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Sommaire du brevet 3030443 

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  • lorsque la demande peut être examinée par le public;
  • lorsque le brevet est émis (délivrance).
(12) Demande de brevet: (11) CA 3030443
(54) Titre français: NANOPARTICULES D'OXYDE METALLIQUE REVETUES DE LIGNINE ET LEUR UTILISATION DANS DES COMPOSITIONS COSMETIQUES
(54) Titre anglais: LIGNIN-COATED METAL OXIDE NANOPARTICLES AND USE THEREOF IN COSMETIC COMPOSITIONS
Statut: Examen demandé
Données bibliographiques
(51) Classification internationale des brevets (CIB):
  • A61K 8/84 (2006.01)
  • B82Y 30/00 (2011.01)
  • B82Y 40/00 (2011.01)
  • A61K 8/11 (2006.01)
  • A61K 8/29 (2006.01)
  • A61Q 17/04 (2006.01)
(72) Inventeurs :
  • MORSELLA, MICHELA (Italie)
  • LANTERNA, ANABEL E. (Canada)
  • SCAIANO, JUAN (Canada)
(73) Titulaires :
  • UNIVERSITY OF OTTAWA (Canada)
  • MORSELLA, MICHELA (Italie)
(71) Demandeurs :
  • UNIVERSITY OF OTTAWA (Canada)
  • MORSELLA, MICHELA (Italie)
(74) Agent: GOWLING WLG (CANADA) LLP
(74) Co-agent:
(45) Délivré:
(86) Date de dépôt PCT: 2017-05-19
(87) Mise à la disponibilité du public: 2017-11-23
Requête d'examen: 2022-05-05
Licence disponible: S.O.
(25) Langue des documents déposés: Anglais

Traité de coopération en matière de brevets (PCT): Oui
(86) Numéro de la demande PCT: PCT/CA2017/050613
(87) Numéro de publication internationale PCT: WO2017/197530
(85) Entrée nationale: 2019-01-10

(30) Données de priorité de la demande:
Numéro de la demande Pays / territoire Date
62/339,603 Etats-Unis d'Amérique 2016-05-20

Abrégés

Abrégé français

Bien que le dioxyde de titane soit couramment utilisé en tant qu'écran solaire, il existe encore des problèmes liés à l'activité catalytique de TiO2 et sa génération d'espèces réactives de l'oxygène (ROS). La présente invention tente de résoudre ces problèmes en fournissant des nanoparticules de TiO2 revêtues de lignine, de façon à obtenir une nanoparticule d'oxyde métallique à revêtement mince. Le revêtement de lignine est supposé piéger les ROS avant qu'elles diffusent depuis les particules et interagissent avec le corps de l'utilisateur ou d'autres composants d'une formulation cosmétique (telle que des écrans solaires organiques). L'invention concerne en outre d'autres oxydes métalliques revêtus de lignine, ainsi que des procédés de préparation de nanoparticules d'oxyde métallique revêtues de lignine.


Abrégé anglais

While titanium dioxide is commonly used as a sunscreen, concerns persist regarding the catalytic activity of TiO2 and its generation of reactive oxygen species (ROS). The present invention attempts to address these concerns by providing TiO2 nanoparticles coated with lignin, yielding a thin-coated metal oxide nanoparticle. The lignin coating is presumed to scavenge ROS before they diffuse away from the particles and interact with the user's body or other components of a cosmetic formulation (such as organic sunscreens). Other metal oxides coated with lignin are also disclosed, as well as methods for preparing lignin coated metal oxide nanoparticles.

Revendications

Note : Les revendications sont présentées dans la langue officielle dans laquelle elles ont été soumises.


WHAT IS CLAIMED IS:
1. A nanoparticle comprising a metal oxide particle coated with lignin to form
coated
particles which have a nanometric size.
2. The nanoparticle of claim 1, wherein the metal oxide is TiO2 or ZnO.
3. The nanoparticle of claim 1, wherein the lignin is cross-linked over a
surface of the metal
oxide particle.
4. The nanoparticle of claim 3, wherein the lignin is cross-linked by UVA
irradiation.
5. The nanoparticle of claim 1, wherein the coating on the metal oxide
particle is from about
1 to 10 nm in thickness.
6. The nanoparticle of claim 1, wherein the coating on the metal oxide
particle is from 2 to 5
nm in thickness.
7. The nanoparticle of claim 1, wherein the coating on the metal oxide
particle is about 3 nm
in thickness.
8. The nanoparticle of claim 1, wherein the lignin is Kraft lignin,
Organosolv lignin, Low
Sulfonate Content (LSC) lignin, Sodium lignin, Sodium lignin without sugars or
Alkali
lignin.
9. The nanoparticle of claim 1, wherein the lignin is a pure source of
lignin or is a source
which includes carbodydrates.
10. A process for preparing a lignin coated nanoparticle, comprising
a. mixing a metal oxide nanoparticle precursor with solubilized lignin to form
a
mixture; and
b. irradiating the mixture with UVA at a wavelength effective to coat the
metal oxide
nanoparticle precursor and form particles which have a nanometric size.

19

11. The process of claim 10, wherein the metal oxide is TiO2 or ZnO.
12. The process of claim 10, wherein the lignin is cross-linked over a surface
of the metal
oxide particle.
13. The process of claim 10, wherein the mixture is irradiated at about 369
nm.
14. The process of claim 10, wherein the irradiating is carried out for a time
effective to
produce a coating on the metal oxide particle from about 1 to 10 nm in
thickness.
15. The process of claim 10, wherein the irradiating is carried out for a time
effective to
produce a coating on the metal oxide particle from 2 to 5 nm in thickness.
16. The process of claim 10, wherein the irradiating is carried out for a time
effective to
produce a coating on the metal oxide particle of about 3 nm in thickness.
17. The process of claim 10, wherein the lignin is Kraft lignin, Organosolv
lignin, Low
Sulfonate Content (LSC) lignin, Sodium lignin, Sodium lignin without sugars or
Alkali
lignin.
18. The process of claim 10, wherein the lignin is a pure source of lignin or
is a source which
includes carbohydrates.
19. The process of claim 10, wherein the metal oxide nanoparticle precursor
and lignin are
combined with an excess of lignin by weight in the mixture.
20. The process of claim 10, wherein the irradiating is carried out for up to
2 hours with
stirring to keep the oxide nanoparticle precursor and lignin in suspension.
21. The process of claim 10, wherein the irradiating is carried out in batch
or continuously.
22. The process of claim 21, wherein the continuous irradiating is carried out
under
continuous flow conditions.


23. The process of claim 22, wherein the continuous irradiating is carried out
under
continuous flow conditions at a flow rate of 1 to 10 mL/sec.
24. The process of claim 23, wherein the continuous irradiating is carried out
under
continuous flow conditions at a flow rate of about 4 mL/sec.
25. The process of claim 10, wherein after irradiation the nanoparticles are
separated from the
mixture by centrifugation and washed.
26. A cosmetic composition comprising as a sunblock agent a nanoparticle as
defined in any
one of claims 1 to 9, or produced according to a process of any one of claims
10 to 21, and
a suitable carrier or excipient.
27. The cosmetic composition according to claim 26, which is a topical skin
care composition.
28. The cosmetic composition according to claim 26, which is a sunscreen, skin
moisturizer,
skin cream, body lotion, body spray, mascara, foundation, rouge, face powder,
eyeliner,
eyeshadow, nail polish, or lipstick.
29. A sunscreen comprising as a sunblock agent a nanoparticle as defined in
any one of claims
1 to 9, or produced according to a process of any one of claims 10 to 21, and
a suitable
carrier or excipient.

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Description

Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.


CA 03030443 2019-01-10
LIGNIN-COATED METAL OXIDE NANOF'ARTICLES AND USE THEREOF IN
COSMETIC COMPOSITIONS
FIELD OF INVENTION
[0001] The present invention relates to nanoparticles, and associated
preparative methods. In
particular, the present invention relates to nanoparticles coated with lignin,
a process to
prepare the coated nanoparticles, and uses thereof, including uses in cosmetic
formulations
and diagnostic applications.
BACKGROUND OF THE INVENTION
[0002] Titanium dioxide (TiO2) is a common ingredient in many sun protection
products,
including sunscreens. In fact TiO2, along with zinc oxide are the two
ingredients that are
allowed in the largest concentrations in sunscreens commercialized in North
America (up to
25% permitted). This is rather surprising, as the same form of TiO2
(predominantly anatase) is
also used for a wide range of light initiated and free radical mediated
processes, including
solar cells, self-sterilizing tiles and treatment and purification of polluted
waters, due to its
high reactivity.
[0003] There have been significant concerns about titanium dioxide safety as a
sunscreen
ingredient. These concerns reflect the known fact that titanium dioxide
generates highly
reactive oxygen species (ROS) when exposed to light in the presence of oxygen
and humidity.
[0004] Accordingly, new compositions which alleviate some of the concerns
relating to TiO2
adverse effects are highly desirable.
SUMMARY OF THE INVENTION
[0005] It is an object of the invention to provide a thin-coated metal oxide
nanoparticle and
process for preparing a thin-coated nanoparticle.
[0006] Accordingly, there is provided herein a nanoparticle comprising a metal
oxide coated
with lignin to form discrete coated particles which have a nanometric size.
The metal oxide
can in certain embodiments be TiO2 or ZnO.

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[0007] In the above described embodiment, the lignin is cross-linked over a
surface of the
metal oxide particle. In certain embodiments, the cross-linking is carried out
by UVA
irradiation.
[0008] In certain embodiments, the coating on the metal oxide particle may be
from about 1
to about 10 nm in thickness. In further embodiments, the coating on the metal
oxide particle
may be from 2 to 5 nm in thickness. ln a specific embodiment, the coating on
the metal oxide
particle is about 3 nm in thickness.
[0009] As described herein, the lignin may be an organo-soluble or water-
soluble source of
lignin. Moreover, the lignin can be a pure source of lignin, or a source which
includes
carbohydrates. In certain specific embodiments, the lignin is Kraft lignin,
Organosolv lignin,
Low Sulfonate Content (LSC) lignin, Sodium lignin, Sodium lignin without
sugars or Alkali
lignin. Other lignin sources with similar properties may also be used.
[0010] Also provided herein is a process for preparing a lignin coated
nanoparticle,
comprising
a. mixing a metal, or a metal oxide nanoparticle precursor with solubilized
lignin
to form a mixture, and
b. irradiating the mixture with UVA at a wavelength effective to form
particles
which have a nanometric size.
[0011] In the described process, the metal oxide may be TiO2 or ZnO.
[0012] In embodiments of the described process, the lignin is cross-linked
over a surface of
the metal oxide particle. In certain embodiments the mixture may be irradiated
at about 369
nm to carry out the cross-linking. More particularly, the irradiating is
carried out for a time
effective to produce a coating on the metal oxide particle that is preferably
from about 1 to 10
nm in thickness, more typically from 2 to 5 nm in thickness, and in specific
embodiments
about 3 nm in thickness.
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[0013] As mentioned above, the lignin used in the process may be an organo-
soluble or
water-soluble source of lignin. Moreover, the lignin can be a pure source of
lignin, or a source
which includes carbohydrates In certain specific embodiments, the lignin is
Kraft lignin,
Organosolv lignin, Low Sulfonate Content (LSC) lignin, Sodium lignin, Sodium
lignin
without sugars or Alkali lignin. Other lignin sources with similar properties
may also be used.
[0014] The process can be carried out, in specific embodiments, by combining
the metal
oxide nanoparticle precursor with an excess of lignin, for example, from about
1:10 to about
1:5 w/w metal oxide:Lignin. In specific non-limiting examples, 1:10 w/w or 1:5
w/w
Ti02:Lignin may be combined in the mixture. In addition, in further
embodiments, the
irradiating may be carried out for up to 2 hours with stiffing to keep the
oxide nanoparticle
precursor and lignin in suspension. The process may also be carried out in
batch or
continuously. For example, if a continuous reaction is used for larger scale
production, the
irradiating may be carried out under continuous flow conditions. For example,
continuous
flow conditions may be carried out at a flow rate of Ito 10 mL/sec, or in a
specific
embodiment, at a flow rate of about 4 mL/sec. In certain embodiments of the
continuous flow
conditions described herein, it is possible to produce gram scale quantities
of lignin-coated
metal oxide nanoparticles. Using a small optical width for the UVA irradiation
can also
increase cross-linking efficiency and reduce reaction times
[0015] In further embodiments, which may be preferred depending upon user
needs, the
process may include a further step after irradiation whereby the nanoparticles
are separated
from the mixture by centrifugation and washed.
[0016] Also provided is a cosmetic composition comprising a nanoparticle as
described
above, or produced according to a process as described above, and a suitable
carrier or
excipient. In certain non-limiting embodiments, the cosmetic composition may
be a sunscreen
or other cosmetic including the nanoparticle defined herein as a sunblock
agent.
[0017] For example, the cosmetic composition may be prepared as a topical skin
care
composition. In such embodiments, the cosmetic composition may be a sunscreen,
skin
moisturizer, skin cream, body lotion, body spray, mascara, foundation, rouge,
face powder,
3

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eyeliner, eyeshadow, nail polish, lipstick, or another personal care
composition in which
sunblock may be included as an ingredient. In one non-limiting embodiment
involving coated
TiO2 nanoparti cies, the lignin coating acts as a sacrificial antioxidant
preventing the free
radical reactions that TiO2 otherwise initiates. It does so while preserving
the sun protection
and light scattering properties of TiO2.
BRIEF DESCRIPTION OF THE DRAWINGS
100181 These and other features of the invention will become more apparent
from the
following description in which reference is made to the appended drawings
wherein:
FIGURE 1 shows ATR-IR spectra of TiO2 (black), Kraft lignin (blue) and
Ti02@Kraft (red);
FIGURE 2 shows DR spectra of TiO2 (black), Kraft lignin (red) and Ti02@Kraft
(blue);
FIGURE 3 shows a TEM image of Ti02@LSC showing organic shell surrounding the
inorganic particle (arrow). Scale bar: 20 nm;
FIGURE 4 shows the percentage of Lignin released (or degraded) upon UVA-UVB
irradiation for 2 h. The plot assumes that the absorption coefficient of
lignin is constant, that
is, insensitive to exposure or release. Data reproducible within +5%;
FIGURE 5 shows the percentage of 2-propanol remaining upon UVA-UVB irradiation
in the
presence of different Particles. (A) TiO2 (black), Ti02@Kraft (blue) and
Ti02@Org (red). (B)
TiO2 (black), Ti02@LSC (blue), Ti02@Sodium (red), Ti02@Sodium without sugars
(green),
and Ti02@Alkali (violet);
FIGURE 6 shows the results of avobenzone photodegradati on using different
amount of
particles: A) 001 %, B) 0.03 % and C) 0 06 %. Percentage of avobenzone
remaining upon
UVA-UVB irradiation in the absence (black) and in the presence of TiO2 (blue),
Ti02@Kraft
(red) and Ti02@Org (green) and Ti02@LSC (violet);
FIGURE 7 shows kinetic traces of the enzymatic activity of ALP acquired at 405
nm for the
dephosphorylation of PNNP. Traces recorded after enzyme pretreatment in
absence of
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particles (black) and in the presence of TiO2 (blue), and Ti02@Kraft (red).
Full circle under
dark conditions and open circle upon UVA irradiation for 30 min;
FIGURE 8 shows initial rates calculated for the enzymatic activity of ALP
under dark and
upon UVA irradiation in the absence (black) and in the presence of TiO2
(blue), Ti02@Kraft
(red) and Ti02@Org (green) and Ti02@_,LSC (violet);
FIGURE 9 shows SPF values measured in vitro using Coppertone sunscreen as
reference (C =
0.3525) before (black) and after UVA-UVB exposure (blue);
FIGURE 10 shows ATR-IR spectra of TiO2, lignin and lignin@Ti02;
FIGURE 11 shows DR spectra of TiO2, L2@Ti02, L3@Ti02, L4@Ti02,
FIGURE 12 shows emission spectrum obtained after combination of UVA-UVB lamps.
DETAILED DESCRIPTION
[0019] The present inventors have developed a novel approach for scavenging
ROS and other
species that may be formed before they diffuse away from metal oxide
particles, such as TiO2,
and cause damage to either biomol ecul es or other important sunblock
ingredients. This
approach is different from the usual modifications of TiO2 using SiO2 or
Al2O3, or from the
known attenuation of radical generation upon encapsulation in large pore
zeolites, and
involves the use of lignin to construct a thin shell around the metal oxide
particles
[0020] Previous work has involved the use of large lignin structures
(micrometers and more)
in which metal oxide (TiO2) nanoparticles have been embedded. In such
formulations, the
nanoparticulate structure of titanium dioxide is lost as it is integrated in a
lignin matrix. The
nanoparticulate structure is a key characteristic in cosmetic and sunscreen
applications as both
its incorporation and light scattering properties are directly affected by the
size and
morphology of the material.
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[0021] The present inventors have found that coating metal oxide nanoparticles
with a thin
shell of lignin (e.g. lignin@Ti02), maintains the nano-structure needed for
effective cosmetic
and sunscreen or sunblock applications Lignin does not necessarily stop the
metal oxide from
making free radicals, but rather acts as a first line of defense, scavenging
the radicals before
they escape the shell or vicinity of the nanoparticle, thus preventing the
interaction of free
radicals with vulnerable biomolecules.
[0022] The lignin coated nanoparticle described herein therefore takes
advantage of the free-
radical scavenging and antioxidant properties of lignin, which is effectively
used as a
sacrificial scavenger for the ROS anticipated from TiO2 and other metal
oxides.
[0023] Different kinds of lignins can be attached to the surface of the metal
oxide
nanoparticles by UVA irradiation. Less than 20% of lignin release has been
found (Figure 4)
upon irradiation with UVA-UVB light showing good particle stability within the
exposure
time expected for sunscreens (2 - 4h). Furthermore, studies carried out with
one type of
particles demonstrate the addition of lignin (LSC) does not affect the SPF
values of TiO2, and
does not deteriorate SPF performance of TiO2 upon UVA-UVB irradiation (Figure
2).
[0024] In addition, the present inventors have tested degradation levels of
avobenzone upon
UVA-UVB irradiation in the presence of different particles and at different
concentrations.
The performance of the coated particles (lignin@Ti02) as avobenzone protectors
is shown
herein to be equal or greater, depending on the particle concentration, than
the pristine TiO2
NPs (Figure 3). This is especially advantageous since avobenzone is one of the
most common
sunscreen ingredients, and is widely employed as a UVA protector. However, it
suffers from
the problem of photodegradation, thus limiting its effectiveness in commercial
formulations.
These results also suggest that these particles can provide additional
protection to other
sunscreen ingredients.
[0025] The coated particles (lignin@Ti02) are also shown, based on experiments
using
horseradish peroxidase (BRP) as a biological indicator of the bioeffects of
TiO2, that
embodiments of the described coated particles can help preserve enzymatic
activity when
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compared with bare TiO2 NPs (Figure 7) This indicates that the coated
particles described
herein can help alleviate certain adverse effects of TiO2.
[0026] Accordingly, as shown in the following experiments, the coated
particles described
herein, in certain embodiments, can reduce free radical damage to
biomolecules, and work
well in conjunction with avobenzone, the most common UVA sunblock, reducing
its level of
photodegradation (the most common problem with avobenzone).
EXPERIMENTS:
[0027] In order to evaluate the effect of a lignin shell on TiO2 reactivity
and a potential
ingredient in sunscreens and cosmetics several types of experiments were
performed. First,
the inventors prepared TiO2gLignin hybrids using various types of lignin and
studied their
properties, including morphology and stability. Second, given that TiO2 is a
good
photocatalyst for the oxidation of alcohols to ketones, the inventors
evaluated to what extent
the oxidation of isopropanol to acetone is inhibited for lignin-modified TiO2.
This provides a
direct measurement of the ability of TiO2 to catalyze oxidations and is rooted
in our
knowledge of the catalytic properties of TiO2. Third, the inventors tested to
what extent lignin
modifications can reduce the extent of TiO2-mediated photochange to enzymes.
For this
purpose, the inventors used the inactivation of Alkaline Phosphatase (ALP) as
a test system.
Fourth, the inventors examined the possible photoprotection of avobenzone by
TiO2 and
lignin modified TiO2. Avobenzone is a widely used UVA ingredient, largely
present in an
enol form that photo-degrades readily upon UVA-UVB exposure. Given the
ubiquitous use of
avobenzone, it was important to establish its compatibility with the new
hybrid materials to
evaluate to what extent they could help with avobenzone's lack of
photostability. The
following sections cover the four types of experiments mentioned above.
Materials & Methods
[0028] Materials: Lignin alkali low sulfonate content, Lignin alkali, Brij 10,
Alkaline
phosphatase (ALP) and p-nitrophenylphosphate (PNNP) were purchased from
Aldrich.
Tetrahydrofuran and 2-propanol were purchased from Fischer Scientific and
Avobenzone
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from Wako. Kraft lignin was purchased from MeadWestVaco and Organosolv lignin
(extracted with 1.1 ethanol/water from mixed hardwoods - aspen, maple and
birch) was
provided by Li gnol Energy Corporation; both were a generous gift from
Professor T. Baker of
Ottawa's Centre for Catalysis Research and Innovation. Lignosulfonate sodium
with and
without sugars were a gift from Burgo company. TiO2 P25 was a gift from Evonik
Degussa.
[0029] Instruments: A High Efficacy 368 nm 11W UV LED Emitter LZ4-00U600 was
used
to synthesize the particles under irradiation. For all irradiation experiments
a Luzchem CCP-
4V customized computer-controlled photoreactor, with temperature control was
used with 10
UVA lamps and 4 UVB lamps (Figure S4). UV-visible spectroscopy was carried out
using a
Cary 100 spectrophotometer. The enzymatic assay was performed in a 96 well-
plate using a
microplate reader SpectraMax M5. NMR spectra were recorded using a Bruker
Avance II 300
spectrometer with an appropriate pulse sequence with a spectral width of -0.5
ppm to 12.5
ppm and with the pre-saturation signal centered at 4.706 ppm (proton water
signal).
Attenuated Total Reflectance Infrared (ATR-IR) spectra were recorded with a
Varian 640
FTIR spectrometer equipped with an ATR accessory in the 500 ¨ 4000 cm-1 range.
Diffuse
reflectance (DR) spectra were recorded in an Agilent Cary 7000
spectrophotometer equipped
with praying mantis (Harrick). The powder X-ray diffraction analysis was
carried out at room
temperature on Rigaku Ultima IV powder diffractometer in Bregg-Brentano
geometry, using
Cu Ka radiation (X, = 1.5418 A). Two theta range of 100 to 1000 was covered
with 0.020 step
width and 1 /min scan speed The percentage of molar mass of the adsorbed
polymer on the
surface of TiO2 was measured by Themiogravimetric analysis (TGA) using a Q5000
IR
instrument (TA Instruments, New Castle, DE, USA) under N2 or air flow (120
mL/min) with
a heating rate of 10 C/min (balance gas with nitrogen 10.0 ml/min; sample gas
with nitrogen
25.0 ml/min). The sample TGA data were analyzed by using TA Instruments
Universal
Analysis 2000 Version 4.5 A. Transmission Electron Microscope (TEM) images
were
acquired with a Jeol JEM-2100F field emission transmission electron
microscope. TEM
samples were prepared by drop casting a water suspension of catalysts onto 400
square mesh
carbon coated copper grids (Electron Microscopy Sciences).
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[0030] Synthesis of particles. Briefly, 10 (or 100 mg) of lignin were
solubilized in 5 mL of
solvent (water or THF, according to the solubility properties of the
corresponding lignin) and
placed together with 10 mg of TiO2. The mixture is kept in the dark overnight
and then
submitted to UVA (368 nm LED) irradiation for 2h under vigorous stirring The
slurry is
separated by centrifugation and washed three times. The resulting particles
are dried at 100-
120 C for at least 1 h. The particles were characterized by ATR-lit, DR, TEM,
and TGA.
[0031] Photocatalytic oxidation of 2-propanol. The photoactivity of the NPs
was observed
using as a reference reaction the photooxidation of 2-propanol to acetone. The
reaction was
carried out at 35-38 C under combined UVA-UVB irradiation (10 UVA lamps and 4
UVB
lamps). Control experiments under dark conditions were also performed (TiO2,
lignin,
Lignin@Ti02) showing no reaction. The conversion of 2-propanol in aqueous
solution (5
mM) under stirring was evaluated in presence of TiO2 and several
Lignin@TiO2NPs. For
this, 1 mL aliquots of particles were used to reach a final concentration of
0.4 mg/mL in 5 mL
and the sample was collected each 1 h for 5 h. Each aliquot was centrifuged at
7000 rpm,
20 C, for 10 min and 800 uL of the supernatant was used to record the 1H NMR
spectrum
using water suppression sequence with pre-saturation signal centered at 4.706
ppm (proton
signal of H20) in presence of 3-(trimethylsily1)-2,2,3,3-tetradeutero
propionic acid (sodium
salt) (TMSP) in D20 as external standard to analyze the degradation of 2-
propanol over
irradiation time using a calibration curve previously done.
[0032] Enzyme inactivation: TiO2-mediated photodamage. Alkaline phosphatase
from
bovine intestinal mucosa (ALP) (0.02 mg,/mL) solution and particles suspension
(0.25
mg/mL) were prepared in cold buffer (1.0 M diethanolamine with 0.50 mM
magnesium
chloride) pH 9.8 at 37 C. The substrate solution ofp-nitro phenylphosphate
(PNPP) was
prepared in water with a concentration of 0.5 mM. The enzyme was submitted to
UVA
irradiation for 30 min in the absence and in the presence of 50 ug/mL TiO2 or
Lignin@TiO2
under stirring. Then, the suspensions were centrifuged at 11,000 rpm for 15
min at 0 C.
Control reactions under dark conditions were also performed. The enzymatic
assay was
performed in a 96 well-plate using the following final concentrations: [PNPP]
= 25 uM and
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[ALP] = 1.5 ng/mL. The enzyme activity was followed by monitoring the
absorbance changes
at 405 nm, where the dephosphorylated product has a maximum absorption.
[0033] Compatibility with Avobenzone. An Avobenzone aqueous solution (24 M)
was
prepared in 1 mM Brij-10 solution (<0.04% of 2-propanol) The mixture was
sonicated for 3
h and stored in the dark one night. The reaction was carried out using 8 mL of
this solution in
a quartz test tube placed in a photoreactor equipped with 10 UVA lamps and 4
UVB lamps
under stirring. TiO2 and several Lignin@TiO2NPs were tested using three
different
Avobenzone/particles ratio: 1/13; 1/41; 1/82 (w/w). Samples (1 mL) were
collected at lh
intervals for 4h and centrifuged at 7000 rpm, 20 C, 10 min. Each aliquot was
analyzed by UV
spectroscopy recording absorbance at 362 nm.
Results and Discussion
Synthesis and characterization
[0034] Different kinds of lignin were used for the synthesis of the new
material ranging from
water soluble lignin to lignins that can only be solubilized in organic
solvents (Table 1).
Briefly, 10 mg of lignin are solubilized in 5 mL of solvent (water or
tetrahydrofuran
according to the solubility properties of the corresponding lignin) and placed
together with 10
mg of TiO2. The mixture is kept under dark overnight and then submitted to UVA
(368 nm
LED) irradiation for 2h under vigorous stirring. The slurry is separated by
centrifugation and
washed with water (or THE, respectively) three times. The resulting particles
are dried at 100-
120 C for at least 1 h. The particles were characterized by Attenuated Total
Reflectance
Infrared (ATR-lR), Diffuse Reflectance (DR), Transmission Electron Microscopy
(TEM), and
Termogravimetry analysis (TGA).
[0035] The particles can thus be synthesized under very mild conditions taking
advantage of
the photocatalytic activity of TiO2. Upon UVA irradiation a mixture of a
lignin solution
(organic or aqueous solution depending on the type of lignin used) in the
presence of TiO2,
lignin can be cross-linked over the particle surface (due to light-induced ROS
generation) and
lead to lignin-coated TiO2nanoparticles within 1-2 hours.

CA 03030443 2019-01-10
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[0036] Figures 1 and 2 show the functionalization of TiO2 using Kraft lignin.
Similar results
were found for the other types of lignin used (Figures 10 and 11). According
to the IR
spectrum of TiO2, the band at 3400 cm-1 is due to the OH stretching, while at
1630 c1i11 OH
bending vibrations can be observed. Between 1000 and 400 cm-1, the broad band
is related to
the Ti-O-Ti stretching bonds. On the other side, the characteristic peaks of
lignin are at 2900
cm-1 for sp3 C-H stretching, 1600 cm-1 for C=0 stretching and below 1500 cm-1
for the
aromatic rings bending. In general, all particles clearly show signals
corresponding to both
TiO2 and lignin. The diffuse reflectance (DR) spectra in Figure 2 show that
the particles can
slightly extend the absorption of TiO2 to the visible light region (typically
below 400 nm) due
to to the presence of lignin. Note the thin lignin coating makes these
compositions cosmetically
acceptable not only in terms of light absorption and scattering but also in
terms of the visible
color and appearance. In fact, lignin@TiO2NPs show a very light tint suitable
for skincare
formulations. Additionally, as the particles are insoluble in water, they are
effectively
waterproof.
[0037] Figure 3 is a HR-TEM image suggesting organic shell surrounds the TiO2
particles,
and more important, that the particles retain their nanometric size (< 50 nm).
Finally, Table 2
shows the amount of lignin found on each particle using TGA. Of note, the
organo-soluble
lignins generate particles with higher loadings, presumably due to the
presence of more
conjugated structures in those types of lignin that can interact better with
the free radicals
generated by TiO2.
Table 1. Different types of lignin used for TiO2 encapsulation
Name Type of Lignin Solubility
Li Kraft Lignin
Organo-soluble
L2 Organosolv Lignin
L3 Low Sulfonate Content (LSC)
L4 Sodium Lignin
Water-soluble
L5 Sodium Lignin without sugars
L6 Alkali Lignin
11

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Table 2. Weight percentage of lignin in each particle found after
thermogravimetric
analysis (TGA).
Particle Lignin (wt %)3 Shell thickness
(nal)*
Ti02 Kraft 43 9.5
13b 3.6
Ti02 Org 18 4.7
8b 2.3
TiOz@LSC 9 2.6
5b 1.5
Ti02 Sodium 8 2.3
Ti02@Sodium no sugars 6 1.8
Ti02 Alkali 3 0.9
a Synthesized using 100 mg of lignin unless otherwise indicated.' Synthesized
using
mg of lignin. *Shell thickness calculated with a density of 3.8 gmL-1 for
anatase
5 (10 mg) and assuming a 50 nm particle size.
Stability test
[0038] The stability of the particles in an aqueous solution upon UVA-UVB
irradiation was
monitored by UV spectroscopy, following the absorption at the wavelength of
maximum
10 absorption of the corresponding lignin. Thus, the absorbance due to
leached or degraded
lignin can be measured in the supernatant of the mixture after 2 h of
irradiation.
[0039] Dried NPs (2.5 mg) were re-suspended in aqueous solutions (10 mL) and
their
stability was analysed at 35-40 C under UVA/UVB irradiation. At specific
times, an aliquot
of 1 mL of each sample was taken and centrifuged at 7000 rpm, 20 C for 10 min.
The
supernatant was analyzed by UV spectroscopy to evaluate the possible presence
of small
compounds originated from leaching and/or degradation of lignin. Control
experiments in
dark conditions show same tendency within the experimental error. As can be
seen in Figure
4, the particles show great stability under UVA-UVB exposure (see also Figure
12). As it was
expected, the % of Kraft and organosolv lignin (organo-soluble lignins L1 and
L2) released
was lower compared with water-soluble lignin.
12

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Photocatalytic oxidation of 2-propanol
[0040] The photoactivity of NPs was observed using as reference reaction the
photooxidation
of 2-Propanol to acetone. The reaction was carried out at 35-38 C under UVA-
UVB
irradiation. Control experiments under dark conditions were also run. The
conversion of 2-
propanol in aqueous solution (5 mM) under stirring was evaluated in presence
of TiO2 and
several Ti02@lignin NPs. For this, 2 mg of particles were used and the sample
was collected
each 1 h for 5 h. Each aliquot was centrifuged at 7000 rpm, 20 C, for 10 min
and 800 1.it of
the supernatant was used to record the 1fINMR spectrum using water suppression
sequence
in presence of 3-(trim ethyl sily1)-2,2,3,3-tetradeutero propionic acid
(sodium salt) (TMSP) in
D20 as external standard to analyze the degradation of 2-propanol over
irradiation time.
Figure 5 shows the photocatalytic activities exhibited by the different
Lignin@TiO2
composites compared to the pristine TiO2. Notice that 2-propanol is totally
consumed after 3 h
of irradiation in the presence of TiO2 but different percentages of alcohol
still remain when
treated with lignin-modified TiO2. Thus, while the strategy used to synthesize
the
lignin@TiO2 is based on the photocatalytic activity of TiO2, the new
composites exhibit the
capacity to inhibit free radical reactions. Particles showing the worst
photocatalytic activity
are indeed the ones chosen as potential sunblock active ingredients. From
Figure 5,
Ti02@Kraft, Ti02@Org and Ti02@LSC were selected for further examination,
although
L4@TiO2 (Sodium Lignin) also shows excellent performance.
Photodegradation of Avobenzone
[0041] In order to determine the compatibility of these new particles with
other sunscreen
ingredients, the photoprotection of avobenzone was tested. Avobenzone is a
widely used
UVA protector, largely present in an enol form that photodegrades readily upon
UVA-UVB
exposure, through a mechanism involving a photo-induced enol-keto
transformation. Other
sunblock agents can stabilize avobenzone, either by competitive light
absorption (or
scattering) or by quenching its excited states. Given the ubiquitous use of
avobenzone, it is
important to establish its compatibility with the new hybrid materials to
evaluate to what
13

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extent they could be involved in the process of photodegradation or
photoprotection of
avobenzone.
[0042] The avobenzone aqueous solution (24 viM) was prepared in 1 mM Brij-10
solution (<
0.04% of 2-propanol). The mixture was sonicated for 3h and stored in dark
overnight The
reaction was carried out using 8 mL of this solution in a quartz test tube
placed in a
photoreactor equipped with 10 UVA lamps and 4 UVB lamps under stirring. TiO2
and several
Ti02@lignin NPs were tested using three different particle concentrations:
0.01; 0.03; and
0.06 wt%. The sample (1 mL) was collected each lh for 4h and centrifuged at
7000 rpm,
20 C, 10 min. Each aliquot was analyzed by UV spectroscopy recording
absorbance at 362
nm. Figure 6 shows the photodegradation of avobenzone at two different times
(2 and 4 h of
UVA-UVB irradiation) and using different amount of particles. Lower particles
concentration
TiO2 can act as a photoprotector (graphs A and B), although when the TiO2
particle
concentration is increased this ability is lost. In contrast, the new
particles retain the
photoprotection ability even at high TiO2 concentrations. These results
clearly show that new
particles not only preserve the photoprotection properties that TiO2 provides
to avobenzone
(graph A) but also prevent the photodegradation of avobenzone when the amount
of TiO2
added generates high concentration of ROS (graph C). This opens the
opportunity to increase
the amount of TiO2 particles in formulations preserving the integrity of other
organic active
ingredients.
Alkaline phosphatase assay
[0043] Alkaline phosphatase from bovine intestinal mucosa (ALP) (0.02 mg/mL)
solution and
particles suspension (0.25 mg/mL) were prepared in cold buffer (1.0 M
Diethanolamine with
0.50 mM Magnesium Chloride) pH 9.8 at 37 C. The substrate solution of p-nitro
phenylphosphate (PNPP) was prepared in water with a concentration of 0.5 mM.
The
enzymatic activity of ALP was performed after enzyme was pre-treated with
different
particles and under different conditions. Briefly, the enzyme was submitted to
UVA
irradiation during 30 min in the absence and in the presence of TiO2 and
Ti02@lignin NPs
14

CA 03030443 2019-01-10
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under stirring. Then, the suspensions were centrifuged at 11,000 rpm for 15
min at 0 C.
Control reactions under dark conditions were also carried out.
[0044] ALP assay was performed in a 96 well-plate with ([PNPP] = 25 uM; [ALP]
= 1.5
ug/mL) and the enzymatic activity was followed by the absorbance changes at
405 nm, where
the dephosphorylated product has an absorption maximum. TiO2 and several
Ti02@lignin
NPs were tested using 50 ug/mL.
[0045] The following equation shows the de-phosphorylation reaction used to
determine the
enzymatic activity, simply by monitoring the formation ofp-nitrophenol using
UV-vis
spectroscopy.
o P-Na4 0
,
0 Na ALP H20 02N 11 OH + 0=P-0 Nn
02N 11 0'
ONa:
[0046] The kinetic traces (Figure 7) acquired at 405 nm for the founation ofp-
nitrophenol are
a reflection of the activity of the enzyme. Each curve is then fitted with the
expression:
A405 nut = a + bt + Ct2
where A is the absorbance and t the time. The coefficients a, b and c are
fitting parameters.
The derivative of this expression with respect tot is given by:
dA
dt = b + ct
which at t = 0 corresponds to b. That is, the first coefficient (b) of the
quadratic fit is the
calculated initial slope. These slopes have been used as a measure of the
enzymatic activity.
[0047] Figure 8 shows the initial rates calculated for the enzymatic activity
of ALP after
treatment with TiO2 and Ti02@lignin particles. As can be noticed, TiO2 can
decrease the
enzymatic activity simply by contact (dark conditions). This inhibition is
increased under
UVA exposure (Light conditions). Coating the TiO2 nanoparticles with any kind
of lignin

CA 03030443 2019-01-10
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PCT/CA2017/050613
prevents the enzyme inactivation even under light conditions. These results
indicate clearly
that the UVA irradiation does not affect the enzyme and, more important,
lignin@TiO2NPs
are innocuous for the enzymatic activity under dark conditions. The TiO2-
mediated
photodamage under UVA irradiation is highly reduced in presence of lignin and
the Li@TiO2
(Kraft Lignin) composite shows no enzyme inactivation. Clearly the changes
that prevent
alcohol photooxidation also inhibit enzyme inactivation.
Sun Protection Factor (SPF) determination in-vitro
[0048] 0.75 mg/cm2 of TiO2 and Ti02@Lignin NPs suspension in glycerol (5%;
10%) were
placed on a quartz slide (9.75 cm2) covered by 3M Transpore Nexcare tape. The
emulsion was
weighed on the slide and evenly distributed on the surface. The sample is left
to dry under air
during 20 min. Five different plates for each sample were analyzed. The light
transmittance of
each sample was measured between 290 to 400 nm before and after exposure to
UVA-UVB
light for 2h. The same protocol was followed using Coppertone sunscreen with
SPF value
informed equal to 30.
[0049] The SPF value was calculated in according to COLIPA standard protocol
by the
following equation:
= 400 UM
f
= 290 ran
SPF. ¨ _ ___________________________
- vitro = 400 nrn
f- A ,())
E (A) * O.) * 10 " *
A = 290 nrn
where E(A) is the erythema action spectrum, I(X) the solar spectral
irradiance, d(X) the spectral
transmittance of the sample, C a coefficient of adjustment and Ao(X)
correspond to the mean
monochromatic absorbance measured per plate of the test product layer before
UV exposure.
Conclusions
[0050] Regardless of its great light absorption and scattering properties,
there are some health
concerns about the use of 'TiO2 because of its intrinsic photocatalytic
properties. Thus, TiO2
16

CA 03030443 2019-01-10
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PCT/CA2017/050613
can generate ROS in the presence of water upon UVA irradiation. The in vitro
studies
reported here demonstrate that TiO2 particles can be modified in order to
decrease their
photocatalytic activity, while retaining the absorption and scattering
properties desirable for
sunscreens and cosmetic uses. Thus, the potential risks from TiO2-mediated
free radical
generation are curtailed by shielding the particles with a good antioxidant.
Here the inventors
have used a non-toxic, biocompatible shell made by lignin that neutralizes the
free radicals by
scavenging them with neutral antioxidants before they exit the new TiO2-lignin
composites,
preserving the scattering and the UV absorption characteristics. For this
purpose, it was
demonstrated that this stable lignin@TiO2 composite plays an important role
reducing the
photocatalytic activity of TiO2 in a chemical and enzymatic reaction,
improving the
photoprotection of the other ingredients even when they are present at high
concentrations. As
such, the particles described here, showing a nanometric size and a very light
color, are
promising candidates as ingredients in skincare formulations, especially for
sunscreens, given
that they are non-toxic and waterproof. Additionally, this approach regarding
the use of a
nontoxic and extremely versatile material, mainly a by-product of the paper
industry, also
contribute to the development of environmentally-friendly processes for the
cosmetic
industry. While illustrated here with lignin, it is clear that the same
strategy could be
implemented with other polymeric or polymerizable antioxidants
[0051] One or more currently preferred embodiments have been described by way
of
example. It will be apparent to persons skilled in the art that a number of
variations and
modifications can be made without departing from the scope of the invention as
defined in the
claims
[0052] The terminology used herein was chosen to best explain the principles
of the
embodiments, the practical application or technical improvement over
technologies found in
the marketplace, or to enable others of ordinary skill in the art to
understand the embodiments
disclosed herein.
[0053] All documents cited in this application are herein incorporated by
reference in their
entirety.
17

CA 03030443 2019-01-10
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REFERENCES:
(1) Jacobs, J. F.; van de Poe!, I.; Osseweijer, P., "Sunscreens with
Titanium Dioxide
(Ti0(2)) Nano-Particles: A Societal Experiment", Nanoethics 2010, 4, 103-113.
(2) Morsella, M.; Giammatteo, M.; Arrizza, L.; Tonucci, L.; Bressan, M.;
d'Alessandro,
N., "Lignin coating to quench photocatalytic activity of titanium dioxide
nanoparticles for
potential skin care applications", RSC Advances 2015, 5, 57453-57461.
(3) Hancock-Chen, T.; Scaiano, J. C., "Enzyme Inactivation by TiO2
Photosensitization
with UFA" , J. Photochem. Photobiol, B: Biol, 2000, 57, 193-196.
(4) Ricci, A.; Chretien, M. N.; Scaiano, J. C., 'TiO2-promoted
mineralization of organic
sunscreens in water suspension and sodium dodecyl sulfate micelles',
Photochem. Photobiol.
Sci. 2003, 2, 487-492.
(5) US 8,445,562
(6) US 2010/0121110
(7) WO 2009/038477
(8) US 2015/0090157
(9) US 2012/0130001
(10) US 2003/0121630
(11) US 2015/0284309
(12) AU 2005/207655
(13) W02005/072680
(14) US 8,632,816
(15) US 2014/0037703
(16) WO 2013/124459
(17) WO 2014/144746
(18) US 8,911,976
(19) US 8,431,143
(20) US 2005/0129634
(21) US 2015/0166836
(22) WO 2014/164418
(23) AU 2008/366085
(24) US 2015/0225531
(25) EP 1 438 361
18

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(86) Date de dépôt PCT 2017-05-19
(87) Date de publication PCT 2017-11-23
(85) Entrée nationale 2019-01-10
Requête d'examen 2022-05-05

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MORSELLA, MICHELA
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