Plant Glucosylceramides and Mosquito Repellent Essential Oils Loaded Nano Carriers for Topical Applications: Formulation, Optimization and In Vitro Release Studies
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Date
2023-09
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Addis Ababa University
Abstract
Skin protects the body against the invasion of external pathogens, allergens, and chemical
substances. It is considered that most of the skin barrier function resides in the epidermis,
particularly in its outermost layer, stratum corneum (SC). The SC is composed of
corneocytes embedded in a lipid-enriched intercellular matrix. Ceramides (CERs), free
fatty acids (FFAs), and cholesterol are the major components of the lipid matrix in an
approximately 1:1:1 molar ratio. The unique molecular organization and the equi-molar
ratio are considered crucial in barrier function and semipermeable nature of the SC.
Compositional and organizational changes in SC lipids such as CERs cause defective skin
barrier function in relation with ageing and skin disorders partly due to altered enzymatic
activity. However, the glucocerebrosidase enzyme responsible for the conversion of
glucosylceramides (GlcCERs) to CERs is not affected by ageing. It has been shown that
direct topical replacement of depleted native skin CERs has beneficial effects in improving
skin barrier function and skin hydration. CERs are obtained from animal, plant and
synthetic sources. Plants are more reliable sources of CERs, as animal and synthetic CERs
have safety/ethical issues and affordability challenge, respectively. Though plants are
preferred, most of the plant sphingolipids (SPLs) are available in a complex form with a
polar head group attached to the CERs. It requires an economical and effective method of
hydrolysis to produce CERs from dominant SPLs such as GlcCERs. Moreover, the poor
solubility and permeability of CERs on top of normal SC barrier property make the delivery
of topical CERs challenging using conventional formulations.
While GlcCERs/CERs are intended to be delivered to the upper layer of the skin, the
present study also considered formulating essential oils having mosquito repellent activity
for topical application. The mosquito repellent potential of plant materials has been
exploited for centuries using different means, including applying essential oils on the skin
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and clothes to combat transmission of malaria. However, due to the volatile nature of
essential oils, the protection against mosquitoes is short-lived.
Therefore, the objectives of this PhD work were to explore potential GlcCER plant sources,
investigate suitable chemical and enzymatic methods of hydrolysis of plant GlcCERs into
CERs and develop an optimized nanoemulgel formulation for topical delivery of the plant
GlcCERs as well as to formulate citronella oil (CO) (Cymbopogon nardus) and palmarosa
oil (PO) (C. martini) in the form of nanoemulgel (NEG) to delay the volatility of the
essential oils upon application.
GlcCERs were isolated from lupin bean (Lupinus albus), mung bean (Vigna radiate) and
naked barley (Hordium vulgare). The GlcCERs were identified using ultra high-
performance liquid chromatography hyphenated with atmospheric pressure chemical
ionization-high resolution tandem mass spectrometer (UHPLC/APCI-HRMS/MS) and
quantified with validated automated multiple development-high performance thin layer
chromatography (AMD-HPTLC) method. The GlcCERs were hydrolyzed into CERs with
mild acid hydrolysis (0.1N HCl) following treatment with an oxidizing agent, NaIO4, and
reducing agent, NaBH4. After securing ethical clearance, excised human skin was
employed to investigate the enzymatic hydrolysis of plant GlcCERs. Homogenized
epidermis in citrate phosphate buffer was incubated with lupin GlcCERs. The total lipid
was extracted with MeOH/CHCl3/H2O solvent mixture and the unhydrolyzed lupin
GlcCERs in the extract was quantified using a UHPLC-QqQ-MS/MS method in MRM
(multiple ion reaction monitoring) mode (m/z 714.5 →696.54→ 262.25). Formulations of
GlcCERs were developed for topical delivery. The Nanoemulsion (NE) components were
screened and pseudo-ternary phase diagrams were constructed at different hydrophilic-
lipophilic balance (HLB) values of surfactant-co-surfactant mixture (Smix). Extreme
vertices mixture design was developed to investigate the impact of percentage
compositions of the independent variables; oil mixture (2-3%), Smix (15-18%) and aqueous
component (79-83%) on the globule size of the NEs. NEG was prepared from optimized
0.25% lupin GlcCER NE and Carbopol 980 gel. The membrane penetration depths of
GlcCERs loaded optimized NEs and NEGs were measured.
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NE for both essential oils were also prepared using ultrasonication technique following
suitable HLB selection of Smix and phase diagram development. NEGs were formulated by
mixing NEs and Carbopol 934 gel. Physicochemical characterizations, including in vitro
release and permeation studies were carried out. The Gas chromatography hyphenated
mass spectrometry method for identification and quantification of essential oils was
validated.
GlcCERs with 4,8-sphingadienine, 8-sphingenine and 4-hydroxy-8-sphingenine sphingoid
bases linked with C14 to C26 α-hydroxylated FAs were identified from all the three plants.
Single GlcCER (m/z 714.5520) was dominant in lupin and mung beans, while five major
GlcCERs species (m/z 714.5520, m/z 742.5829, m/z 770.6144, m/z 842.6719 and m/z
844.56875) were obtained from naked barley. The GlcCERs contents of the three plants
were comparable. However, lupin bean contained predominantly a single GlcCER (m/z
714.5520). CER species bearing 4,8-sphingadienine and 8-sphingenine sphingoid bases
attached to C14 to 24 FAs were found after mild acid hydrolysis. CER species with m/z
552.4992 was the main component in the beans while CER with m/z 608.5613 was
dominant in the naked barley. However, CERs with 4-hydroxy-8-sphingenine sphingoid
base were not detected with UHPLC-HRMS/MS. Following skin enzyme hydrolysis
investigation, the characteristic signals of GlcCER fragments (m/z 696.54, 552.49 and
534.48) due to in-source fragmentation were detected in the MS spectra of the skin extract.
The MS2 fragmentation of the dominant fragment (m/z 696.54) ion provided the target
product ion (m/z 262.25) which was integrated to quantify GlcCER. The LC-MS method
was selective, precise and accurate. It was also free from matrix and carryover effects. The
unhydrolyzed lupin GlcCER amount decreased with time suggesting hydrolysis of
GlcCERs by the skin enzymes. An optimized NE formulation was prepared. The HLB
values of 13.5 and 12 provided broader NE regions for Miglyol and isopropyl myristate,
respectively. The analysis of variance of the quadratic model showed suitability of the
model with R2 of 99.80% and non-significant lack of fit (F value= 17.06). The optimized
percentage compositions of oil phase, Smix and aqueous phase were 2.15%, 16.39% and
81.46%, respectively, with predicted globule size of 23.96 nm. Accordingly, the optimized
NE globule size, polydispersity index and zeta potential were 23.93 ± 0.25 nm, 0.069 ±
0.017 and 23.95± 1.20 mV, respectively. The oil globules were spherical and distributed
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uniformly without aggregation. The NE exhibited Newtonian flow with a viscosity of 6.75
mPa.s, while NEGs showed non-Newtonian flow with shear thinning property. The amount
of lupin GlcCER released and penetrated into each model membrane layer at different time
points was in the order of NEs > basic cream > NEGs. After 180 min, 51%, 84% and 96%
of lupin GlcCER was released and penetrated into the model membrane layers from NEG,
basic cream and NEs, respectively.
The formulation developed for the topical application of essential oils employed CO and
PO as the oil components. Tween 80, Transcutol P and distilled water with 40% glycerol
were selected as surfactant, co-surfactant and aqueous components of the NE, respectively.
HLB values of 11 and 12 were found to be appropriate for CO and PO NEs preparation,
respectively. Clear CO and PO NEs with an average globule size of 131.34 nm and 120.77
nm, respectively, were obtained. The NEG preparations of both essential oils were
kinetically stable, and the formulation components were chemically compatible with each
other.
In conclusion, considering affordability, GlcCER content and yield, lupin bean would be
the preferred alternative commercial source of GlcCERs. The mild acid hydrolysis method
is economical and effective, mainly for plant GlcCERs carrying dihydroxy sphingoid
bases. Plant GlcCER hydrolysis in skin homogenate, being investigated for the first time,
the findings pave the way for a new mode of skin barrier function enhancement modality.
Taking into account the drawbacks of animal and synthetic CERs on top of enzymatic and
chemical methods of plant GlcCER hydrolysis limitations, topical administration of plant
GlcCER to enhance skin barrier function would be the preferred alternative. The NEG
formulation developed for dermal delivery of lupin GlcCERs prolonged the release and
slowed down the penetration of GlcCER into the multilayer membrane model which is
crucial to limit its penetration into the epidermal skin layer. Therefore, NEGs could be
considered as an option for the delivery of plant GlcCER into the upper part of the skin
after further ex-vivo investigation, as the study is the first of its kind.
Regarding formulations of the essential oils, NEGs prolonged the release of both CO and
PO up to 24 h and significantly reduced the percentage permeation via cellulose acetate
membrane as compared to NEs. Therefore, NEG could be an alternative formulation to
prolong the mosquito repellent effect of essential oils.
Description
Keywords
Ceramide, Glucosylceramide, Lupinus albus, Vigna radiate, Hordium vulgare, Mild acid hydrolysis, Enzyme hydrolysis, LC-HRMS/MS, Structural characterization, Method validation, Topical delivery, Nanoemulsion, Nanoemulgel, Optimization, Extreme vertices mixture design, Mosquito repellent, Essential oil, Cymbopogon nardus, Cymbopogon martini, Ultrasonication