PCE_ PN-II-ID-PCE-2012-4-0111
PROJECT Number 80/02.09.2013
FUNDING SOURCE: Executive Unit for Financing Higher Education,
Research, Development and Innovation (UEFISCDI)
BIO-ACTIVE LIPID NANOCARRIERS FOR CO-ENCAPSULATION OF
SELECTIVE VEGETAL EXTRACTS
WITH ENHANCED PHARMACO-COSMETIC PERFORMANCE
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Hederacozide C (from Ivy leaf extract, Ile) |
Salicin (from Willow bark extract, Wbe) |
PROJECT MANAGER: Prof. dr. Aurelia MEGHEA
Implementation Team
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Associate
prof. Ioana Lacatusu |
Associate
prof. Nicoleta Badea |
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Dr.
eng. Gabriela Badea |
Prof. dr. Raluca Stan |
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Lecturer
dr. Cristina Ott |
Dr. eng. Elena Mitrea |
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Lecturer dr. Marcela Barbanta Patrascu |
Dr. eng. Alina Popa |
Implementation period: 02.09.2013 – 30.09.2016
Budget
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Year |
Project Value [RON] |
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2013 |
127.500 |
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2014 |
278.261 |
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2015 |
322.699 |
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2016 |
396.180 |
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Total |
1.125.000 |
Abstract
General
objective of this project is development of novel pharmaco-cosmetic
nanostructured formulations based on vegetable oils and medicinal plant
extracts with enhanced therapeutically performance. The basic idea of the
researches performed in this project is to follow several innovative steps by
joining the “soft nanotechnology” principles with capitalization of vegetal
resources rich in bioactive components. In this context the research activities
deployed in this project are: (1) Stepwise replacing some synthetic active
substances by natural mixtures of bioactive components obtained from selective
plant extracts. This means the use of phytochemicals not only as bioactives encapsulated at nanoscale, but also as main
components of lipid nanocarrier matrices; (2)
Co-encapsulation of two or many natural bioactive compounds from selective
plant extracts (e.g. ivy leaves extract and willow bark extract),
thus taking advantage of their synergistic and/or complementary therapeutic
effects. For the synthesis of bioactive lipidic nanocarriers various types of natural oils have been chosen
(e.g. rasberries,
grape and pomegranate seed oils, and rice brain oil). These free and loaded lipidic
nanocarriers are characterized from porpho-structural point of view by using various adequate
techniques (UV-VIS, FT-IR, HPLC,
GC-MS, DSC, HR-TEM), for their photoprotective
properties and photostability, and also for sustained
delivery of bioactives by using Franz diffusion
cells. In order to evaluate their therapeutic performance, the developed
topical formulations will be examined by specific in vitro and in vivo
tests so that to determine both cosmetic efficacy (antioxidative
and anti-UV properties) and pharmacological activity (antimicrobial, anti-inflammatory
and skin healing properties.
General OBJECTIVES of project
I. The multiple co-encapsulation of vegetal extracts into complex
nanostructured bio-active lipid carriers that
present enhanced biological activity and are able for sustained – release of two bio-active principles with synergistic
effects. The accomplishment
of this objective requires corroboration of several specific goals:
I.1. Characterization of
natural raw materials (e.g. raspberries, grape and pomegranate seed oils, rice
brain oil) used for obtaining of new efficient bio-active lipid nanocarriers (BLNs) with antioxidant and anti-UV
properties, and of their vegetal
bio-active extracts (e.g. ivy leaves extract - ILE and willow bark extract - WBE), by spectral and chromatografic
tehniques and in vitro analysis.
I.2. Obtaining of free-BLNs
with selected natural oils and of BLNs loaded with ILE and WBE,
achieved by a modified melting emulsification coupled with high shear
homogenization technique.
I.3. Physical – chemical characterization of the newly obtained
BLNs which co-encapsulate the vegetal extracts, by using
specific analysis techniques: electrokinetic
potential, DLS, FT-IR, UV-VIS, HPLC, DSC, HR-TEM.
I.4. Evaluation of the biological efficiency of designed
vegetal extracts – BLNs, achieved by specific in vitro analysis (determination
of antioxidant capacity, anti-UV blocking effect, anti-microbial properties)
and in vivo tests (the skin healing and anti-inflammatory properties).
I.5. The sustained-release behaviour of vegetal extracts-BLNs. Testing the delivery and release strategies of two
bio-active compounds from the BLNs, by using in vitro experiments in
Franz cells.
II.
Developing of new pharmaco-cosmetic formulations
based on nanostructured vegetal oils and medicinal extracts with amplified therapeutic performance. The specific goals are:
II.1. Conditioning the BLNs loaded with ivy leaves extract and willow bark extract into topical formulations (e.g. creams,
ointments, hydrogels).
II.2. Cosmetic efficacy of developed topical formulations, by interpreting the
specific parameters (e.g. SPF, UVA/UVB ratio, rheological behaviour,
determination of hydration effect).
II.3. Exploring and testing of therapeutic performance of
topical formulations based on BLNs loaded with ivy leaves
extract and willow bark extract, by specific in vitro and in vivo tests.
EXPECTED RESULTS
· Developing of an experimental method
used at pilot scale
for obtaining new lipid bio-nanocarriers based
on vegetable oils;
· Nomination of efficient models of
nanocarrier systems able to co-encapsulate complex mixtures of extracts;
· Development of an experimental process
for assessing the functionality of
lipid bio-nanocarriers;
· Development of preliminary conditioned formulations (cream/hydrogel)
based on biocompatible nanocarriers loaded with vegetable
extracts;
· Participation in international
conferences;
· Publication of 4 articles in ISI
journals with cumulative FI > 7.
STAGE 1 (September 2012 – November 2013)
OBJECTIVES and ACTIVITIES foreseen and achieved
in 2013
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Objective 1. Characterization of natural raw materials used in the
production of further new bioactive
lipid nanocarriers (BLN)
with effective antioxidant
and anti-UV properties. · Preliminary characterization
of natural lipids by UV-VIS and FTIR
(e. g. raspberry seed oil, grape seed oil,
pomegranate seed oil and rice bran oil)
and plant extracts (ivy leaf extract,
willow bark extract). · The
development of the experimental conditions for
determining the fatty acid
composition (e.g. omega-3,
5, 6 and 9) of
the vegetable oil (gas chromatographic methods). · Evaluation
of anti-UV properties of
raspberry seed oil, pomegranate oil and
rice bran (in vitro tests). ·
In vitro determination of
antioxidant activity of natural oils
and plant extracts selected (chemiluminescence technique). |
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SELECTED ASPECTS of research in 2013
During September 2012 – November 2013 stage an advanced characterization of plant materials (grape seed oil – GSO, rice bran oil –
RBO, raspberry seed oil –
RSO, pomegranate seed oil –
PSO, willow bark extract and ivy leaf extract) was performed, based on
the setting of the four complex compositions of vegetable oils (by gas
chromatography), the identification of
the characteristic bands of the IR and UV-Vis, and
the in vitro determination of some specific
properties, that have as a direct result obtaining the
information on the effectiveness of
these oils and extracts for subsequent
synthesis of nanostructures
with high lipid complex
natural mixtures.
STAGE 2 (December
2013 – November 2014)
OBJECTIVES and ACTIVITIES foreseen and achieved
in 2014
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Objective 1. Obtaining of free
bioactive lipid nanocarriers (BLN) using different natural lipids. ·
Synthesis experiments of lipid
nanocarriers in the aqueous phase
using a modified technique with high
shear homogenization. ·
Evaluation of bioactive
lipid nanocarriers stability by determining the zeta potential. ·
Effect of composition
of natural oils on particle size distribution of lipid nanocarriers. ·
Dimensional analysis by DLS. ·
Evaluation of crystallinity
and polymorphism of lipid nanocarriers
prepared with different natural oils (DSC analysis). |
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Objective 2. Synthesis and physico-chemical characterization of BLN loaded with ivy
leaf extract. ·
Co-encapsulation
experiments of ivy leaves extract into
bio-active nanocarriers, by two synthesis procedure
(HSH and HPH). ·
Determination
of physical stability (electrokinetic potential)
and average diameters and polydispersity index (DLS). ·
Establishing
of ivy leaves extract – lipid
matrix interaction, by evaluation of spectral characteristics (FT-IR,
UV-VIS-NIR). ·
Crystallinity and
polymorphism evaluation of lipid matrices (with/without loaded ivy leaves extract) by DSC analysis. ·
Morphologic characterization
of loaded nanocarriers
with ivy leaves extract by
electron microscopy (TEM, Single molecule spectroscopy). |
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Objective 3. Assessment of biological efficiency of ivy leaf extract (Ile) co-encapsulated
into BLN. · The comparative in vitro evaluation of
antioxidant capacity of ivy leaves extract – BLNs prepared
with different natural oils. · In vitro antiproliferation studies
on different cultures (MTT tests or System x CELLigence). |
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Expected and performed
RESULTS in Step 2014
·
Experimental methods for obtaining new lipid bionanocarriers based vegetable oils;
·
Effective models of nanocarrier systems able to co-encapsulate complex
vegetable mixtures;
·
Participation in two international conferences;
·
Publication of 2 articles
in ISI journals
with cumulative FI
> 3;
·
Annual scientifically and economical Report.
SELECTIVE ASPECTS of 2014 research stage
During December 2013 – November
2014 stage novel bioactive lipid
nanostructures (BLN) based on vegetable oils rich in omega 3, 5, 6
and 9, free and loaded with ivy leaf extract (Ile) were obtained. Assessment of biological efficiency of ivy leaf
extract co-encapsulated BLN was
another key aspect of the research, this being
achieved in a first step by determining in vitro
antioxidant activity and in vitro cytotoxicity
tests of formulations developed.
Exploration of a variety of vegetable
oils containing different
concentrations in omega-3,
omega-5, omega-6 and
omega-9 (e.g. raspberry seed oil,
pomegranate oil, grape
seed oil, oil germs of rice) in combination
with two types of surfactant mixtures (mainly ionic
- 2% sodium
cholate or predominantly
non-ionic - 1.75%
Tween 20) resulted in obtaining valuable results associated with the
development of bio- lipid nanocarriers with average diameters between 105 and 145 nm.
By DLS measurements was observed that the position and number of double bonds in vegetable oils studied showed no significant influence on the Zave of lipid nanocarriers, instead the encapsulation of ivy leaf extract resulted in a decrease in average diameters.
In present research, zeta potential
values are strongly electronegative (-37 ÷
-47 mV), which shows a good stability of
these lipid nanocarriers prepared with various vegetable
oils associated with reduced
occurrence of aggregation phenomena
of lipid particles in aqueous suspension.
During encapsulation
of complex mixture of ivy leaf extract a slight deceleration
of endothermic peak, of the crystallinity index and of melting point values have
been observed for NLC containing
7.4% EI, suggesting a minor disturbance of
lipid network formed
by the solid lipids together with
vegetable oil. The combination of vegetable oils with the presence of an
initial concentration of 1% EI led to a predominant
distribution of vegetable
extract in surfactants
mixture shell.
UV-Vis characterization
of lipid nanoacrriers free and loaded with EI highlighted the existence
of a broad absorption domain which may be
associated with overlapping absorption bands of functional
groups existing in the main components of
EI as well as of unsaturated fatty acids from GSO/PSO/RBO or RSO. By
IR deconvolution in the 1600-1700 cm-1 area specific bands of the principal component of EI, respectively carbonyl groups
of Hederacoside C and double bonds of
hederagenina aglycone
were identified.
For solid formulations of lipid bio-nanocarriers that include 22.2% raspberry/rice/grape or pomegranate oil and
7.4% ivy leaf extract a capacity more than 98% of
scavenging free radicals in situ generated using a chemiluminescence system
has been identified.
The results obtained by testing proliferative vs.
cytotoxic of BLNs were varied according to the composition, the concentration,
time of treatment, and the murine cell line, a normal cell (L929), and the
other of tumor cells (B16). BLN that encapsulate 1% Ile and 3% rice brain oil had a more pronounced proliferative
effect on L929 cells.
For the purpose of comparison in real time of
proliferative versus cytotoxic
capacity of bionanocarriers prepared with rice bran
oil, raspberry seed oil and pomegranate seed oil on tumor cells B16, was used
the RTCA test. RTCA results obtained on the melanoma B16 cells confirm the data
obtained by MTS colorimetric technique. A cytotoxicity of 50% can be achieved
in the B16 cell line following a treatment for 24h with much higher
concentration of BLN-Ile prepared
with pomegranate seed oil (688 mg/mL) as compared to the BLN-Ile prepared with raspberry seed oil (352
mg/mL).
STAGE 3 (December 2014 – November 2015)
OBJECTIVES and ACTIVITIES foreseen and achieved
in 2015
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Objective 1. Evaluation
of the biological efficiency of ivy
leaves extract
co-encapsulated into BLNs (part II) ·
Determination
of encapsulation efficiency (EE%) by
spectrophotometric or chromatographic methods (UV-VIS, HPLC). ·
In
vitro determination of UV
blocking effect (assessing of SPF index and photostability
by simulated irradiation tests on UV-A and UV-B). |
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Objective 2.
Synthesis
and structural characterization of BLNs
loaded with willow bark extract or carotenoids extract · Co-encapsulation
experiments of willow
bark extract or carotenoids extract into
bio-active nanocarriers, by using the optimized
synthesis procedure · The structural and morphologic characterization of lipid nanocarriers loaded with willow bark extract or carotenoids extract (DLS,
electrokinetic potential, DSC, UV-VIS/HPLC, TEM). |
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Objective 3. The evaluation of
biological activity of BLNs
loaded with willow bark extract or marigold/carotenoids
flower extract ·
The in vitro evaluation of antioxidant
activity and anti-UV properties of lipid nanocarriers
loaded with willow bark extract or carotenoids
extract ·
Microbiologic tests achieved on BLNs – willow
bark extract/carotenoids extract (specific in vitro tests on
cellular cultures; cell apoptosis by flow cytometry). |
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Objective 4. Pharmacokinetic
behaviour on optimized ivy leaves extract,
willow bark extract or marigold flower extract (carotenoids
extract) – BLNs · The release strategies of bio-active compounds
from the BLNs (in vitro release experiments in Franz diffusion cells).
HPLC or UV determinations. · Investigation of the kinetic release, as
function of liquid and solid lipids mixture and surfactants type. |
Expected and performed RESULTS
·
Preliminary conditioned formulations based on lipid bionanocarriers loaded with vegetable
extracts;
·
Experimental procedures for evaluation of
lipid bionanocarriers functionality;
·
Participation to one international conferences;
·
Publication of one article
in ISI journals
with FI > 2;
·
Annual scientific and economic reports.
SELECTIVE ASPECTS of 2015 research stage
Experimental research conducted during December 2014 and
December 2015 has
considered the continuing of characterization of previous systems of lipid bio-nanocarriers (BLN) loaded with ivy leaves extract and
reaching another important objective of the project – the synthesis and
structural, biological and pharmacokinetic characterization of other lipid
nanostructures prepared on the basis of raspberry
seed oil (Rso), rice bran oil (Rbo),
hemp seed oil (Hso) and amaranth oil (Ao). The new BLN systems
were subsequently used for encapsulation of plant
mixtures rich in bioactive components with remarkable therapeutic effects: one
lipophilic carotenoids extract, Ce
(derived from cold pressing of Marigold flowers)
known mainly for its role in combating free radicals and one hydrophilic
extract that shows potential anti-inflammatory effect – willow bark extract, Wbe and/or
associated with a synthetic anti-inflammatory drug (Indometacin, Ind).
Objective 1
Characterization of the lipid
bio-nanocarriers with a content
of 30% raspberry seed oil/ grape seed oil/seed oil, pomegranate seed oil/rice bran oil and loaded with 7.6% ivy leaf extract continued in
stage 2015 with
the determination of encapsulation efficiency of hederacoside C (the main
component present in the ivy leaf extract. HPLC results
have highlighted an efficient capacity of BLN to provide host spaces for accommodation of hydrophilic
mixture (e.g.
encapsulation efficiency up to
80%).
To evaluate the BLN –
ivy extract potential to block UV
radiation, the BLN were conditioned as
hydrogel formulations. The results have shown that hydrogels based on BLN-ivy
extract did not manifest anti-UV
properties, having an SPF and FP-UVA
= 1.
Objective 2
The synthesis experiments in aqueous phase of new
BLN types were carried out using the technique of high-shear homogenization
(HSH) coupled to the high pressure homogenization (HPH). The size
characteristics of aqueous BLN systems prepared with Hso/blend of Ao and Hso were evaluated based on DLS technique.
The BLN-average diameter (e.g. 112.2
and 131.4 nm for BLN prepared Hso; 123.3 and
110.1 nm for BLN prepared with blend of vegetable oils) was slightly higher
than those of their corresponding unloaded BLN systems.
Similar results were obtained for BLN systems
prepared with Rso and Rbo, despite the fact that the type and nature
of bio-active encapsulated components have been entirely changed. By
co-encapsulation of the two active ingredients, natural and synthetic – Wbe and Ind, the obtained average diameters were of
134.9 nm/PdI = 0.172 (for systems synthesized with Rso) and of 140.4
nm/PdI = 0.176 (for systems synthesized with Rbo). The narrow
size distribution of nanocarries with PdI< 0.16 was achieved for BLN that encapsulates only
the hydrophilic mixture
In terms of BLN-Ce
stability, the extract encapsulation
of variable concentrations of
carotenoids has not significantly
affected the zeta potential value,
only a slow increase being observed for BLN-Ce compared
with those of unloaded/free BLN. A
decrease of Hso
content resulted in higher values of zeta potential
(e.g. from -38.1 mV to -34.5 mV for BLN prepared Hso and from -37.2 mV to
-33.4 mV for
BLN prepared mixture
of Hso and Ao.
Encapsulation of the Wbe or mixture of Wbe and Ind into bio-nanocarriers,
resulted in BLN presenting excellent physical stability with zeta potential
values between -40 and - 42 mV (for BLN synthesized with Rbo) and -43 and
-48 mV (for BLN obtained with Rso).
Disruption of lipid network after capturing the
vegetable mixture of lipophilic (Ce)
or hydrophilic nature (Wbe),
and of the mixture of Wbe
with Ind
was evident by comparing the calorimetry behavior of BLN-free and those that
co-encapsulate the selected active principles. BLN synthesized using varying
amounts of vegetable oils are characterized by a lipid network with many
imperfections, and therefore, with a significant effect on encapsulating
efficiency of plant extract. The quantitative results obtained on the BLN
prepared with blend of Hso
and Ao
proved an independent behavior of crystallinity index versus encapsulation efficiency. The best capacity to capture the
extract carotenoids, with an efficiency of 83.5% was encountered in the case of
bio-nanocarriers prepared by mixing 1.5% Hso with 1.5 of Ao%.
A comparative analysis of the DSC results achieved
on BLN-Wbe
and BLN-Wbe-Ind, emphasized the occurrence of significant changes in
the lipid networks synthesized with Rso and Rbo, reported at the
first view to the displacement of melting points compared to BLN-free. Also the
presence of Ind
and Wbe
caused a decrease in the melting enthalpy, suggesting a lower level of
organization in the amorphous network of BLN that co-encapsulates both natural
and synthetic active ingredients.
Objective 3
For this part of experiments,
vegetable oils, free lipid bio-nanocarriers,
and BLN loaded with marigold extract and willow
bark extract with/without inflammatory
synthetic drug, have been subjected to
the action of oxygen free radicals
generated in situ
in a chemiluminescence system. Mean antioxidant
activity obtained for BLN prepared with Hso ranged from 94.9 to 97.1%,
while those detected for BLN synthesized
with mixture of Hso and Ao, ranged between
93.4 and 97%. The
percentage of antioxidant activity increase was proportional to the vegetable oil used in bio-nanocarriers synthesis. The highest antioxidant activity was achieved for BLN-Ce 1 (AA = 98.1%) and BLN-Ce
4 (AA = 97%).
Changing of the lipid core of BLN systems by using
other two vegetable oils – Rso and Rbo, together with
the introduction of a hydrophilic plant extract – Wbe resulted in a clear
distinction of effectiveness of the two selected oils. Although the two native
oils show comparable antioxidant activity values, BLN systems prepared with Rso have demonstrated a better capacity to
capture free radicals, most likely composition of Rso having a key role in inducing
the antioxidant properties (e.g. AA BLN-Wbe 1
= 94.4% and AA BLN-Wbe-Ind 2 = 89.9%).
The determinations achieved by flow cytometry point out the induced effect of each BLN
tested on the process of apoptosis. The total
apoptosis induced
by BLN varied between 8.4 and 20.5% (e.g. 18.9% for BLN-free, 17.2%
for BLN-Wbe 2 and 20.5% for BLN-Wbe-Ind 1), but much lower as compared with the
apoptosis values induced by 50 mM H2O2
(29%) or 100 mM (46.2%).
Objective
4
The results of in
vitro release profiles of carotenoids achieved on three representative bio-nanocarriers loaded with various amounts of marigold extract, using diffusion cells
Franz, revealed a faster release of carotenoid from BLN prepared with one
vegetable oil – Ao
than those determined for BLN prepared with mixture of Hso and Ao. In the first case, a large
amount of released carotenoids, 76% has beed
determined after only 1h. Instead, BLN synthesized with blend of Hso and Ao ensured a
gradual release of carotenes into acceptor environment consisting of phosphate
buffer and ethanol. BLN-Ce 6 released
~29% carotenoids after 24 hours, while BLN-Ce
5 reached a cumulative amount of ~ 44% carotenoids detected in the receiver medium.
Conclusion
The research carried
out during the stage 2015 has
demonstrated that – by association of the biological and nutritional properties
of vegetable oils with those of plant extract containing antioxidant and
anti-inflammatory compounds with unique features of soft nanotechnology – valuable lipid
bio-nanocarriers (BLN) with multiple therapeutic benefices
have been successfully developed.
The encapsulation of natural plant mixtures, e.g. lyophilic extract derived from
Marigold plant (carotenoids extract, Ce),
hydrophilic extract isolated from willow (willow
bark extract, Wbe) into unique nanocarriers formulation based on various vegetable oils
(e.g. raspberry seed oil, hemp seed oil,
rice bran oil, amaranth oil etc.) resulted in highly efficient precursors
of health products, particularly for pharmaceutical industry. For instance, the
level of the antioxidant activity was ranged between 90 and 98% for almost all
the synthesized bio-nanocarriers, being proportional
with the extent of vegetable oils. BLN synthesized with a blend of Hso and Ao have been assured a slow release of
carotenoids (e.g. BLN-Ce
6 released ~29% carotenoids
after 24h, while BLN-Ce 5 has reached about 44% carotenoids detected
into the receptor environment).
The developed lipid bio-nanocarriers offer advantages of a minimum carrier cytotoxicity, good storage stability, synergistic effects, antioxidant and sustained release benefices, easy to scale up production.
STAGE 4 (December 2015 – November 2016)
OBJECTIVES and ACTIVITIES foreseen and achieved
in 2016
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Objective 1.
Pharmacokinetic behaviour on optimized BLN loaded with ivy
leaves extract or willow bark extract (part II) ·
Achievement of experimental models for conditioning
of loaded BLNs into topical formulations (e.g.
creams or hydrogel formulations). ·
Comparative evaluation of release profiles in skin simulation conditions. |
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Objective 2. Cosmetic efficacy of
developed topical formulations · Interpreting of the
specific parameters (e.g. SPF, UVA/ UVB ratio and critical wavelenghts - λc). ·
In vitro photostability
tests carried out before and after controlled irradiation on both UV-A and
UV-B domains. ·
Rheological behaviour and determination of hydration
effect of bioactive formulations based on vegetal extract – BLNs. |
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Objective 3.
Exploring therapeutic performance of some topical
formulation based on BLNs loaded with ivy leaves extract and willow bark
extract ·
In vitro evaluation of sustained –
release of the two main active principles from two types of conditioning
forms (cream or hydrogel). ·
Biological characterization of the optimized topical
formulations. In vitro cell viability study (comparative tests for
cytotoxicity assay). ·
In vitro tests for identification of some pharmaco-cosmetic formulations as new safety
bio-active hydrogels/creams based on natural principles. |
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SELECTIVE ASPECTS of 2016 research stage
The 2016 stage aimed
the investigation and conditioning some bio-nanostructured
lipids carriers (BLN) based on various vegetable oils, by developing topical
formulations with cosmetic profile that exhibit multiple functionalities. Among
the bio-nanocarriers previously synthesized, those
with superior performance, respectively BLN that co-encapsulates a natural
active principle - willow bark extract
(Wbe) and a
synthetic one - indomethacin (Ind) were conditioned as carbopol hydrogels,
and their therapeutic ability was evaluated based on in vitro release tests of both categories of natural and synthetic
active ingredients, and the healing properties of the skin, by assessing the in vitro anti-inflammatory effect. To
identify formulations with effective cosmetics on skin layers new types of nanocarriers with carrot oil (Co) and pomegranate seed oil (Pso) that show the ability to
co-encapsulate three active ingredients - a plant extract (willow bark extract - Wbe, ivy leaves extract
- Ile) and two sunscreens acting as
anti-UVA and anti-UVB (e.g.
2-ethylhexyl-2-cyano-3,3-diphenylacrylate, or octocrylene - OCT
and butyl metoxidibenzoilmetan - BMDBM) were synthesized. Exploring the cosmetic efficiency of
hydrogels containing BLN- Wbe /Ile-OCT-BMDBM
took into account to determine the in
vitro protection factor on both UVB and UVA areas (e.g. SPF and UVA-PF), the achieving of in-vitro photostability tests, before and
after a controlled UV-A and UV-B irradiation, and also the rheological behavior
tracking and determining of the hydration effect of bioactive formulations
based BLN with vegetable extract and sunscreens.
Objective 1
· Achievement of experimental models for conditioning of
loaded BLNs into topical formulations
Prior to the conditioning stage of BLNs
as hydrogel, synthesis of new types of bio-nanocarriers
co-loaded with ivy extract/willow bark extract
and sunscreens - OCT and BMDBM were conducted using the high shear homogenization
(HSH) coupled with high pressure homogenization (HPH).
Size characterization of new bio-nanocarriers,
based on DLS technique, highlighted the obtaining of free and loaded-BLNs with average
diameters between 73 and 171 nm. The use of carrot oil led to obtaining BLN with
average diameters smaller than when using pomegranate seed oil. The combination
of carrot oil with solid lipids resulted in a narrow size distribution of lipid
particle population, most of the BLNs formulations showing PDI values < 0.14.
Regarding the physical stability of
aqueous dispersions of BLN-Wbe/Ile-sunscreens,
the development of negative charges on the lipid particles surface was
highlighted for bio-nanocarriers that co-encapsulate
the three - vegetable and
synthetic - active principles, the zeta potential decreasing significantly
compared to the BLNs without plant extract and sunscreens (e.g. -51 ± 0.819 mV for BLN-Pso-Wbe-OCT-BMDBM versus -34.8 ± 0.306 mV, for BLN-Pso).
Structural characterization of new BLNs, based on DSC and UV-Vis analyses
revealed a good correlation between DSC parameters and encapsulation efficiency
of the two sunscreens.
Structural rearranging of lipid core
after capturing the plant extract and sunscreens was highlighted by comparing
DSC behavior of free BLNs and those that co-encapsulate the selected active
ingredients. A decrease in melting point and a change in melting enthalpy of
BLN loaded with active ingredients were detected by reducing the area under the
DSC curves.
Notably, a significant disruption of
the lipid core composed of carrot oil, cetyl
palmitate and glycerol monostearate was observed;
depletion of the endothermic peak seen in BLN-Co justifies the existence of a lipid core reorganization after Wbe/Ile and sunscreens encapsulation. This last statement is supported
by the appearance of the second melting shoulder (located at approx. 53oC)
that was not encountered in case of BLN-free.
Quantitative determinations have revealed greater efficiency to capture
BMDBM in all BLNs prepared with 30% Pso/Co (relative
to the total lipids concentration) compared to OCT, ranging from 76.4% to 89.5%
for BMDBM and between 57.8 to 84.4% for OCT. The best capture capability of the
two sunscreens was found in the case of NLC-Pso-Wbe-OCT-BMDBM, where
efficiencies of 89.50% for BMDBM and 84.4% for OCT have been determined.
After the characterization stages of BLNs,
the development of an experimental conditioning model of the lipid bio-nanocarriers in the form of a topical hydrogel formulation
has been achieved. Conditioning was carried out in two stages: preparation of carbopol hydrogel and incorporation of lyophilized BLNs into
the hydrogel. The hydrogel was prepared by adding a rheology-modifying agent - Carbopol 940 in an aqueous solution containing glycerol and ethanol. After 24h of
stirring at room temperature, the aqueous solution was added to a solution of triethanolamine in water, getting carbopol
hydrogel. An optimum ratio of 2:1 lyophilized BLN-Wbe/Ile-sunscreens to hydrogel was used for the development of topical
formulations.
· Comparative evaluation of release profiles in skin
simulation conditions.
The in vitro
release profiles of OCT and BMDBM from the topical hydrogels was investigated
for 24 hours. Hydrogels based BLN-willow bark
extract showed a slow release profile, with maintaining of high concentrations of OCT and BMDBM inside the nanocarriers.
At first view, a slower release of
BMDBM compared to OCT was observed in all developed topical formulations. The slow
release of BMDBM from BLN and subsequently from hydrogel
suggests its homogenous capturing inside the lipid core, also demonstrated by
the higher encapsulation efficiency values obtained for BMDBM compared to those
of OCT.
Regarding the influence of vegetable
oil, the amount of sunscreen released was significantly higher from BLN
prepared with carrot oil (e.g 20.3% OCT in BLN-Co, compared to 14.8% OCT in BLN-Pso, after 8h experiments).
Kinetic parameters, expressed through
the release rate constant (k), the release
exponent (n) of the OCT and BMDBM from
hydrogels based on bio-nanocarriers and the
correlation coefficient (R2)
demonstrated that all tested hydrogels correspond to the Higuchi model, which
describes a diffusion process governed by Fick's law.
Objective 2
· Interpretation of the specific parameters. In vitro
photostability tests carried out before and after
controlled irradiation on both UV-A and UV-B domains.
In order to determine the in vitro UV blocking effect of synthesized bio-nanocarriers
containing the willow bark extract
and two sunscreens acting on both UV-A and UV-B radiations, cosmetic
formulations of the hydrogel type were prepared in a BLN : carbopol
hydrogel 1 : 2. The hydrogel contains OCT 1.6%, 1.15% BMDBM and 6.92% carrot
oil or pomegranate seed oil.
The results demonstrated that
hydrogels based on BLN present significant anti-UV properties. SPF values (sun
protection factor in the UVB domain) and FP-UVA values (sun protection factor in
the UVA domain) determined for two of the four developed hydrogel formulations
demonstrate the benefits of using these systems which combine carrot oil and ivy leaves extract/willow bark extract in
association with minimal amounts of synthetic sunscreens.
Photoprotective behavior of the hydrogels containing BLNs under irradiation has
changed as follows:
ü UVB protection significantly
increased after irradiation for all four hydrogel formulations developed;
ü for hydrogels containing BLNs
prepared with carrot oil, a slight decrease in the UVA-PF was observed,
compared to initial value, while systems prepared with pomegranate seed oil, had
manifested a superior anti-UVA behavior after irradiation.
Results obtained at the determination of critical wavelength (representing
the wavelength at which 90% of the area under the curve absorbance is reached
in the 290-400 nm domain), have highlighted the fact that all hydrogels
containing BLNs shows a wide photoprotection spectrum,
the most effective being the BLNs-hydrogels prepared with carrot oil, which has
λc = 281 nm.
· Rheological behaviour and determination of hydration
effect of bioactive formulations based on vegetal extract
In order to evaluate the rheological behavior and
the hydration effect of synthesized bio-nanocarriers containing
willow bark extract and two anti-UV-A
and UV-B sunscreens, the same hydrogel formulations previously
developed were subjected to testing.
Assigning the rheological
behavior of the hydrogels was estimated based on the stress and frequency sweep tests. The results have shown that all hydrogels are
stable to about 5Pa.
For both hydrogels containing BLN-willow
bark extract prepared with carrot oil/ pomegranate seed oil, the results
have shown that the elastic modulus (G ',
Pa) is greater than the modulus of the leakage (G’’, Pa), which proves that the elastic component
is dominant over the viscous
one in the investigated frequency range.
To determine the hydration effect of topical formulations based
on BLN-willow bark extract, specific
equipment that has a smart system for measuring moisture levels in the skin was
used. By applying the two hydrogels onto the skin, an encouraging hydration potential
was determined for hydrogel formulations. The best result was recorded when the
hydrogel containing BLN-Pso-Wbe-OCT-BMDBM was tested; it shows
a dehydration prevent degree net superior compared with a control sample.
Objective 3
Among the categories of bio-nanocarriers synthesized
in previous 2015 and 2016 stages two suitable BLNs systems based on raspberry seed oil-Rso/rice bran
oil-Rbo were
selected for providing an advanced therapeutic potential: BLNs that
co-encapsulates a mix of active plant extract (willow bark extract) and a synthetic drug (indomethacin - Ind) to supplement the therapeutic effect. These bio-nanocarriers incorporated into carbopol
hydrogel were undergoing to in vitro
release tests, evaluated in terms of cell viability and were also subjected to
ELISA test for assigning the anti-inflammatory properties.
·
In vitro evaluation of sustained – release of the two main active
principles from two types of conditioning forms
Release profiles of indomethacin (Ind) and salicin (Sal), obtained
from in vitro release tests conducted
in Franz diffusion cells, had a biphasic pattern with a relatively quick
release in the first hour (most obvious in the case of salicin),
followed by a sustained release for the next 8h of experiment. The release rate
of Sal from BLN-hydrogels higher than those of Ind,
is due to a distribution thereof in the surfactants coating and also due to higher
solubility of Sal in the release medium consisting of phosphate buffer : alcohol.
In
vitro study of the
release kinetics of the two hydrogels based on BLN-Rso/Rbo-willow bark extract-indomethacin,
has shown that salicin and Indomethacin release
corresponds to the kinetic model of order 1. The kinetics of order 1 found in these
systems ensure continuous drug release rate in a diminished concentration (via controlled diffusion processes).
· Biological characterization of optimized topical formulations. The in vitro study of cellular viability
Assessment of proliferative versus cytotoxic character
of BLN systems loaded with willow
bark extract and Ind
was done by MTS colorimetric method. The results for bio-nanocarriers
showed a cell proliferation of free-BLN systems prepared with raspberry seed oil, the proliferation
also retrieved for very low concentrations of BLN-Wbe-Ind prepared with raspberry seed oil/rice
bran oil (12.5 mg/mL). For the other five
concentrations, ranging between 25 and 400 mg/mL,
the cell viability was maintained at values higher than 90%, which indicates a
lack of cytotoxicity induced by treatment of L 929 dermal cells with bio-nanocarriers developed in the stage 2015.
For the purpose of real time
comparison of proliferative versus cytotoxic capacity of BLN
systems on L929 cells,
a more appropriate RTCA
test was performed. To evidence these
parameters, the results were normalized to untreated cells. The
results have highlighted different actions of BLN
systems depending on the vegetable oil
type, treatment time and the type of active principles co-encapsulated
into lipid nanocarriers.
·
In vitro tests for identification of some pharmaco-cosmetic formulations as new safety bio-active
hydrogels/creams based on natural principles.
Anti-inflammatory activity was assessed
by evaluating the production of pro-inflammatory cytokines (IL-6, TNF-alpha) in
a cellular model that consisted in stressing the L929 cells by treatment with hydrogen
peroxide. When the treatment with H2O2 was applied over
the cells previously treated with BLNs, production of pro-inflammatory
cytokines TNF-alpha and IL-6 was inhibited as compared with the single
treatment with H2O2. The anti-inflammatory effect of BLNs
was dose-dependent; treatments with concentrations of 200 mg/mL
lead to a greater reduction in the release of TNF-alpha than those with 50 mg/mL, higher dose more efficiently
counteracting the oxidative activity of H2O2.
Conclusions
Research conducted within the "Bio-active lipid nanocarriers for co-encapsulation of selective vegetal
extracts with enhanced pharmaco-cosmetic performance" project has been
finalized with the development of topical hydrogel formulations containing
different categories of bio-active
lipid nanocarriers (BLN):
ü BLNs based on carrot oil and
oil pomegranate that co-encapsulate three active ingredients - willow bark extract/ivy extract and two anti-UVA
and anti-UVB sunscreens (2-ethylhexyl-2-cyano-3, 3-diphenylacrylate and butyl metoxidibenzoil-metan);
ü BLNs based on raspberry seed oil
and rice bran oil that co-encapsulate a natural active ingredient - willow extract and one synthetic active
- indomethacin.
The incorporation of active natural
and synthetic ingredients into lipid bio-nanocarriers
and their formulation in a semi-solid vehicle with application in the topical
treatment of skin lesions can significantly enhance the therapeutic efficacy of
the plant extract and sunscreens in addition to reducing the side effects
caused by high concentrations of synthetic filters commonly used in commercial
cosmetic formulations that exhibit a high degree of irritability.
Topical treatment with BLNs for
different skin diseases that involve the occurrence of inflammation, has the
advantage that a high level of active substance can be provided at the affected
tissue, while the systemic side effects can be reduced compared to
administration by oral or parenteral administration.
Size and structural aspects, physicochemical properties
of the carrier system completed by the type of vegetable actives encapsulated
and their specific biological effects, are considered
to be the main features that provide differentiated distribution of active compounds
into the skin and a desired therapeutic action. The nanocarriers
systems by BLNs type were investigated in this project to create so-called
"enabling environment" for the development of cosmetic formulations
for topical application that lead to improved treatment of skin diseases such
as atopic eczema, psoriasis, and other skin inflammation.
RESULTS DISSEMINATION
|
Stages |
Foreseen in the project proposal |
Achieved |
|
2013-2014 |
Papers: 2 (FI > 3) Patents: 0 Conferences: 2 |
Papers: 3 Patents: 1 Conferences: 2 |
|
2015 |
Papers: 1 (FI > 2) Conferences: 1 |
Papers: 4 Conferences: 4 |
|
2016 |
Papers: 1 (FI > 2) Conferences: 1 |
Papers: 1 Conferences: 2 |
|
TOTAL |
Papers: 4 (FI > 7) International conferences: 4 Patents: 0 |
Papers: 8 (FI = 19.238) International conferences: 8 |
Papers published in ISI journals:
1.
Lacatusu I., Badea N., Niculae G., Bordei N., Stan R., Meghea A.,
Lipid nanocarriers based on natural compounds: an evolving role in further
plant extracts delivery, Eur. J. Lipid
Sci. Tehnol., 116(12), 2014, 1708-1717 (IF =
1.953; WOS: 000346068700013).
2.
Mitrea E., Ott C., Meghea A., New approaches on the synthesis of effective
nanostructured lipid carriers, Rev. Chim., 2014, 65, 1
(IF = 0.956).
3.
Mitrea E., Lacatusu I., Badea N., Ott C., Oprea O., Meghea A., New Approach to Prepare Willow Bark Extract – Lipid Based Nanosystems
with Enhanced Antioxidant Activity, Journal of Nanoscience and Nanotechnology, 2015, 15(6), 4080-4089
(IF = 1.338; WOS: 000347435300009).
4.
Lacatusu I., Badea N., Badea G., Oprea O., Mihaila M.A., Kaya D.A., Stan R., Meghea A. Lipid nanocarriers based on
natural oils with high activity against oxygen free radicals and tumor cell
proliferation, Materials Science and Eng. C, 2015, 56, 88-94
(IF = 3.420; WOS:
000359873900011).
5.
Badea G., Lacatusu I., Ott C., Badea N., Grafu I., Meghea, A. Integrative approach in prevention
and therapy of basal cellular carcinoma by association of three actives loaded
into lipid nanocarriers, J. of Photochem. and Photobiol. B: Biology, 2015, 147, 1-8
(IF = 3.035; WOS: 000354156000001).
6.
Badea G., Lacătusu I., Badea N., Ott C., Meghea A., Use of various vegetable oils in designing photoprotective nanostructured formulations for UV
protection and antioxidant activity, Ind.
Crops and Products, 2015, 67,
18-24 (IF = 3.449; WOS: 000352039600004).
7.
Badea G., Bors A., Lacatusu
I., Oprea O., Ungureanu C.,
Stan R., Meghea A., Influence of basil oil
extract on antioxidant and antifungal activity of nanostructured
carriers loaded with Nystatin, CR Chim., 2015,
18, 668-667 (IF: 1.798; WOS: 000357703400012).
8.
Lacatusu I., Badea N.,
Badea G.,
Brasoveanu L., Stan R., Ott C., Oprea O., Meghea A.,
Ivy leaves extract based – lipid nanocarriers
and their bioefficacy on antioxidant and antitumor
activities, RSC
Advances, 2016, 6,
77243-77255 (IF = 3.289, WOS: 000382482200015).
Patents:
Patent application A/005821
(since 05.12.2013) „Nanostructured
lipid carriers based on vegetal oils with photoprotective
and antioxidant properties”, Authors: Gabriela
Niculae, Ioana Lăcătuşu, Nicoleta
Badea, Raluca Stan, Aurelia Meghea.
International
conferences:
1. N. Badea, I. Lacatusu, G. Niculae, R. Stan, A. Meghea, “Antioxidant effect of Laurel leaf extract loaded into bioactive lipid nanostructure”, 55th International Conference on the Bioscience of Lipids, 22 – 28.06.2014, Aberdeen, Scoţia.
2. A. Bors, I. Lacatusu, N. Badea, R. Stan, A. Meghea, Effect of savory oil on the antioxidant and antifungal properties of Nystatin loaded lipid nanocarriers, 4th International Colloids Conference, 14 – 19.06.2014, Madrid, Spania.
3.
R. Stan, I.
Lacatusu, N. Badea, G. Niculae, D. Istrati, A. Meghea Influence of various vegetable oils on designing
appropriate lipid nanocarriers for encapsulation of ivy
leaves extract, 13th Int. Conf. on Frontiers of Polymers and Advanced Materials
(ICFPAM 2015), 30.03- 02.04.2015,
Maroc.
4. C. Ott, I. Lacatusu, N.
Badea, G. Badea, R. Stan,
A. Meghea, The valuable effect of vegetable oils on the improvment of hydrophyl extract
encapsulation into lipid nanocarriers”, Conf.
Int. RICCCE 19, 02– 05.09.2015, Sibiu, Romania.
5. A. Meghea, G. Badea, I. Lacatusu, N. Badea, „Novel nanostructured lipid carriers for drug
delivery by co-encapsulating synthetic and natural bioactive compounds”, BIT’s 5th Annual Symposium of
Drug Delivery System-2015 New Horizons in Improved Drug Delivery Technology,
20-22.10.2015, Beijing, China.
6. A. Meghea, G. Badea, I. Lacatusu, N. Badea, 13th
Annual Congress of International Drug Discovery Science & Technology, Terapy and Expo - IDDST 2015, 20-22 Octomber, Beijing, China.
7.
N. Badea, I. Lacatusu, G. Badea, A. Meghea, The efficiency of lipid
nanocarriers in accommodation of hydrophilic plant
mixture, Nanotexnology 2016, 13th
Int. Conference on Nanosciences &
Nanotechnologies (NN16), 2-9.07.2016,
Thessaloniki, Grecia,
pag 229.
8.
A. Meghea, I. Lacatusu, N. Badea, G. Badea, Novel nanostructured lipid carriers for
co-encapsulation of selective plant extracts with enhanced pharmaco-cosmetic
performance, 14th Int.
Conference on Frontiers of Polymers and Advanced Materials (14th
ICFPAM), 29.10.2016-06.11.2016, Daejeon, Coreea,
pag. 155.