocellulose modifications
modifications were were performed
performed with cetyltrime-
with cetyltrimethylam-
thylammonium
monium bromidebromide
(CTAB),(CTAB), tannic
tannic acid andacid and decylamine
decylamine (TADA),
(TADA), and and by TEMPO-mediated
by TEMPO-mediated oxidation
oxidation
(TEMPO). (TEMPO).
Characteristic cellulose vibrations were found in all nanocellulose samples, namely

at: 3200–3400 cm−1 (O-H stretching), 2900 cm−1 (C-H stretching), 1600–1640 cm−1 (O-H
bending), 1430 cm−1 (CH2 bending), 1110 cm−1 (C-O-C stretching) and 900 cm−1 (C-H
rocking), indicating that cellulose was preserved in all modification processes. Similar
vibrations in the wavelength positions observed here have been reported in the literature
for CNF [44] and CNC [45–47], but also for TEMPO-mediated [36,48,49], CTAB [28,35] and
TADA [4,38] modified celluloses.
Figure 1. ATR-FT-IR spectra of the modified and unmodified cellulose nanocrystals (CNC) and cel-
lulose nanofiber (CNF) materials. Nanocellulose modifications were performed with cetyltrime-
thylammonium bromide (CTAB), tannic acid and decylamine (TADA), and by TEMPO-mediated
oxidation (TEMPO).
Figure
Figure 2. Illustration
2. Illustration ofof
(a)(a) the
the nanocellulose-basedproduct
nanocellulose-based product obtained
obtained by
by TEMPO-mediated
TEMPO-mediatedoxida-
oxidation;
tion; and the interaction of nanocellulose (NC) with (b) cetyltrimethylammonium bromide (CTAB)
and the interaction of nanocellulose (NC) with (b) cetyltrimethylammonium bromide (CTAB) and
and (c) tannic acid and decylamine (TADA).
(c) tannic acid and decylamine (TADA).
After oxidation, both CNC-TEMPO and CNF-TEMPO exhibited a new vibration at

1600 cm−1 , corresponding to C=O stretching in carboxyl functional groups bound to a
sodium ion (COONa) (Figure 2a). Similar vibrations have been reported in the literature for
TEMPO mediated oxidized CNF [48,49] and CNC [36]. In general, characteristic vibrations
on the spectra of CTAB modified nanocelluloses exhibited similar spectral properties
with the unmodified CNC and CNF materials. Similar results have been reported in the
literature by Qing et al. (2016) for CTAB modified CNC [35]. Only very small vibrations
can be noticed at 2850 cm−1 and 2925 cm−1 , also reported by Zainuddin et al. (2017) [28],
which correspond to the symmetric and asymmetric CH2 stretching vibrations of the alkyl
chain in CTAB. Modification with CTAB involves the physical adsorption of the surfactant
molecules onto the surface of nanocellulose via electrostatic attraction of opposite charges
(Figure 2b). Strong electrostatic interactions were due to the binding of the cationic charged
head of CTAB with the negatively charged NC surfaces [28]. As the surfactant was non-
covalently bound with nanocellulose, no major differences in the ATR-FT-IR spectra of
modified and unmodified CNC and CNF were observed.
For the TADA-modified samples, the presence of asymmetrical and symmetrical CH2
stretches from the C10 alkyl chain at 2850 cm−1 and 2925 cm−1 suggests the attachment
of decylamine onto CNC and CNF [38]. Notable changes across the broad band in the
3200–3500 cm−1 region were also observed, with more prominent peaks at 3330 cm−1 and
3345 cm−1 , which have been ascribed to N=H stretching [4]. Furthermore, new peaks at
1460 cm−1 corresponding to C=C aromatic stretching vibration, at 1470 cm−1 attributed
to secondary N–H bending (indicating Michael addition occurred), and at 1650 cm−1
assigned to C-N stretching were detected, confirming the introduction of quaternary
ammonium groups to CNC and CNF [4,38]. Moreover, the vibration intensity of the ether
bond at 1055 cm−1 was stronger for all TADA-modified samples, which also indicates
the attachment of long alkyl groups to the nanocellulose [28]. Similar vibrations in the
wavelength positions observed here have been reported in the literature for TADA-modified
nanocellulose [4,38]. The chemical structure of the tannic acid primer, containing catechol
groups, and decylamine, which imparts hydrophobicity, is shown in Figure 2c. In step 1,
tannic acid reacts with NC at pH 8, as under these conditions polyphenol oxidation and
to C-N stretching were detected, confirming the introduction of quaternary ammonium
groups to CNC and CNF [4,38]. Moreover, the vibration intensity of the ether bond at 1055
cm−1 was stronger for all TADA-modified samples, which also indicates the attachment of
long alkyl groups to the nanocellulose [28]. Similar vibrations in the wavelength positions
observed here have been reported in the literature for TADA-modified nanocellulose
Pharmaceutics 2023, 15, 981 [4,38]. The chemical structure of the tannic acid primer, containing catechol groups, and 8 of 19
decylamine, which imparts hydrophobicity, is shown in Figure 2c. In step 1, tannic acid
reacts with NC at pH 8, as under these conditions polyphenol oxidation and oligomeriza-
tion is known to occur in the presence of available dissolved oxygen. This produces qui-
oligomerization is known to occur in the presence of available dissolved oxygen. This
nones that in the second step of the modification react with primary amine groups in dec-
produces quinones that in the second step of the modification react with primary amine
ylamine via a Schiff-base reaction and/or Michael addition, as depicted in Figure 2c
groups in decylamine via a Schiff-base reaction and/or Michael addition, as depicted in
[38,50]. As strong covalent bonds are formed during this modification, clear and multiple
Figure 2c [38,50]. As strong covalent bonds are formed during this modification, clear and
changes in the ATR-FT-IR spectra of CNC and CNF were observed.
multiple changes in the ATR-FT-IR spectra of CNC and CNF were observed.
3.1.2.
3.1.2.PXRD
PXRDAnalysis
Analysis
Commercial CNC, CNF and CNF-TEMPO, and prepared CNC-TEMPO, CNC-CTAB,
Commercial CNC, CNF and CNF-TEMPO, and prepared CNC-TEMPO, CNC-CTAB,
CNF-CTAB, CNC-TADA and CNF-TADA were structurally characterized in terms of
CNF-CTAB, CNC-TADA and CNF-TADA were structurally characterized in terms of
crystallographic structure using PXRD. The PXRD patterns and crystallinity indices are
crystallographic structure using PXRD. The PXRD patterns and crystallinity indices are
shown in Figure 3.
shown in Figure 3.
Figure
Figure3.3.Powder
PowderX-Ray
X-rayDiffraction analyses
Diffraction analyses of of
thethe
modified andand
modified unmodified
unmodifiedcellulose nanocrystals
cellulose nanocrystals
(CNC) and cellulose nanofiber (CNF) materials. Nanocellulose modifications have
(CNC) and cellulose nanofiber (CNF) materials. Nanocellulose modifications have been been performed
performed
with cetyltrimethylammonium bromide (CTAB), tannic acid and decylamine (TADA), and by
with cetyltrimethylammonium bromide (CTAB), tannic acid and decylamine (TADA), and by TEMPO-
TEMPO-mediated oxidation (TEMPO). (Averages followed by the same letters in the same column
mediated
do oxidation
not differ (TEMPO).
statistically (Averages
by Tukey’s test (p > followed
0.05). by the same letters in the same column do not
differ statistically by Tukey’s test (p > 0.05).
All modified and unmodified nanocellulose samples exhibited cellulose characteristic

reflections at 2θ = 15.0◦ /16.5◦ , 22.5◦ and 34.5◦ , corresponding to the (1-10)/(110), (200) and
(004) planes of cellulose I, respectively [46,51], demonstrating that the cellulose structure
was generally maintained. Nevertheless, TEMPO-mediated oxidation showed a statistically
significant (p < 0.05) decrease in crystallinity (from 70–75% to 53–56%), which was sup-
ported by the less defined reflections at 2θ = 15.0◦ /16.5◦ and 34.5◦ , indicating an increase
in the amorphous part. Similar results have been reported by Isogai et al. (2019) and
Kim et al. (2021) [52,53].
For TADA-modified samples, cellulose peaks prevailed, but there was some broaden-
ing due to the modification, which was also observed by Hu et al. (2017) [38]. As expected,
the crystallinity index significantly (p < 0.05) decreased after modification, for both CNC
(from 75% to 61%) and CNF (from 70% to 59%). On the contrary, the attachment of CTAB
to the surface of NC gave similar PXRD patterns to unmodified NC, implying that the
underlying particle crystallinity was unchanged by the modification, as the surfactant
was only electrostatically bound to nanocellulose. Even so, the crystallinity significantly
(p < 0.05) decreased for CNC (from 75 to 70%) after CTAB modification. Decreases in CI of
up to 5% from CNC to CNC-CTAB have also been obtained in the literature, which was
been attributed to the disordered of the cellulose crystalline structure during modification,
and the attachment of the bulky structure of a long alkyl chain of CTAB, which may increase
the amorphous phase of the material [28,35].
3.1.3. Zeta Potential

Dispersions with zeta potentials high in absolute value are electrically stabilized
(repulsion exceeds attraction), while dispersions with low zeta potentials (attraction ex-
ceeds repulsion) tend to coagulate or flocculate [54]. According to Simões et al. (2020)
and Ghalandari et al. (2015), a colloidal system is considered stable when showing ZP
values above +30 mV or below −30 mV, thus meaning that the charge between particles
(i.e., repulsion) is enough to prevent aggregation [55,56]. All nanocellulose suspensions
exhibited negative zeta potential, with values ranging from −17 to −45 mV (Table 1). Both
unmodified CNC (ZP = −45 mV) and CNF (ZP = −34 mV) originated stable suspensions,
as ZP > ±30 mV. The extra negative charge of CNC indicates the attachment of negative
sulfate groups (–OSO3 − ) on its surface, considering CNC is generally prepared by acid
hydrolysis with H2 SO4 [57]. TEMPO-mediated oxidized CNC and CNF also exhibited
negative and stable suspensions (ZP = −31 to −34 mV), mostly due to the presence of
negative carboxylate (COO−) groups at the surface of NC generated during the oxidative
treatment. The same has been observed in works by Gamelas et al. (2015) and Zhou
et al. (2018) [36,58]. Nanocellulose modification with CTAB and TADA, which targets the
hydroxyl (OH-) groups on NC surface (to which negative sulfate groups are also attached
in the case of CNC), led to a less electronegative zeta potential (p < 0.05), with results of
−28 mV to −17 mV for NC-CTAB and −25 mV to −22 mV for NC-TADA. Similar results
were reported by Jackson et al. (2011) for NC modification with CTAB 2 mM, and Foo
et al. (2019) for TADA modification, demonstrating modified CNC structures with ZP ca.
−30 mV and −15 mV, respectively [4,34].
Table 1. Zeta potential results of modified and unmodified CNC and CNF.
Sample Zeta Potential (mV)

CNC −45.03 ± 0.79 a
CNF −34.20 ± 1.03 b
CNC-TEMPO −34.37 ± 1.04 b
CNF-TEMPO −30.90 ± 1.68 c
CNC-CTAB −27.97 ± 2.01 c
CNF-CTAB −17.47 ± 2.25 d
CNC-TADA −22.03 ± 0.84 d,e
CNF-TADA −24.90 ± 1.37 e
Legend: CNC—cellulose nanocrystals; CNF—cellulose nanofibers; TEMPO—oxidized by 2,2,6,6-
tetramethylpiperidine 1-oxyl; CTAB—modified with cetyltrimethylammonium bromide; TADA—modified
with tannic acid and decylamine. a–e Means within the same column, labeled with the same letter do not differ
statistically by Tukey’s test (p > 0.05).
3.2. Curcumin Binding Analysis

3.2.1. Preliminary Curcumin:Carrier Ratio Screening
An initial screening test was performed in order to optimize the curcumin:carrier ratio
in the encapsulated systems. CNC was used as a carrier model for the screening. The ratios
tested were 1:2, 1:3, 1:5, 1:10 and 1:15 (w/w) curcumin:carrier. The loading capacity and
encapsulation efficiency results obtained are presented in Table 2.
The curcumin encapsulation efficiency was found to be a function of curcumin concen-
tration between ratios 1:15 and 1:3 curcumin:CNC (w/w), with EE increasing significantly
(p < 0.05) from 75 to 81% when the amount of added curcumin increased from 1:15 to
1:3 curcumin:CNC (w/w). A higher binding efficiency was also achieved when a higher
amount of curcumin was added to nanocellulose materials in a work conducted by Zainud-
din et al. (2017), who attributed this phenomenon to the increased number of active sites on
curcumin molecular structure [28]. A higher curcumin:carrier ratio also allowed a higher
loading capacity, with experimental values being consistent with the theoretical values for
the ratios 1:10 to 1:3 (w/w). However, when an excessive amount of curcumin was added, a
statistically significant (p < 0.05) decrease in EE and LC (in relation to the theoretical value)
was observed, assessing from the results of the 1:2 curcumin:CNC (w/w) ratio. This can
be attributed to a lack of ability from CNC to bind more curcumin molecules, suggesting
that there is a limit to curcumin encapsulation by nanocellulose materials. From the data
showed in Table 1, the maximum curcumin encapsulation efficiency (81.16% ± 0.21) oc-
curred for the 1:3 curcumin:CNC (w/w) ratio, which corresponds to a loading capacity of
ca. 25%. This curcumin:carrier ratio was used for further encapsulation studies.
Table 2. Curcumin encapsulation efficiency (EE) and loading capacity (LC) (both experimental and
theoric) of nanocellulose systems at different curcumin to nanocellulose ratios.
Ratio Curcumin:CNC EE (%) LC (%) Theoric LC (%)

1:2 56.64 ± 0.76 a 26.90 ± 0.83 a 33.33
1:3 81.26 ± 0.21 b 24.57 ± 0.30 b 25.00
1:5 79.46 ± 0.25 c 16.04 ± 0.42 c 16.67
1:10 78.31 ± 0.19 d 8.82 ± 0.23 d 9.09
1:15 74.97 ± 0.83 e 3.13 ± 0.86 e 6.25
Legend: CNC—cellulose nanocrystals; EE—encapsulation efficiency; LC—loading capacity. The theoretical LC (%)
was expressed as the ratio of the amount of curcumin added and the mass of the starting materials (cellulose-based
carrier material and curcumin). a–e Means within the same column, labeled with the same letter do not differ
3.2.2. ATR-FT-IR Analysis of the Loaded Nanostructures

Pharmaceutics 2023, 15, x FOR PEER REVIEWFigure 4 shows the ATR-FT-IR spectra of unmodified and modified nanocellulose (with
11 of 20
CTAB, TADA and by TEMPO mediated oxidation) systems encapsulating curcumin, as well
as the spectra of pure curcumin. The curcumin spectra showed characteristic vibrations
at 1610 cm−1 , 1500 cm−1 , 1275 cm−1 and 1150 cm−1 , which correspond to the stretching
stretching
vibrationsvibrations of thering,
of the benzene benzene ring, C-O C-H
C-O stretching, stretching, C-Hand
stretching stretching and C–O–C
C–O–C stretching, re-
stretching, respectively [59–61]. The same vibrations can be observed in the
spectively [59–61]. The same vibrations can be observed in the nanocellulose encapsulation nanocellulose
encapsulation systems,
systems, indicating theindicating
successfulthe successful
loading loadingonto
of curcumin of curcumin
the systems.onto Moreover,
the systems.
the
Moreover, the curcumin characteristic peaks were not significantly
curcumin characteristic peaks were not significantly shifted in curcumin loaded shifted in curcumin
systems,
loaded systems,
revealing revealing
that there was no that thereinwas
change theno changecomposition/structure
chemical in the chemical composition/structure
of curcumin after
ofthe
curcumin
loading process. Nevertheless, the intensity of thethe
after the loading process. Nevertheless, intensity
bands of the bandssystems
on encapsulation on encap-
was
sulation systems was lower than in pure curcumin, suggesting the incorporation
lower than in pure curcumin, suggesting the incorporation of curcumin into the delivery of cur-
cumin intoasthe
systems, deliveryby
discussed systems, as discussed
Iurciuc-Tincu by Iurciuc-Tincu
et al. (2020) [62] et al. (2020) [62]
Figure
Figure4.4.FT-IR
FT-IRspectra
spectraofofthe
themodified
modifiedand
andunmodified
unmodifiedcellulose
cellulosenanocrystals
nanocrystals(CNC)
(CNC)and
andcellulose
cellulose
nanofiber
nanofiber(CNF)
(CNF)systems
systemsencapsulating
encapsulatingcurcumin,
curcumin,asaswell
wellasaspure
purecurcumin.
curcumin.Nanocellulose modi-
Nanocellulose mod-
fications
ifications have been performed with cetyltrimethylammonium bromide (CTAB), tannic acidand
have been performed with cetyltrimethylammonium bromide (CTAB), tannic acid and
decylamine
decylamine(TADA),
(TADA),and
andby byTEMPO-mediated
TEMPO-mediatedoxidation
oxidation(TEMPO).
(TEMPO).
3.2.3. Curcumin Loading

Curcumin loading into modified CNC and CNF (with CTAB, TADA and by TEMPO-
mediated oxidation) was examined and compared against the unmodified CNC and CNF.
Yield, encapsulation efficiency, loading capacity and zeta potential results are presented
in Table 3.
3.2.3. Curcumin Loading

Curcumin loading into modified CNC and CNF (with CTAB, TADA and by TEMPO-
mediated oxidation) was examined and compared against the unmodified CNC and CNF.
Yield, encapsulation efficiency, loading capacity and zeta potential results are presented in
Table 3.
Table 3. Yield, encapsulation efficiency (EE), loading capacity (LC) and zeta potential (ZP) of modified
and unmodified nanocellulose systems encapsulating curcumin.
Delivery System Yield (%) EE (%) LC (%) ZP (mV)

CNC 68.01 ± 3.22 a 84.29 ± 3.06 a 31.00 ± 3.41 a −49.83 ± 2.75
CNF 91.22 ± 3.64 b 84.81 ± 4.79 a 24.72 ± 2.12 b −19.67 ± 5.52
CNC-TEMPO 43.33 ± 4.01 c 54.52 ± 3.29 b 31.50 ± 3.45 a −26.13 ± 0.58
CNF-TEMPO 68.90 ± 2.73 a 85.39 ± 0.28 a 25.41 ± 2.70 b −25.13 ± 0.90
CNC-CTAB 90.36 ± 3.24 b,d 90.23 ± 1.58 c 24.78 ± 3.47 b −21.97 ± 1.86
CNF-CTAB 68.95 ± 3.40 a 81.27 ± 2.97 a 26.98 ± 3.29 b −15.37 ± 1.18
CNC-TADA 83.06 ± 2.89 d 99.85 ± 0.23 d 30.10 ± 1.06 a −20.90 ± 2.43
CNF-TADA 93.25 ± 2.16 b 99.84 ± 0.31 d 26.80 ± 2.29 b −21.91 ± 1.96
Legend: CNC—cellulose nanocrystals; CNF—cellulose nanofibers; TEMPO—oxidized by 2,2,6,6-
tetramethylpiperidine 1-oxyl; CTAB—modified with cetyltrimethylammonium bromide; TADA—modified with
tannic acid and decylamine. a–d —Means within the same column, labeled with the same letter do not differ
Unmodified CNC and CNF were able to bind ca. 85% of the added curcumin, with no
significant differences (p > 0.05) found between the two systems. The oxygen of hydroxyl on
the benzene ring of curcumin is a potential hydrogen donor and an important binding site
for nanocellulose hydroxyl groups, and it is possible that an interaction occurred between
curcumin and nanocellulose molecules through hydrogen bonding (Figure 5a) [59]. The
large surface area of nanocellulose materials further potentiates the binding of curcumin
molecules onto nanocellulose [11]. Lower curcumin EE of ca. 27% and 55% have been
reported in the literature for unmodified nanocellulose materials, which may be due to the
considerably lower amount of curcumin added in these studies, with curcumin:CNC ratios
of 1:16 and 1:10 (w/w), respectively, as discussed in the ratio screening section [4,28].
Modifications of CNF with CTAB and by TEMPO mediated oxidation did not show
significant differences (p > 0.05) in curcumin encapsulation from the unmodified CNF
system, which might indicate that curcumin loading to CNF may be more related to fiber
network entanglement and entrapment than actual molecule binding to CNF, as has been
previously suggested by Kolakovic et al. [63]. On the other hand, when CNC has been
modified by TEMPO-mediated oxidation, curcumin encapsulation significantly (p < 0.05)
decreased to ca. 55%, indicating that the modification of hydroxyl groups to carboxyl
groups decreased the ability of CNC to bind curcumin.
On the contrary, when hydrophobic modifications were employed to CNC, statistically
significant (p < 0.05) higher curcumin encapsulation efficiencies were observed. CNC-
CTAB was able to bind ca. 90% of added curcumin, with a LC of ca. 25%, which matches
the theoretical value. Similar results of curcumin EE of 80–96% have been reported in
the literature for CNC-CTAB [28]. Due to its structure and hydrophobicity properties,
curcumin may show the interaction between its benzene rings with the hydrophobic region
of the modified CNC-CTAB (C-H moieties) via electrostatic and hydrophobic interactions
(Figure 5b). These interactions in combination with the hydrogen bonding described earlier,
are more effective in curcumin binding and encapsulation [4]. Furthermore, the adsorbed
surfactant monomers can reorganize and induce hydrophobic interactions between the
alkyl chains of the surfactant to form surfactant clusters, further favoring curcumin binding
(Figure 5c) [28]. The binding capacity of nanocellulose significantly (p < 0.05) increased
after TADA modification, achieving EE of ca. 99.8%, for both modified CNC and CNF.
The increased level of hydrophobicity in NC-TADA favored the hydrophobic interactions
between the hydrophobic phenolic moieties of curcumin and the long alkyl chain of
Unmodified CNC and CNF were able to bind ca. 85% of the added curcumin, with
no significant differences (p > 0.05) found between the two systems. The oxygen of hy-
droxyl on the benzene ring of curcumin is a potential hydrogen donor and an important
Pharmaceutics 2023, 15, 981 binding site for nanocellulose hydroxyl groups, and it is possible that an interaction 12 ofoc-
19
curred between curcumin and nanocellulose molecules through hydrogen bonding (Fig-
ure 5a) [59]. The large surface area of nanocellulose materials further potentiates the bind-
ing of
DA curcumin
(Figure molecules
5b), resulting in onto nanocellulose
remarkable [11]. Lower efficiencies.
curcumin-binding curcumin EE of ca. 27% and
Furthermore, the
55% havebinding
excellent been reported
capacityin
of the literature
NC-TADA hasfor
beenunmodified
ascribed tonanocellulose
the emergencematerials, which
of entanglement
may
of be due toand
NC-TADA the the
considerably
formation lower amount of curcumin
of crosslinked-like networks,added in thesethe
facilitating studies,
bindingwithof
curcumin:CNC
curcumin ratios
onto the of 1:16
surface and 1:10 (w/w),
of nanocellulose respectively,
[4]. Curcumin EE as discussed in the
ranging from 95 ratio
to 99%screen-
have
ing section
also [4,28]. in the literature for TADA modified CNC [4].
been reported
structures;
Figure 5. (a) Representation of hydrogen bonding between curcumin and nanocellulose structures;
Representationof
(b) Representation ofhydrophobic
hydrophobicinteractions
interactionsbetween
betweencurcumin
curcuminandand hydrophobic
hydrophobic modified
modified nano-
nanocel-
cellulose;
lulose; (c) (c) Schematic
Schematic illustration
illustration of CTAB
of CTAB andand nanocellulose
nanocellulose interactions.
interactions.
Generally,
Modifications higher yields
of CNF were
with observed
CTAB forTEMPO
and by CNF systems
mediated (with CNF anddid
oxidation CNF-TADA
not show
showing
significant yields > 90%),(pindicating
differences that the smaller
> 0.05) in curcumin size of CNC
encapsulation from themayunmodified
have led toCNF a loss of
sys-
carrier particles
tem, which might during the encapsulation
indicate that curcuminprocessloading(mostto CNFlikely during
may centrifugation).
be more The
related to fiber
fact that the
network loading capacity
entanglement was also usually
and entrapment higher
than actual than thebinding
molecule theoretical valueasofhas
to CNF, 25% for
been
CNC systems also suggests that a loss of carrier materials may
previously suggested by Kolakovic et al. [63]. On the other hand, when CNC has been have occurred. However,
for the CNC-CTAB-modified
modified by TEMPO-mediated system this tendency
oxidation, curcumin was not observed,
encapsulation exhibiting a(pyield
significantly of
< 0.05)
90.36% and a LC of 24.78%, which supports the success of this
decreased to ca. 55%, indicating that the modification of hydroxyl groups to carboxylencapsulation approach.
groupsZeta potentialthe
decreased results show
ability that unmodified
of CNC CNC encapsulating curcumin produced a
to bind curcumin.
very Onstable
thesuspension
contrary, when −30 mV), while
(ZP <hydrophobic all the otherwere
modifications systems were moderately
employed stable
to CNC, statisti-
(ZP ca. − 20 mV). The zeta potential became slightly less electronegative
cally significant (p < 0.05) higher curcumin encapsulation efficiencies were observed. from the free
nanocellulose materials (Table 1) to the curcumin-bound nanocellulose
CNC-CTAB was able to bind ca. 90% of added curcumin, with a LC of ca. 25%, which systems (Table 3).
This increase has been observed in previous works and might be due
matches the theoretical value. Similar results of curcumin EE of 80–96% have been re- to interactions between
the charged
ported in theNC surfacefor
literature groups and curcumin
CNC-CTAB [28]. Due[35]. Thestructure
to its fact thatand NC hydrophobicity
zeta potentials do not
prop-
suffer major alterations provides an indication that no strong bonds
erties, curcumin may show the interaction between its benzene rings with the hydropho- are formed between
carrier
bic regionandofcurcumin,
the modifiedmeaning that curcumin
CNC-CTAB conserved
(C-H moieties) via its chemical structure
electrostatic and can
and hydrophobic
potentially
interactionsbe(Figure
released in These
5b). active interactions
form from the innanocellulose
combination with materials.
the hydrogen bonding
described
3.3. earlier,
Curcumin are Profile
Release more effective in curcumin binding and encapsulation [4]. Further-
The curcumin release profile from the nanocellulose delivery systems was studied in
simulated gastric fluid for 2 h, followed by simulated intestinal fluid for 6 h, both at 37 ◦ C,
in order to simulate gastrointestinal conditions. Curcumin release profiles from the different
nanocellulose systems (modified and unmodified) are shown in Figure 6. Unmodified
CNC and CNF showed a slow release of curcumin, (18–23% after 8 h), while the NC
TEMPO-mediated oxidized forms released ca. 15% over 8 h, which was a slight decrease in
comparison to the unmodified NC structures. CNF-CTAB showed a similar release profile

to unmodified CNF, with ca. 18% of curcumin being released after 8 h. On the other hand,
CNC-CTAB showed an increased, but still sustained curcumin release capability, with
ca. 48% of curcumin being released over 8 h, which was the highest amount of curcumin
released in this study. This may be due to the different hydrophobicity of the carriers, allied
to the non-covalent interactions between CNC and the surfactant, which may facilitate
curcumin release from the carrier system, as opposed to NC-TADA. Studies available in the
literature showed that CTAB modification enabled a sustained release of lipophilic bioactive
compounds of 45–75% over 1–2 d in PBS [34,35]. The faster release of curcumin observed
in the present study might be related to the simulated gastric conditions, in which the
acidic medium might have helped in curcumin unbinding and faster release, as observed
by Iurciuc-Tincu et al. (2020) [62]. Hydrophobic modification with TADA showed a small
amount of curcumin released, since only ca. 2–4% curcumin was detected after 8 h, for both
CNC-TADA and CNF-TADA. Despite the excellent encapsulation efficiencies obtained
for this type of material, this system hindered the release of the bioactive compound
Pharmaceutics 2023, 15, x FOR PEER REVIEW
in
14 of 20
simulated biological fluids, and so it does not seem a promising curcumin delivery system
for pharmaceutical and nutraceutical applications.
Figure 6. Curcumin
Curcumin release
release profile from modified and unmodified cellulose nanocrystals (CNC) and
cellulose nanofiber
cellulose nanofiber (CNF)
(CNF) delivery
deliverysystems
systemsininsimulated
simulatedgastric
gastricfluid
fluid (SGF,
(SGF, pHpH 3) and
3) and simulated
simulated in-
intestinal fluid (SIF, pH 6.8). Nanocellulose modifications have been performed with cetyltrime-
testinal fluid (SIF, pH 6.8). Nanocellulose modifications have been performed with cetyltrimethylam-
thylammonium
monium bromidebromide
(CTAB),(CTAB), tannic
tannic acid andacid and decylamine
decylamine (TADA), (TADA), and by TEMPO-mediated
and by TEMPO-mediated oxidation
oxidation (TEMPO).
(TEMPO).
3.4. Cytotoxicity Evaluation

Considering a potential gastrointestinal delivery of the developed developed nanocellulose
nanocellulose de-de-
livery systems within pharmaceutical or nutraceutical applications, the cytotoxicity of the
systems was assayed against the intestinal
intestinal cell
cell line Caco-2,
Caco-2, in order
order to determine
determine the
the maxi-
max-
mum
imumconcentration
concentrationthat thatisisnot
notcytotoxic.
cytotoxic. The
The results
results are
are presented
presented in Figure 7a,b for CNF
and CNC systems
systems (0.03–0.5
(0.03–0.5 g/L), respectively, alongside
g/L), respectively, alongside pure
pure curcumin
curcumin(0.01–0.17
(0.01–0.17g/L,
g/L, the
same concentration that was present in the encapsulated systems). Considering
same concentration that was present in the encapsulated systems). Considering a thresh- a threshold
for
old cell cytotoxicity
for cell of <30%
cytotoxicity metabolic
of <30% metabolicinhibition (according
inhibition to ISO
(according 10993-5:2),
to ISO curcumin
10993-5:2), curcu-
proved to be cytotoxic to cells at all tested concentrations (metabolic inhibitions
min proved to be cytotoxic to cells at all tested concentrations (metabolic inhibitions of 55–95%).
of
Literature studies reported
55–95%). Literature studies areported
concentration-dependent curcumincurcumin
a concentration-dependent cytotoxicity, with cell
cytotoxicity,
viability
with cellbeing less being
viability than 80% lessfor concentrations
than above 0.009 above
80% for concentrations g/L (i.e., 25 µM)
0.009 [64,65].
g/L (i.e., 25 µM)
[64,65].
Delivery systems composed of CNF (Figure 7a) had no impact upon cells’ metabolic
activity, with all CNF systems (unmodified and modified) presenting either no alterations
or small promotions in the cell’s metabolism at all the concentrations tested. When con-
CNF and CNC systems under safe concentrations was able to reduce curcumin cytotoxi-
city, as encapsulated curcumin did not exhibit metabolic inhibition as opposed to pure
Pharmaceutics 2023, 15, 981
curcumin. The use of encapsulation techniques to overcome ingredient toxicity is a14valu-
of 19
able approach to promote the applicability of interesting bioactive compounds [67,68].
Figure
Figure 7.7. Impact
Impact of
ofpure
pure curcumin
curcumin and
and modified
modified and
and unmodified
unmodified cellulose
cellulose nanofiber
nanofiber (CNF)
(CNF) (a)
(a) and
and
cellulose nanocrystal (CNC) (b) systems encapsulating curcumin upon Caco-2 cells metabolic activ-
cellulose nanocrystal (CNC) (b) systems encapsulating curcumin upon Caco-2 cells metabolic activity.
ity. Nanocellulose modifications have been performed with cetyltrimethylammonium bromide
Nanocellulose modifications have been performed with cetyltrimethylammonium bromide (CTAB),
(CTAB), tannic acid and decylamine (TADA), and by TEMPO-mediated oxidation (TEMPO). The
tannic acid
dotted and decylamine
line represents (TADA),
the 30% and bylimit
cytotoxicity TEMPO-mediated oxidation
as defined by the (TEMPO). The dotted line
ISO 10993-5:2.
represents the 30% cytotoxicity limit as defined by the ISO 10993-5:2.
3.5. Morphological Analysis
Delivery systems composed of CNF (Figure 7a) had no impact upon cells’ metabolic
Thewith
activity, morphological properties
all CNF systems of freeze-dried
(unmodified free CNC-CTAB
and modified) presentingand CNC-CTAB
either no alterationsen-
capsulating curcumin,inwhich
or small promotions proved
the cell’s to be theatmost
metabolism all thepromising delivery
concentrations system
tested. in terms
When con-
of encapsulation
sidering and release
the cytotoxicity properties,
results of CNCwere analyzed
systems (Figure by SEM (Figure
7b), the 8) and
scenario TEM (Fig-
is somewhat
ure 9). SEM
different. images showed
Unmodified thatCNC-
CNC and the cellulosic
TEMPOstructures
also exhibited formno ansignificant
aggregated polymeric
impact upon
laminar matrix during
cells metabolic activity,the freeze-drying
with no metabolic process, as has
inhibition beenobserved.
being previously observed
CTAB in the
and TADA
literature [69]. Small
modifications, however,particles
proveddeposited on the to
to be cytotoxic surface of the encapsulating
cells depending structures
on the concentration
(Figure
tested. 8b) are likely
Curcumin to represent curcumin
concentrations higher than particles,
0.25 g/L suggesting the binding
for CNC-CTAB and of curcumin
0.50 g/L for
onto CNC-CTAB.
CNC-TADA led toThese particles
significant were not
reductions in observed in the free
cell metabolism, withcellulose-based material
metabolic inhibitions of
65–100%.
(Figure 8a).Cytotoxicity
The lamellar concerns about
structures formthemultiple
use of surfactants
layers around and the
amino compounds
entrapped have
curcumin
arisen in the
particles, in aliterature, and a previous
similar morphology to thestudy has shown
one observed bythat modified-NC
Kolakovic withfor
et al. (2012) cationic
CNF
surfactant CTAB
entrapping can cause drugs
the hydrophobic a significant reduction
itraconazole in cellular (fibroblasts
and indomethacin [70] On the 3T3) viability
other hand,
and proliferation
TEM images were[66]. ableHowever, despite the inhibitions
to show individualized particlesobserved in the present
with a needle-like study, no
morphology
cytotoxic
and effect was
dimensions detected
in the up to range.
nanometric 0.125 g/L for CNC-CTAB
Similar morphologies andhave
0.25 been
g/L for CNC-TADA
reported in the
encapsulating
literature curcumin,
for CNC meaning
modified that up[28].
with CTAB to this
Afterconcentration, the delivery system
curcumin encapsulation, is not
the rod-like
cytotoxic of
structure towards
CNC was intestinal cells. It(Figure
not affected is interesting to note thatdescribed
9b), as previously curcumin by encapsulation into
Foo et al. (2019)
CNF and CNC systems under safe concentrations was able to reduce
[4]. However, the average diameter and length of the nanoparticles appears to have in- curcumin cytotoxicity,
as encapsulated
creased, suggestingcurcumin did notofexhibit
the binding curcuminmetabolic inhibition as opposed to pure curcumin.
onto CNC-CTAB.
The use of encapsulation techniques to overcome ingredient toxicity is a valuable approach
to promote the applicability of interesting bioactive compounds [67,68].
3.5. Morphological Analysis

The morphological properties of freeze-dried free CNC-CTAB and CNC-CTAB en-
capsulating curcumin, which proved to be the most promising delivery system in terms
of encapsulation and release properties, were analyzed by SEM (Figure 8) and TEM (Fig-
ure 9). SEM images showed that the cellulosic structures form an aggregated polymeric
laminar matrix during the freeze-drying process, as has been previously observed in the
literature [69]. Small particles deposited on the surface of the encapsulating structures
(Figure 8b) are likely to represent curcumin particles, suggesting the binding of curcumin
onto CNC-CTAB. These particles were not observed in the free cellulose-based material
(Figure 8a). The lamellar structures form multiple layers around the entrapped curcumin
particles, in a similar morphology to the one observed by Kolakovic et al. (2012) for CNF
entrapping the hydrophobic drugs itraconazole and indomethacin [70] On the other hand,
TEM images were able to show individualized particles with a needle-like morphology
and dimensions in the nanometric range. Similar morphologies have been reported in
the literature for CNC modified with CTAB [28]. After curcumin encapsulation, the rod-
like structure of CNC was not affected (Figure 9b), as previously described by Foo et al.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 16 of 20
(2019)
Pharmaceutics 2023, 15, x FOR PEER REVIEW
[4]. However, the average diameter and length of the nanoparticles appears to have
16 of 20
increased, suggesting the binding of curcumin onto CNC-CTAB.
Figure 8. Scanning electron microscopy images of (a) free cellulose nanocrystals modified with
Figure
Figure 8. Scanning electron
8. Scanning electron microscopy
microscopy images
images of (a)
(a) free
ofand free cellulose
cellulose nanocrystals
nanocrystals modified
modified with
with
cetyltrimethylammonium bromide (CNC-CTAB) (b) CNC-CTAB encapsulating curcumin
cetyltrimethylammonium
cetyltrimethylammonium bromide
bromide (CNC-CTAB) and
(CNC-CTAB)
(3000×, 5 kV, 1.0 Pa, scale bar equals 50 µm). (b)
and CNC-CTAB
(b) encapsulating
CNC-CTAB curcumin
encapsulating (3000
curcumin ×,
5(3000×,
kV, 1.05Pa,
kV,scale
1.0 Pa,
barscale bar50equals
equals µm). 50 µm).
Figure 9. Transmission electron microscopy images of (a) free cellulose nanocrystals modified with
Figure 9. Transmission electron
cetyltrimethylammonium microscopy
bromide images and
(CNC-CTAB) of (a) freeCNC-CTAB
cellulose nanocrystals modified with
Figure 9. Transmission electron microscopy images of(b)(a) free celluloseencapsulating
nanocrystals curcumin
modified
cetyltrimethylammonium
(100,000×, 120 kV, scale barbromide
equals (CNC-CTAB)
100 nm). and (b) CNC-CTAB encapsulating curcumin
with cetyltrimethylammonium bromide (CNC-CTAB) and (b) CNC-CTAB encapsulating curcumin
(100,000×, 120 kV, scale bar equals 100 nm).
(100,000×, 120 kV, scale bar equals 100 nm).
4. Conclusions
4. Conclusions
4. Conclusions
In the present study, nanocellulose (cellulose nanocrystals and cellulose nanofibers)
In the
In the as
was tested present
present study,
carrierstudy,
for thenanocellulose
nanocellulose (celluloseananocrystals
(cellulose
delivery of curcumin, nanocrystals and cellulose
and
model liposoluble cellulose
compound.nanofibers)
nanofibers)
Hydro-
was
was tested
tested as
as carrier
carrier for
forthe
thedelivery
delivery of curcumin,
of curcumin, a model
a modelliposoluble compound.
liposoluble
phobic modifications with the surfactant CTAB and tannic acid/decylamine (TADA) and compound. Hydro-
Hy-
phobic
drophobicmodifications
modifications with the
with surfactant
the CTAB
surfactant CTABand tannic
and acid/decylamine
tannic (TADA)
acid/decylamine
modification by TEMPO-mediated oxidation, were also tested and compared with the un- and
(TADA)
modification
and
modified by TEMPO-mediated
modification
nanocellulose structures. oxidation,
by TEMPO-mediated Structural were
oxidation, alsoalso
were tested and
tested
characterizations compared
and with
compared
confirmed the the un-
with
success the
of
modified
unmodified nanocellulose
nanocellulose
cellulose modifications, structures.
and Structural
structures.
the bindingStructuralcharacterizations
characterizations
of curcumin confirmed the success
confirmedsystems.
onto the nanocellulose of
the success
The
cellulose
of
results modifications,
cellulose modifications,
showed andand
that all systemsthethe
binding of curcumin
binding
encapsulatedof curcumin ontoonto
significant the nanocellulose systems.
the nanocellulose
amounts of curcumin, The
systems.
ranging
results
from 54showed
to 99%, that all systems
depending encapsulated
on the significant
structure used. amountsthat
We concluded of curcumin, ranging
modification with
from 54 to 99%, depending on the structure used. We concluded that
CTAB and TADA resulted in effective curcumin encapsulation efficiencies of 90–99%. modification with
CTAB and TADAwas
While NC-TADA resulted
unableintoeffective curcumin in
release curcumin encapsulation efficiencies offluids
simulated gastrointestinal 90–99%.
(2–
While NC-TADA was unable to release curcumin in simulated gastrointestinal
4% released in 8 h), CNC-CTAB allowed for a curcumin sustained release of ca. 50% in fluids (2–8
The results showed that all systems encapsulated significant amounts of curcumin, rang-
ing from 54 to 99%, depending on the structure used. We concluded that modification
with CTAB and TADA resulted in effective curcumin encapsulation efficiencies of 90–99%.
While NC-TADA was unable to release curcumin in simulated gastrointestinal fluids (2–4%
released in 8 h), CNC-CTAB allowed for a curcumin sustained release of ca. 50% in 8 h,
demonstrating to be the most promising delivery system. This system showed no cyto-
toxic effect towards intestinal cells up to 0.125 g/L. Furthermore, curcumin encapsulation
allowed to reduce curcumin cytotoxicity, highlighting the potential of curcumin encapsula-
tion into nanocellulose delivery systems. This work suggests nanocellulose as a promising
sustainable delivery system, while cationic modification with CTAB proved to be a capable
approach to alter its surface properties for more efficient binding and release of liposoluble
compounds, increasing the range of curcumin concentrations that could be employed in
pharmaceutical or nutraceutical applications. These findings also highlight the potential
added-value applications of cellulose, the world’s most abundant natural polymer, which
can be obtained from several renewable and sustainable sources, such as lignocellulosic
biomass from industrial and agricultural wastes.
Author Contributions: Conceptualization: Ó.L.R., F.C., C.F.P. and A.B.R.; Methodology: F.C., C.F.P.,
A.B.R. and E.M.C.; Writing—Original Draft Preparation: F.C.; Writing—Review and Editing: C.F.P.,
Ó.L.R., A.B.R., R.F., E.M.C., P.M.C., J.C.F. and M.P.; Supervision: Ó.L.R.; Validation: Ó.L.R., J.C.F. and
M.P.; Resources: Ó.L.R., J.C.F. and M.P.; Project Administration: M.P.; Funding Acquisition: M.P. All
authors have read and agreed to the published version of the manuscript.
Funding: Project co-financed by the European Regional Development Fund (ERDF), through the
Operational Program for Competitiveness and Internationalization (POCI) supported by Amyris Bio
Products Portugal, Unipessoal Ltda and Escola Superior de Biotecnologia—Universidade Católica
Portuguesa through the Alchemy project “Capturing High Value from Industrial Fermentation Bio
Products” (POCI-01-0247-FEDER-027578). We would also like to thank the scientific collaboration
under the FCT project UID/Multi/50016/2020.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Acknowledgments: The authors gratefully thank Ana Oliveira and the Project TEX4WOUNDS
(POCI-01-0247-FEDER-047029), financed under the Incentive System for Research and Technological
Development, R&DT Projects, in co-promotion (Notice SI/17/2019) for the SEM. The authors also
acknowledge the support of the i3S Scientific Platform HEMS, member of the national infrastructure
PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122) for the TEM.
Conflicts of Interest: The authors declare no conflict of interest.
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Pharmaceutics - Cellulose Nanostructures

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Pharmaceutics - Cellulose Nanostructures

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CBQF—Centro de Biotecnologia e Química Fina–Laboratório Associado, Escola Superior de Biotecnologia,

Pharmaceutics 2023, 15, 981. https://doi.org/10.3390/pharmaceutics15030981 https://www.mdpi.com/journal/pharmaceutics

2. Materials and Methods

2.2. Production of CNC-TEMPO

2.3. CNC and CNF Modification with CTAB Surfactant

2.4. CNC and CNF Modification with TA and DA

2.5.2. Powder X-ray Diffraction

2.5.3. Zeta Potential Determination

2.5.4. Microscopy Analysis

2.6. Curcumin Loading

into CNF and functionalized materials (CNC-TEMPO, CNF-TEMPO, CNC-CTAB, CNF-

2.7. Curcumin Quantification

2.8. Encapsulation Efficiency, Loading Capacity and Yield

2.9. Curcumin Release Profile

2.10. Cytotoxicity Evaluation

2.11. Statistical Analysis

Characteristic cellulose vibrations were found in all nanocellulose samples, namely

After oxidation, both CNC-TEMPO and CNF-TEMPO exhibited a new vibration at

All modified and unmodified nanocellulose samples exhibited cellulose characteristic

3.1.3. Zeta Potential

Sample Zeta Potential (mV)

3.2. Curcumin Binding Analysis

Ratio Curcumin:CNC EE (%) LC (%) Theoric LC (%)

3.2.2. ATR-FT-IR Analysis of the Loaded Nanostructures

3.2.3. Curcumin Loading

3.2.3. Curcumin Loading

Delivery System Yield (%) EE (%) LC (%) ZP (mV)

comparison to the unmodified NC structures. CNF-CTAB showed a similar release profile

3.4. Cytotoxicity Evaluation

able approach to promote the applicability of interesting bioactive compounds [67,68].

3.5. Morphological Analysis

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